CDK5 as a Therapeutic Target in Prostate Cancer Metastasis

National Research Council Canada - National Science Library

Nelkin, Barry D

2008-01-01

We have recently found that CDK5 is active in prostate cancer cell lines and in almost all human metastatic prostate cancers, and inhibition of CDK5 activity resulted in reduction of spontaneous metastases by 79...

CDK5 as a Therapeutic Target in Prostate Cancer Metastasis

National Research Council Canada - National Science Library

Nelkin, Barry

2007-01-01

We have recently found that CDK5 is active in prostate cancer cell lines and in almost all human metastatic prostate cancers, and inhibition of CDK5 activity resulted in reduction of spontaneous metastases by 79...

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

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

2012-01-01

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

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

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Grassmann Ralph

2005-09-01

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

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

International Nuclear Information System (INIS)

Zhang, Yue; Li, Hongbo; Hao, Jun; Zhou, Yi; Liu, Wei

2014-01-01

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

Critical role of CDK11p58 in human breast cancer growth and angiogenesis

International Nuclear Information System (INIS)

Chi, Yayun; Huang, Sheng; Peng, Haojie; Liu, Mengying; Zhao, Jun; Shao, Zhiming; Wu, Jiong

2015-01-01

A capillary network is needed in cancer growth and metastasis. Induction of angiogenesis represents one of the major hallmarks of cancer. CDK11 p58 , a Ser/Thr kinase that belongs to the Cell Division Cycle 2-like 1 (CDC2L1) subfamily is associated with cell cycle progression, tumorigenesis, sister chromatid cohesion and apoptotic signaling. However, its role in breast cancer proliferation and angiogenesis remains unclear. Tumorigenicity assays and blood vessel assessment in athymic mice were used to assess the function of CDK11 p58 in tumor proliferation and angiogenesis. CCK-8 assay was used to detect breast cancer cell growth. Immunohistochemistry was used to detect the expression of vascular endothelial growth factor (VEGF), CD31 and CD34 in CDK11 positive patient breast cancer tissues. Dual-Luciferase array was used to analyze the function of CDK11 p58 in the regulation of VEGF promoter activity. Western blot was used to detect related protein expression levels. CDK11 p58 inhibited breast cancer growth and angiogenesis in breast cancer cells and in nude mice transplanted with tumors. Immunohistochemistry confirmed that CDK11 p58 was negatively associated with angiogenesis-related proteins such as VEGF, CD31 and CD34 in breast cancer patients. Real-time PCR and dual-luciferase assay showed CDK11 p58 inhibited the mRNA levels of VEGF and the promoter activity of VEGF. As CDK11 p58 is a Ser/Thr kinase, the kinase-dead mutant failed to inhibit VEGF mRNA and promoter activity. Western blot analysis showed the same pattern of related protein expression. The data suggested angiogenesis inhibition was dependent on CDK11 p58 kinase activity. This study indicates that CDK11 p58 inhibits the growth and angiogenesis of breast cancer dependent on its kinase activity. The online version of this article (doi:10.1186/s12885-015-1698-7) contains supplementary material, which is available to authorized users

Targets downstream of Cdk8 in Dictyostelium development

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Skelton Jason

2011-01-01

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

Dopamine signaling negatively regulates striatal phosphorylation of Cdk5 at tyrosine 15 in mice.

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Yukio eYamamura

2013-02-01

Full Text Available Striatal functions depend on the activity balance between the dopamine and glutamate neurotransmissions. Glutamate inputs activate cyclin-dependent kinase 5 (Cdk5, which inhibits postsynaptic dopamine signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa at Thr75 in the striatum. c-Abelson tyrosine kinase (c-Abl is known to phosphorylate Cdk5 at Tyr15 (Tyr15-Cdk5 and thereby facilitates the Cdk5 activity. We here report that Cdk5 with Tyr15 phosphorylation (Cdk5-pTyr15 is enriched in the mouse striatum, where dopaminergic stimulation inhibited phosphorylation of Tyr15-Cdk5 by acting through the D2 class dopamine receptors. Moreover, in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine mouse model, dopamine deficiency caused increased phosphorylation of both Tyr15-Cdk5 and Thr75-DARPP-32 in the striatum, which could be attenuated by administration of L-3,4-dihydroxyphenylalanine and imatinib (STI-571, a selective c-Abl inhibitor. Our results suggest a functional link of Cdk5-pTyr15 with postsynaptic dopamine and glutamate signals through the c-Abl kinase activity in the striatum.

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

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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

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

DEFF Research Database (Denmark)

Kunick, Conrad; Lauenroth, Kathrin; Wieking, Karen

2004-01-01

',2':4,5]pyrrolo[3,2-d][1]benzazepine. The best statistical values for the CoMSIA were obtained for the CDK1-models (r(2)() = 0.929 and q(2)() = 0.699), which were clearly superior to the models for CDK5 (r(2)() = 0.874 and q(2)() = 0.652) and GSK-3 (r(2)() = 0.871 and q(2)() = 0.554)....... data of 52 paullone entities, which were aligned by a docking routine into the ATP-binding cleft of a CDK1/cyclin B homology model. Variation of grid spacing and column filtering were used during the optimization of the models. The predictive ability of the models was shown by a leave-one-out cross...

Cdk7 Is Required for Activity-Dependent Neuronal Gene Expression, Long-Lasting Synaptic Plasticity and Long-Term Memory

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Guiqin He

2017-11-01

Full Text Available In the brain, de novo gene expression driven by learning-associated neuronal activities is critical for the formation of long-term memories. However, the signaling machinery mediating neuronal activity-induced gene expression, especially the rapid transcription of immediate-early genes (IEGs remains unclear. Cyclin-dependent kinases (Cdks are a family of serine/threonine kinases that have been firmly established as key regulators of transcription processes underling coordinated cell cycle entry and sequential progression in nearly all types of proliferative cells. Cdk7 is a subunit of transcriptional initiation factor II-H (TFIIH and the only known Cdk-activating kinase (CAK in metazoans. Recent studies using a novel Cdk7 specific covalent inhibitor, THZ1, revealed important roles of Cdk7 in transcription regulation in cancer cells. However, whether Cdk7 plays a role in the regulation of transcription in neurons remains unknown. In this study, we present evidence demonstrating that, in post-mitotic neurons, Cdk7 activity is positively correlated with neuronal activities in cultured primary neurons, acute hippocampal slices and in the brain. Cdk7 inhibition by THZ1 significantly suppressed mRNA levels of IEGs, selectively impaired long-lasting synaptic plasticity induced by 4 trains of high frequency stimulation (HFS and prevented the formation of long-term memories.

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

Science.gov (United States)

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.

Cdk1 Activates Pre-Mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem cells

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Baffet, Alexandre D.; Hu, Daniel J.; Vallee, Richard B.

2015-01-01

Summary Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells, and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2, via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell cycle regulated, and identify the trigger mechanism for apical nuclear migration in the brain. PMID:26051540

An ERK/Cdk5 axis controls the diabetogenic actions of PPARγ.

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Banks, Alexander S; McAllister, Fiona E; Camporez, João Paulo G; Zushin, Peter-James H; Jurczak, Michael J; Laznik-Bogoslavski, Dina; Shulman, Gerald I; Gygi, Steven P; Spiegelman, Bruce M

2015-01-15

Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ (peroxisome proliferator-activated receptor γ) at serine 273 by cyclin-dependent kinase 5 (Cdk5) stimulates diabetogenic gene expression in adipose tissues. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic drugs that bind PPARγ, such as the thiazolidinediones and PPARγ partial agonists or non-agonists. For a better understanding of the importance of this obesity-linked PPARγ phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. These mice have both a paradoxical increase in PPARγ phosphorylation at serine 273 and worsened insulin resistance. Unbiased proteomic studies show that extracellular signal-regulated kinase (ERK) kinases are activated in these knockout animals. Here we show that ERK directly phosphorylates serine 273 of PPARγ in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MAP kinase/ERK kinase (MEK). Importantly, pharmacological inhibition of MEK and ERK markedly improves insulin resistance in both obese wild-type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPARγ function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes.

Enhancement of radioresponse by combined treatment with flavopiridol, a cycline dependent kinase (CDK) inhibitor, in oral cancer cells

International Nuclear Information System (INIS)

Mihara, Mariko; Mano, Takamitsu; Ueyama, Yoshiya; Shintani, Satoru; Li, Syunnann; Klosek, S.; Hamakawa, Hiroyuki

2005-01-01

Cyclin dependent kinases (CDKs) play a pivotal role in cell cycle regulation. Flavopiridol is known to potently inhibit such CDKs as CDK1, CDK2, CDK4, CDK7. We already reported that flavopiridol inhibited the growth of oral squamous cell carcinoma (OSCC) cells and induced apoptosis in OSCC cells. In the present study, we investigated whether the treatment with flavopiridol improves the response to radiosensitivity in OSCC cell lines. In an in vitro study, there was a cooperative antiproliferative effect of combined treatment with flavopiridol and radiation in OSCC cell lines. Tumor xenograft studies demonstrated that the combination of flavopiridol and radiation caused growth inhibition and tumor regression of well-established OSCC tumor in athymic mice. Overall, we concluded that flavopiridol enhances tumor radioresponse and it is considered a suitable candidate drug in the treatment of oral cancer. (author)

CDK9-dependent RNA polymerase II pausing controls transcription initiation.

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Gressel, Saskia; Schwalb, Björn; Decker, Tim Michael; Qin, Weihua; Leonhardt, Heinrich; Eick, Dirk; Cramer, Patrick

2017-10-10

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

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

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Kumari Ratna

2010-07-01

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

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

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Xu Tang

2016-01-01

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

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

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Hemant Varma

2007-12-01

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

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

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Franziska Graf

2009-01-01

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

A cdk1 gradient guides surface contraction waves in oocytes.

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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.

Iron chelators ICL670 and 311 inhibit HIV-1 transcription

International Nuclear Information System (INIS)

Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R.; Ray, Patricio E.; Gordeuk, Victor R.; Nekhai, Sergei

2007-01-01

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

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.

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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.

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

International Nuclear Information System (INIS)

Irazoqui, Ana P.; Heim, Nadia B.; Boland, Ricardo L.; Buitrago, Claudia G.

2015-01-01

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

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

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Sanghoon Lee

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

Anticancer screening of medicinal plant phytochemicals against Cyclin-Dependent Kinase-2 (CDK2: An in-silico approach

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Wajahat Khan

2017-08-01

Full Text Available Background: Cyclin-Dependent Kinase-2 (CDK2 is a member of serine/threonine protein kinases family and plays an important role in regulation of various eukaryotic cell division events. Over-expression of CDK2 during cell cycle may lead to several cellular functional aberrations including diverse types of cancers (lung cancer, primary colorectal carcinoma, ovarian cancer, melanoma and pancreatic carcinoma in humans. Medicinal plants phytochemicals which have anticancer potential can be used as an alternative drug resource. Methods: This study was designed to find out anticancer phytochemicals from medicinal plants which could inhibit CDK2 with the help of molecular docking technique. Molecular Operating Environment (MOE v2009 software was used to dock 2300 phytochemicals in this study. Results: The outcome of this study shows that four phytochemicals Kushenol T, Remangiflavanone B, Neocalyxins A and Elenoside showed the lowest S-score (-17.83, -17.57, -17.26, -17.17 respectively and binds strongly with all eight active residues Tyr15, Lys33, Ileu52, Lys56, Leu78, phe80, Asp145 and Phe146 of CDK2 binding site. These phytochemicals could successfully inhibit the CDK2. Conclusion: These phytochemicals can be considered as potential anticancer agents and used in drug development against CDK2. We anticipate that this study would pave way for phytochemical based novel small molecules as more efficacious and selective anti-cancer therapeutic compounds.

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

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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.

Altered expression of the Cdk5 activator-like protein, Cdk5α, causes neurodegeneration, in part by accelerating the rate of aging

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Joshua Spurrier

2018-03-01

Full Text Available Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. Here, we develop a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. We applied this metric to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5α, the ortholog of mammalian p35. Cdk5α-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, our results suggest that Cdk5α-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.

Analysis list: CDK9 [Chip-atlas[Archive

Lifescience Database Archive (English)

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-03-27

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

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

Science.gov (United States)

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.

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

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Pinhero Reena

2004-01-01

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

Anti-diabetes drug pioglitazone ameliorates synaptic defects in AD transgenic mice by inhibiting cyclin-dependent kinase5 activity.

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Jinan Chen

Full Text Available Cyclin-dependent kinase 5 (Cdk5 is a serine/threonine kinase that is activated by the neuron specific activators p35/p39 and plays many important roles in neuronal development. However, aberrant activation of Cdk5 is believed to be associated with the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease (AD and Parkinson's disease (PD. Here in the present study, enhanced Cdk5 activity was observed in mouse models of AD; whereas soluble amyloid-β oligomers (Aβ, which contribute to synaptic failures during AD pathogenesis, induced Cdk5 hyperactivation in cultured hippocampal neurons. Inhibition of Cdk5 activity by pharmacological or genetic approaches reversed dendritic spine loss caused by soluble amyloid-β oligomers (Aβ treatment. Interestingly, we found that the anti-diabetes drug pioglitazone could inhibit Cdk5 activity by decreasing p35 protein level. More importantly, pioglitazone treatment corrected long-term potentiation (LTP deficit caused by Aβ exposure in cultured slices and pioglitazone administration rescued impaired LTP and spatial memory in AD mouse models. Taken together, our study describes an unanticipated role of pioglitazone in alleviating AD and reveals a potential therapeutic drug for AD curing.

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

DEFF Research Database (Denmark)

Baumer, Nicole; Tickenbrock, Lara; Tschanter, Petra

2011-01-01

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

Cdk5 Is Essential for Amphetamine to Increase Dendritic Spine Density in Hippocampal Pyramidal Neurons

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Soledad Ferreras

2017-11-01

Full Text Available Psychostimulant drugs of abuse increase dendritic spine density in reward centers of the brain. However, little is known about their effects in the hippocampus, where activity-dependent changes in the density of dendritic spine are associated with learning and memory. Recent reports suggest that Cdk5 plays an important role in drug addiction, but its role in psychostimulant’s effects on dendritic spines in hippocampus remain unknown. We used in vivo and in vitro approaches to demonstrate that amphetamine increases dendritic spine density in pyramidal neurons of the hippocampus. Primary cultures and organotypic slice cultures were used for cellular, molecular, pharmacological and biochemical analyses of the role of Cdk5/p25 in amphetamine-induced dendritic spine formation. Amphetamine (two-injection protocol increased dendritic spine density in hippocampal neurons of thy1-green fluorescent protein (GFP mice, as well as in hippocampal cultured neurons and organotypic slice cultures. Either genetic or pharmacological inhibition of Cdk5 activity prevented the amphetamine–induced increase in dendritic spine density. Amphetamine also increased spine density in neurons overexpressing the strong Cdk5 activator p25. Finally, inhibition of calpain, the protease necessary for the conversion of p35 to p25, prevented amphetamine’s effect on dendritic spine density. We demonstrate, for the first time, that amphetamine increases the density of dendritic spine in hippocampal pyramidal neurons in vivo and in vitro. Moreover, we show that the Cdk5/p25 signaling and calpain activity are both necessary for the effect of amphetamine on dendritic spine density. The identification of molecular mechanisms underlying psychostimulant effects provides novel and promising therapeutic approaches for the treatment of drug addiction.

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

Science.gov (United States)

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).

A novel role for the cell cycle regulatory complex cyclin D1-CDK4 in gluconeogenesis

OpenAIRE

Hosooka, Tetsuya; Ogawa, Wataru

2016-01-01

Dysregulation of gluconeogenesis is a key pathological feature of type 2 diabetes. However, the molecular mechanisms underlying the regulation of gluconeogenesis remain unclear. Bhalla et?al. recently reported that cyclin D1 suppresses hepatic gluconeogenesis through CDK4?dependent phosphorylation of PGC1alpha and consequent inhibition of its activity. The cyclin D1?CDK4 might thus serve as an important link between the cell cycle and control of energy metabolism through modulation of PGC1alp...

Novel Alternative Splice Variants of Mouse Cdk5rap2.

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Nadine Kraemer

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

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

Science.gov (United States)

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.

Targeting the AKT/GSK3β/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy

International Nuclear Information System (INIS)

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

2011-01-01

Purpose: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3β-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3β/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Methods and Materials: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Results: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Conclusion: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor eradication in

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

International Nuclear Information System (INIS)

Boudra, M.T.

2011-12-01

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

Cdk phosphorylation of the Ste11 transcription factor constrains differentiation-specific transcription to G1

DEFF Research Database (Denmark)

Kjaerulff, Søren; Andersen, Nicoline Resen; Borup, Mia Trolle

2007-01-01

Eukaryotic cells normally differentiate from G(1); here we investigate the mechanism preventing expression of differentiation-specific genes outside G(1). In fission yeast, induction of the transcription factor Ste11 triggers sexual differentiation. We find that Ste11 is only active in G(1) when...... Cdk activity is low. In the remaining part of the cell cycle, Ste11 becomes Cdk-phosphorylated at Thr 82 (T82), which inhibits its DNA-binding activity. Since the ste11 gene is autoregulated and the Ste11 protein is highly unstable, this Cdk switch rapidly extinguishes Ste11 activity when cells enter...... S phase. When we mutated T82 to aspartic acid, mimicking constant phosphorylation, cells no longer underwent differentiation. Conversely, changing T82 to alanine rendered Ste11-controlled transcription constitutive through the cell cycle, and allowed mating from S phase with increased frequency...

Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1

International Nuclear Information System (INIS)

Wierstra, Inken; Alves, Juergen

2008-01-01

FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27

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

International Nuclear Information System (INIS)

Vanhara, Petr; Bryja, Vitezslav; Horvath, Viktor; Kozubik, Alois; Hampl, Ales; Smarda, Jan

2007-01-01

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

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

Science.gov (United States)

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.

Knockdown of CDK2AP1 in human embryonic stem cells reduces the threshold of differentiation.

Directory of Open Access Journals (Sweden)

Khaled N Alsayegh

Full Text Available Recent studies have suggested a role for the Cyclin Dependent Kinase-2 Associated Protein 1 (CDK2AP1 in stem cell differentiation and self-renewal. In studies with mouse embryonic stem cells (mESCs derived from generated mice embryos with targeted deletion of the Cdk2ap1 gene, CDK2AP1 was shown to be required for epigenetic silencing of Oct4 during differentiation, with deletion resulting in persistent self-renewal and reduced differentiation potential. Differentiation capacity was restored in these cells following the introduction of a non-phosphorylatible form of the retinoblastoma protein (pRb or exogenous Cdk2ap1. In this study, we investigated the role of CDK2AP1 in human embryonic stem cells (hESCs. Using a shRNA to reduce its expression in hESCs, we found that CDK2AP1 knockdown resulted in a significant reduction in the expression of the pluripotency genes, OCT4 and NANOG. We also found that CDK2AP1 knockdown increased the number of embryoid bodies (EBs formed when differentiation was induced. In addition, the generated EBs had significantly higher expression of markers of all three germ layers, indicating that CDK2AP1 knockdown enhanced differentiation. CDK2AP1 knockdown also resulted in reduced proliferation and reduced the percentage of cells in the S phase and increased cells in the G2/M phase of the cell cycle. Further investigation revealed that a higher level of p53 protein was present in the CDK2AP1 knockdown hESCs. In hESCs in which p53 and CDK2AP1 were simultaneously downregulated, OCT4 and NANOG expression was not affected and percentage of cells in the S phase of the cell cycle was not reduced. Taken together, our results indicate that the knockdown of CDK2AP1 in hESCs results in increased p53 and enhances differentiation and favors it over a self-renewal fate.

Pharmacological targeting of CDK9 in cardiac hypertrophy.

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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.

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-10-01

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

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

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

2018-03-30

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

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

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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

Memory Enhancement by Targeting Cdk5 Regulation of NR2B

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

2014-01-01

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

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

International Nuclear Information System (INIS)

Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G.; Bosnjak, Zeljko J.

2014-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-10-31

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

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

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

2009-02-15

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

Inactivation of TGF-β signaling in lung cancer results in increased CDK4 activity that can be rescued by ELF

International Nuclear Information System (INIS)

Baek, Hye Jung; Kim, Sang Soo; Silva, Fabio May da; Volpe, Eugene A.; Evans, Stephen; Mishra, Bibhuti; Mishra, Lopa; Blair Marshall, M.

2006-01-01

Escape from TGF-β inhibition of proliferation is a hallmark of multiple cancers including lung cancer. We explored the role of ELF, crucial TGF-β adaptor protein identified from endodermal progenitor cells, in lung carcinogenesis and cell-cycle regulation. Interestingly, elf -/- mice develop multiple defects that include lung, liver, and cardiac abnormalities. Four out of 6 lung cancer and mesothelioma cell lines displayed deficiency of ELF expression with increased CDK4 expression. Immunohistochemistry and Western blot analysis of primary human lung cancers also showed decreased ELF expression and overexpression of CDK4. Moreover, rescue of ELF in ELF-deficient cell lines decreased the expression of CDK4 and resulted in accumulation of G1/S checkpoint arrested cells. These results suggest that disruption in TGF-β signaling mediated by loss of ELF in lung cancer leads to cell-cycle deregulation by modulating CDK4 and ELF highlights a key role of TGF-β adaptor protein in suppressing early lung cancer

Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

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Hylsová, Michaela; Carbain, Benoit; Fanfrlík, Jindřich; Musilová, Lenka; Haldar, Susanta; Köprülüoğlu, Cemal; Ajani, Haresh; Brahmkshatriya, Pathik S; Jorda, Radek; Kryštof, Vladimír; Hobza, Pavel; Echalier, Aude; Paruch, Kamil; Lepšík, Martin

2017-01-27

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R 2  = 0.49). However, the addition of the active-site waters resulted in significant improvement (R 2  = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Jon H Chung

2010-02-01

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

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

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Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

2016-05-09

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mutations in CDK5RAP2 cause Seckel syndrome.

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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

Science.gov (United States)

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

Expression of cdk4 and p16 in Oral Lichen Planus.

Science.gov (United States)

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.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

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Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

2016-01-01

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase. PMID:28077999

Overview of CDK9 as a target in cancer research.

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Morales, Fatima; Giordano, Antonio

2016-01-01

CDK9 is a protein in constant development in cancer therapy. Herein we present an overview of the enzyme as a target for cancer therapy. We provide data on its characteristics and mechanism of action. In recent years, CDK9 inhibitors that have been designed with molecular modeling have demonstrated good antitumoral activity in vitro. Clinical studies of the drugs flavopiridol, dinaciclib, seliciclib, SNS-032 and RGB-286638 used as CDK9 inhibitors are also reviewed, with their additional targets and their relative IC50 values. Unfortunately, treatment with these drugs remains unsuccessful and involves many adverse effects. We could conclude that there are many small molecules that bind to CDK9, but their lack of selectivity against other CDKs do not allow them to get to the clinical use. However, drug designers currently have the tools needed to improve the selectivity of CDK9 inhibitors and to make successful treatment available to patients.

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

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Dipankar Ray

2011-05-01

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

Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

International Nuclear Information System (INIS)

Orba, Yasuko; Sunden, Yuji; Suzuki, Tadaki; Nagashima, Kazuo; Kimura, Takashi; Tanaka, Shinya; Sawa, Hirofumi

2008-01-01

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

Suppression of wheat TaCDK8/TaWIN1 interaction negatively affects germination of Blumeria graminis f.sp. tritici by interfering with very-long-chain aldehyde biosynthesis.

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Kong, Lingyao; Chang, Cheng

2018-01-01

Wheat TaCDK8 interacts with TaWIN1 to regulate very-long-chain aldehyde biosynthesis required for efficient germination of Blumeria graminis f.sp. tritici. Powdery mildew caused by Blumeria graminis f.sp. tritici (Bgt) is a devastating disease of common wheat (Triticum aestivum L.). Bgt infection initiates with its conidia germination on the aerial surface of wheat. In this study, we isolated the cyclin-dependent kinase 8 (TaCDK8) from wheat cultivar Jing411 and found that silencing of TaCDK8 impeded Bgt germination. The biochemical and molecular-biological assays revealed that TaCDK8 interacts with and phosphorylates the wheat transcription factor wax inducer 1 (TaWIN1) to stimulate the TaWIN1-dependent transcription. Bgt conidia on the leaves of TaWIN1-silenced plants also showed reduced germination. Gas chromatographic analysis revealed that knockdown of TaCDK8 or TaWIN1 resulted in decreases of wax components and cutin monomers in wheat leaves. Moreover, Bgt germination on leaves of TaCDK8 or TaWIN1 silenced plants could be fully restored by application of wild-type cuticular wax. In vitro studies demonstrated that very-long-chain aldehydes absent from the cuticular wax of the TaCDK8 or TaWIN1 silenced plants were capable of chemically stimulating Bgt germination. These results implicated that the suppression of TaCDK8/TaWIN1 interaction negatively affects Bgt germination by interfering with very-long-chain aldehyde biosynthesis required for efficient fungal germination.

Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

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Yan Li

2015-04-01

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

Structure-based virtual screening of molecular libraries as cdk2 inhibitors

International Nuclear Information System (INIS)

Riaz, U.; Khaleeq, M.

2011-01-01

CDK2 inhibitor is an important target in multiple processes associated with tumor growth and development, including proliferation, neovascularization, and metastasis. In this study, hit identification was performed by virtual screening of commercial and in-house compound libraries. Docking studies for the hits were performed, and scoring functions were used to evaluate the docking results and to rank ligand-binding affinities. Subsequently, hit optimization for potent and selective candidate CDK2 inhibitors was performed through focused library design and docking analyses. Consequently, we report that a novel compound with an IC50 value of 89 nM, representing 2-Amino-4,6-di-(4',6'-dibromophenyl)pyrimidine 1, is highly selective for CDK2 inhibitors. The docking structure of compound 1 with CDK2 inhibitor disclosed that the NH moiety and pyrimidine ring appeared to fit tightly into the hydrophobic pocket of CDK2 inhibitor. Additionally, the pyrimidine NH forms a hydrogen bond with the carboxyl group of Asp348. These results confirm the successful application of virtual screening studies in the lead discovery process, and suggest that our novel compound can be an effective CDK2 inhibitor candidate for further lead optimization. (author)

Identification of Atuveciclib (BAY 1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer.

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Lücking, Ulrich; Scholz, Arne; Lienau, Philip; Siemeister, Gerhard; Kosemund, Dirk; Bohlmann, Rolf; Briem, Hans; Terebesi, Ildiko; Meyer, Kirstin; Prelle, Katja; Denner, Karsten; Bömer, Ulf; Schäfer, Martina; Eis, Knut; Valencia, Ray; Ince, Stuart; von Nussbaum, Franz; Mumberg, Dominik; Ziegelbauer, Karl; Klebl, Bert; Choidas, Axel; Nussbaumer, Peter; Baumann, Matthias; Schultz-Fademrecht, Carsten; Rühter, Gerd; Eickhoff, Jan; Brands, Michael

2017-11-08

Selective inhibition of exclusively transcription-regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY-958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Cell cycle control by a minimal Cdk network.

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Claude Gérard

2015-02-01

Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

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

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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.

Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

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John S Y Park

Full Text Available CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2 or an alternatively spliced variant form (hCDK5RAP2-V1 that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species.

Caspase 8/10 are not mediating apoptosis in neuroblastoma cells treated with CDK inhibitory drugs

OpenAIRE

Ribas i Fortuny, Judit; Gómez Arbonés, Javier; Boix Torras, Jacint

2005-01-01

Olomoucine and Roscovitine are pharmacological inhibitors of cyclin-dependent kinases (CDK) displaying a promising profile as anticancer agents. Both compounds are effective inductors of apoptosis in a human neuroblastoma cell line, SH-SY5Y. The characterization of this process had suggested the involvement of an extrinsic pathway [Ribas, J., Boix, J., 2004. Cell differentiation, Caspase inhibition, and macromolecular synthesis blockage, but not Bcl-2 or Bcl-XL proteins, protect SH-SY5Y cells...

Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties

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Helder Veras Ribeiro Filho

2018-02-01

Full Text Available Peroxisome proliferator-activated receptor gamma (PPARγ is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879’s mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an

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

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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.

Development of mice without Cip/Kip CDK inhibitors

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

2012-10-19

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

A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

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Miriam Sansó

Full Text Available Transcript elongation by RNA polymerase II (RNAPII is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1 plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb, in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell

Cyclin D-Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization.

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Muntean, Andrew G; Pang, Liyan; Poncz, Mortimer; Dowdy, Steven F; Blobel, Gerd A; Crispino, John D

2007-06-15

Endomitosis is a unique form of cell cycle used by megakaryocytes, in which the latter stages of mitosis are bypassed so that the cell can increase its DNA content and size. Although several transcription factors, including GATA-1 and RUNX-1, have been implicated in this process, the link between transcription factors and polyploidization remains undefined. Here we show that GATA-1-deficient megakaryocytes, which display reduced size and polyploidization, express nearly 10-fold less cyclin D1 and 10-fold increased levels of p16 compared with their wild-type counterparts. We further demonstrate that cyclin D1 is a direct GATA-1 target in megakaryocytes, but not erythroid cells. Restoration of cyclin D1 expression, when accompanied by ectopic overexpression of its partner Cdk4, resulted in a dramatic increase in megakaryocyte size and DNA content. However, terminal differentiation was not rescued. Of note, polyploidization was only modestly reduced in cyclin D1-deficient mice, likely due to compensation by elevated cyclin D3 expression. Finally, consistent with an additional defect conferred by increased levels of p16, inhibition of cyclin D-Cdk4 complexes with a TAT-p16 fusion peptide significantly blocked polyploidization of wild-type megakaryocytes. Together, these data show that GATA-1 controls growth and polyploidization by regulating cyclin D-Cdk4 kinase activity.

Cyclin D–Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization

Science.gov (United States)

Muntean, Andrew G.; Pang, Liyan; Poncz, Mortimer; Dowdy, Steven F.; Blobel, Gerd A.

2007-01-01

Endomitosis is a unique form of cell cycle used by megakaryocytes, in which the latter stages of mitosis are bypassed so that the cell can increase its DNA content and size. Although several transcription factors, including GATA-1 and RUNX-1, have been implicated in this process, the link between transcription factors and polyploidization remains undefined. Here we show that GATA-1–deficient megakaryocytes, which display reduced size and polyploidization, express nearly 10-fold less cyclin D1 and 10-fold increased levels of p16 compared with their wild-type counterparts. We further demonstrate that cyclin D1 is a direct GATA-1 target in megakaryocytes, but not erythroid cells. Restoration of cyclin D1 expression, when accompanied by ectopic overexpression of its partner Cdk4, resulted in a dramatic increase in megakaryocyte size and DNA content. However, terminal differentiation was not rescued. Of note, polyploidization was only modestly reduced in cyclin D1–deficient mice, likely due to compensation by elevated cyclin D3 expression. Finally, consistent with an additional defect conferred by increased levels of p16, inhibition of cyclin D-Cdk4 complexes with a TAT-p16 fusion peptide significantly blocked polyploidization of wild-type megakaryocytes. Together, these data show that GATA-1 controls growth and polyploidization by regulating cyclin D-Cdk4 kinase activity. PMID:17317855

Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

International Nuclear Information System (INIS)

Yang, Zhengmin; Cao, Yonghao; Zhu, Xiaoyan; Huang, Ying; Ding, Yuqiang; Liu, Xiaolong

2009-01-01

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

Residual Cdk1/2 activity after DNA damage promotes senescence

Czech Academy of Sciences Publication Activity Database

Müllers, E.; Cascales, H.S.; Burdová, Kamila; Macůrek, Libor; Lindqvist, A.

2017-01-01

Roč. 16, č. 3 (2017), s. 575-584 ISSN 1474-9726 R&D Projects: GA ČR GA13-18392S Institutional support: RVO:68378050 Keywords : Cdk1 * Cdk2 * cell cycle * checkpoint recovery * DNA damage response * G2phase * p21 * senescence Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

Science.gov (United States)

Cánepa, Eduardo T; Scassa, María E; Ceruti, Julieta M; Marazita, Mariela C; Carcagno, Abel L; Sirkin, Pablo F; Ogara, María F

2007-07-01

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

Design and synthesis of selective CDK8/19 dual inhibitors: Discovery of 4,5-dihydrothieno[3',4':3,4]benzo[1,2-d]isothiazole derivatives.

Science.gov (United States)

Ono, Koji; Banno, Hiroshi; Okaniwa, Masanori; Hirayama, Takaharu; Iwamura, Naoki; Hikichi, Yukiko; Murai, Saomi; Hasegawa, Maki; Hasegawa, Yuka; Yonemori, Kazuko; Hata, Akito; Aoyama, Kazunobu; Cary, Douglas R

2017-04-15

To develop a novel series of CDK8/19 dual inhibitors, we employed structure-based drug design using docking models based on a library compound, 4,5-dihydroimidazolo[3',4':3,4]benzo[1,2-d]isothiazole 16 bound to CDK8. We designed various [5,6,5]-fused tricyclic scaffolds bearing a carboxamide group to maintain predicted interactions with the backbone CO and NH of Ala100 in the CDK8 kinase hinge region. We found that 4,5-dihydrothieno[3',4':3,4]benzo[1,2-d]isothiazole derivative 29a showed particularly potent enzymatic inhibitory activity in both CDK8/19 (CDK8 IC 50 : 0.76nM, CDK19 IC 50 : 1.7nM). To improve the physicochemical properties and kinase selectivity of this compound, we introduced a substituted 3-pyridyloxy group into the scaffold 8-position. The resulting optimized compound 52h showed excellent in vitro potency (CDK8 IC 50 : 0.46nM, CDK19 IC 50 : 0.99nM), physicochemical properties, and kinase selectivity (only 5 kinases showed DMG activation loop. In vitro pharmacological evaluation of 52h revealed potent suppression of phosphorylated STAT1 in various cancer cells. The high oral bioavailability found for this compound enabled in vivo studies, in which we demonstrated a mechanism-based in vivo PD effect as well as tumor growth suppression in an RPMI8226 human hematopoietic and lymphoid xenograft model in mouse [T/C: -1% (2.5mg/kg, qd)]. Copyright © 2017 Elsevier Ltd. All rights reserved.

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

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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.

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

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Mohammed S. I. Makki

2014-01-01

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

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

Science.gov (United States)

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

2016-06-01

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

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

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Han Jin Cho

2016-02-01

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

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

International Nuclear Information System (INIS)

He, Licheng; Lu, Na; Dai, Qinsheng; Zhao, Yue; Zhao, Li; Wang, Hu; Li, Zhiyu; You, Qidong; Guo, Qinglong

2013-01-01

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

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6.

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Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-07-15

To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection.

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

International Nuclear Information System (INIS)

Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Rice, Andrew P.

2010-01-01

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

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

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Sang Hoon Ha

2016-02-01

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

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

Science.gov (United States)

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

Growth inhibition induced by antiprogestins RU-38486, ORG-31710, and CDB-2914 in ovarian cancer cells involves inhibition of cyclin dependent kinase 2.

Science.gov (United States)

Goyeneche, Alicia A; Seidel, Erin E; Telleria, Carlos M

2012-06-01

Antiprogestins have been largely utilized in reproductive medicine, yet their repositioning for oncologic use is rapidly emerging. In this study we investigated the molecular mediators of the anti-ovarian cancer activity of the structurally related antiprogestins RU-38486, ORG-31710 and CDB-2914. We studied the responses of wt p53 OV2008 and p53 null SK-OV-3 cells to varying doses of RU-38486, ORG-31710 and CDB-2914. The steroids inhibited the growth of both cell lines with a potency of RU-38486 > ORG-31710 > CDB-2914, and were cytostatic at lower doses but lethal at higher concentrations. Antiprogestin-induced lethality associated with morphological features of apoptosis, hypodiploid DNA content, DNA fragmentation, and cleavage of executer caspase substrate PARP. Cell death ensued despite RU-38486 caused transient up-regulation of anti-apoptotic Bcl-2, ORG-31710 induced transient up-regulation of inhibitor of apoptosis XIAP, and CDB-2914 up-regulated both XIAP and Bcl-2. The antiprogestins induced accumulation of Cdk inhibitors p21(cip1) and p27(kip1) and increased association of p21(cip1) and p27(kip1) with Cdk-2. They also promoted nuclear localization of p21(cip1) and p27(kip1), reduced the nuclear abundances of Cdk-2 and cyclin E, and blocked the activity of Cdk-2 in both nucleus and cytoplasm. The cytotoxic potency of the antiprogestins correlated with the magnitude of the inhibition of Cdk-2 activity, ranging from G1 cell cycle arrest towards cell death. Our results suggest that, as a consequence of their cytostatic and lethal effects, antiprogestin steroids of well-known contraceptive properties emerge as attractive new agents to be repositioned for ovarian cancer therapeutics.

Rising cyclin-CDK levels order cell cycle events.

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Catherine Oikonomou

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

Discovery of potent and selective CDK8 inhibitors through FBDD approach.

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

2009-01-01

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

A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

DEFF Research Database (Denmark)

Lukas, C; Kramer, E R; Peters, J M

2001-01-01

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits...... the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism...... of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified...

Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation

International Nuclear Information System (INIS)

Lohberger, Birgit; Leithner, Andreas; Stuendl, Nicole; Kaltenegger, Heike; Kullich, Werner; Steinecker-Frohnwieser, Bibiane

2015-01-01

Chondrosarcoma is characterized for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and low rates of survival. Research within the field of development and expansion of new treatment options for unresectable or metastatic diseases is of particular priority. Diacerein, a symptomatic slow acting drug in osteoarthritis (SYSADOA), implicates a therapeutic benefit for the treatment of chondrosarcoma by an antitumor activity. After treatment with diacerein the growth behaviour of the cells was analyzed with the xCELLigence system and MTS assay. Cell cycle was examined using flow cytometric analysis, RT-PCR, and western blot analysis of specific checkpoint regulators. The status for phosophorylation of mitogen-activated protein kinases (MAPKs) was analyzed with a proteome profiler assay. In addition, the possible impact of diacerein on apoptosis was investigated using cleaved caspase 3 and Annexin V/PI flow cytometric analysis. Diacerein decreased the cell viability and the cell proliferation in two different chondrosarcoma cell lines in a dose dependent manner. Flow cytometric analysis showed a classical G2/M arrest. mRNA and protein analysis revealed that diacerein induced a down-regulation of the cyclin B1-CDK1 complex and a reduction in CDK2 expression. Furthermore, diacerein treatment increased the phosphorylation of p38α and p38β MAPKs, and Akt1, Akt2, and Akt 3 in SW-1353, whereas in Cal-78 the opposite effect has been demonstrated. These observations accordingly to our cell cycle flow cytometric analysis and protein expression data may explain the G2/M phase arrest. In addition, no apoptotic induction after diacerein treatment, neither in the Cal-78 nor in the SW-1353 cell line was observed. Our results demonstrate for the first time that the SYSADOA diacerein decreased the viability of human chondrosarcoma cells and induces G2/M cell cycle arrest by CDK1/cyclin B1 down-regulation

Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

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Pierre Hentges

2014-12-01

Full Text Available Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs: homologous recombination (HR and nonhomologous end-joining (NHEJ. DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1 activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2Cdk1 provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.

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

International Nuclear Information System (INIS)

Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae; Lee, Janet; Lee, Chang-Woo; Yang, Kwangmo; Lee, Chang Geun

2013-01-01

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

Cycling Towards Progress: Ribociclib, CDK 4/6 inhibitor for Breast Cancer.

Science.gov (United States)

Spring, Laura; Bardia, Aditya

2018-04-23

Ribociclib is an orally active, highly selective inhibitor of cyclin-dependent kinase (CDK) 4 and 6. It is the second CDK 4/6 inhibitor approved for hormone receptor-positive breast cancer. The addition of ribociclib to an aromatase inhibitor has resulted in marked improvements in progression-free survival for patients with metastatic breast cancer. Copyright ©2018, American Association for Cancer Research.

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

Science.gov (United States)

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

2006-11-24

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

[The role of balanced low-protein diet in inhibition of predialysis chronic kidney disease progression in patients with systemic diseases].

Science.gov (United States)

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.

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

NARCIS (Netherlands)

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,

AbetaPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576.

Science.gov (United States)

Otth, Carola; Concha, Ilona I; Arendt, Thomas; Stieler, Jens; Schliebs, Reinhard; González-Billault, Christian; Maccioni, Ricardo B

2002-10-01

Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimer's type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.

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

Science.gov (United States)

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.

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

Energy Technology Data Exchange (ETDEWEB)

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

2009-08-28

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

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

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Hye-Eun Kim

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

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

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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.

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

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Chad M. Toledo

2015-12-01

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

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

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Hong-Hai Zhang

Full Text Available 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.

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

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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.

Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

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Zhuo Zhan

Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

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

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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.

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

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Xi-Nan Shi

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

Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

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Guo, Fang-Zi; Zhang, Lian-Shuang; Wei, Jia-Liu; Ren, Li-Hua; Zhang, Jin; Jing, Li; Yang, Man; Wang, Ji; Sun, Zhi-Wei; Zhou, Xian-Qing

2016-10-01

Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.

Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells.

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Hashimoto, Takashi; He, Zhiwei; Ma, Wei-Ya; Schmid, Patricia C; Bode, Ann M; Yang, Chung S; Dong, Zigang

2004-05-01

Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

Design and Development of a Series of Potent and Selective Type II Inhibitors of CDK8

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2016-01-01

Using Sorafenib as a starting point, a series of potent and selective inhibitors of CDK8 was developed. When cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode. PMID:27326333

Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability.

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Yusuke Mori

Full Text Available The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs. Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding-deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.

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

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Pasquale Pisani

Full Text Available 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

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer

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Chiara Francavilla

2017-03-01

Full Text Available Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer.

CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

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Hwang, Chae Young; Lee, Seung-Min; Park, Sung Sup; Kwon, Ki-Sun

2012-01-01

Highlights: ► H 2 O 2 differently adjusted senescence and proliferation in normal and cancer cells. ► H 2 O 2 exposure transiently decreased PCNA levels in normal cells. ► H 2 O 2 exposure transiently increased CDK2 activity in cancer cells. ► p21 Cip1 is likely dispensable when H 2 O 2 induces senescence in normal cells. ► Suggestively, CDK2 and PCNA play critical roles in H 2 O 2 -induced cell fate decision. -- Abstract: Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H 2 O 2 decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H 2 O 2 increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H 2 O 2 -induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21 Cip1 /PCNA complex plays an important role as a regulator of cell fate decisions.

Abemaciclib: a CDK4/6 inhibitor for the treatment of HR+/HER2– advanced breast cancer

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Corona SP

2018-02-01

Full Text Available Silvia Paola Corona,1 Daniele Generali2 1Radiation Oncology Department, Peter MacCallum Cancer Centre, Bentleigh East, VIC, Australia; 2Department of Medical, Surgery and Health Sciences, University of Trieste, Trieste, Italy Abstract: Although early breast cancer (BC is highly curable, advanced or metastatic disease poses numerous challenges in terms of medical management and treatment decisions and is associated with significantly worse prognosis. Among the new targeted agents, anticancer drugs exploiting the cell-cycle machinery have shown great potential in preclinical studies. CDK4/6 inhibitors target the cyclin D/CDK/retinoblastoma signaling pathway, inducing cell-cycle arrest, reduced cell viability and tumor shrinking. As the cyclin D/CDK complex is activated downstream of estrogen signaling, the combination of CDK4/6 inhibitors with standard endocrine therapies represents a rational approach to elicit synergic antitumor activity in hormone receptor-positive BC. The results of clinical trials have indeed confirmed the superiority of the combination of CDK4/6 inhibitors plus endocrine therapies over endocrine therapy alone. Currently approved are three compounds that exhibit similar structural characteristics as well as biological and clinical activities. Abemaciclib is the latest CDK4/6 inhibitor approved by the US Food and Drug Administration (FDA in view of the results of the MONARCH 1 and 2 trials. Further trials are ongoing as other important questions await response. In this review, we focus on abemaciclib to examine preclinical and clinical results, describing current therapeutic indications, open questions and ongoing clinical trials. Keywords: CDK4/6 inhibitor, abemaciclib, breast cancer, hormone receptor-positive BC, metastatic BC, mBC

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

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McGrath, Denise A.; Fifield, Bre-Anne; Marceau, Aimee H.; Tripathi, Sarvind; Porter, Lisa A.; Rubin, Seth M. (UCSC); (Windsor)

2017-06-30

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 changes 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.

Ursodeoxycholic acid inhibits the proliferation of colon cancer cells by regulating oxidative stress and cancer stem-like cell growth

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Kim, EuiJoo

2017-01-01

Introduction The regulation of reactive oxygen species (ROS) exists as a therapeutic target for cancer treatments. Previous studies have shown that ursodeoxycholic acid (UDCA) suppresses the proliferation of colon cancer cells. The aim of this study was to evaluate the effect of UDCA upon the proliferation of colon cancer cells as a direct result of the regulation of ROS. Method Colon cancer cell lines (HT29 and HCT116) were treated with UDCA. The total number of cells and the number of dead cells were determined using cell counters. A fluorescein isothiocyanate-bromodeoxyuridine flow kit was used to analyze cell cycle variations. Upon exposure to UDCA, the protein levels of p27, p21, CDK2, CDK4 and CDK6 were determined using western blotting, and qRT-PCR was used to determine levels of mRNA. We preformed dichlorofluorescindiacetate (DCF-DA) staining to detect alteration of intracellular ROS using fluorescence activated cell sorting (FACS). Colon cancer stem-like cell lines were generated by tumorsphere culture and treated with UDCA for seven days. The total number of tumorspheres was determined using microscopy. Results We found that UDCA reduced the total number of colon cancer cells, but did not increase the number of dead cells. UDCA inhibited the G1/S and G2/M transition phases in colon cancer cells. UDCA induced expression of cell cycle inhibitors such as p27 and p21. However, it was determined that UDCA suppressed levels of CDK2, CDK4, and CDK6. UDCA regulated intracellular ROS generation in colon cancer cells, and induced activation of Erk1/2. Finally, UDCA inhibited formation of colon cancer stem-like cells. Conclusion Our results indicate that UDCA suppresses proliferation through regulation of oxidative stress in colon cancer cells, as well as colon cancer stem-like cells. PMID:28708871

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

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Najmeh Fahham

2009-01-01

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

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

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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.

MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

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Xu, Shun; Huang, Haijiao; Li, Nanhong; Zhang, Bing; Jia, Yubin; Yang, Yukun; Yuan, Yuan; Xiong, Xing-dong; Wang, Dengchuan; Zheng, Hui-ling [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China); Liu, Xinguang, E-mail: xgliu64@126.com [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China)

2016-05-13

MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.

MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

International Nuclear Information System (INIS)

Xu, Shun; Huang, Haijiao; Li, Nanhong; Zhang, Bing; Jia, Yubin; Yang, Yukun; Yuan, Yuan; Xiong, Xing-dong; Wang, Dengchuan; Zheng, Hui-ling; Liu, Xinguang

2016-01-01

MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.

Targeting RNA transcription and translation in ovarian cancer cells with pharmacological inhibitor CDKI-73.

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Lam, Frankie; Abbas, Abdullahi Y; Shao, Hao; Teo, Theodosia; Adams, Julian; Li, Peng; Bradshaw, Tracey D; Fischer, Peter M; Walsby, Elisabeth; Pepper, Chris; Chen, Yi; Ding, Jian; Wang, Shudong

2014-09-15

Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and down-regulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4E-mediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPK-mediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers.

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

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Sebastian Jessberger

2008-11-01

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

Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

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Kaldis, Philipp; Aleem, Eiman

2005-11-01

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

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

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Francavilla, Chiara; Lupia, Michela; Tsafou, Kalliopi; Villa, Alessandra; Kowalczyk, Katarzyna; Rakownikow Jersie-Christensen, Rosa; Bertalot, Giovanni; Confalonieri, Stefano; Brunak, Søren; Jensen, Lars J; Cavallaro, Ugo; Olsen, Jesper V

2017-03-28

Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

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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 chronic alcoholism group (P chronic alcoholism group( P chronic alcoholism group were significantly increased compared with control group (P chronic alcoholism group ( P chronic alcoholism damage may interrelate with regulation of Cdk5 kinase activity.

CDK4 and miR-15a comprise an abnormal automodulatory feedback loop stimulating the pathogenesis and inducing chemotherapy resistance in nasopharyngeal carcinoma

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Liu, Zhen; Cheng, Chao; Luo, Xiaojun; Xia, Qiong; Zhang, Yejie; Long, Xiaobing; Jiang, Qingping; Fang, Weiyi

2016-01-01

In previous investigation, we reported that stably knocking down cyclin-dependent kinase 4(CDK4) induced expression of let-7c, which further suppressed cell cycle transition and cell growth by modulating cell cycle signaling in nasopharyngeal carcinoma (NPC). In this study, we further explored the molecular function and mechanism of CDK4 modulating miRNAs to stimulate cell cycle transition, cell growth, and Cisplatin (DDP) -resistance on in NPC. We identified changes in miRNAs by miRNA array and real-time PCR and the effect on DDP after knocking down CDK4 in NPC cells. Further, we investigated the molecular mechanisms by which CDK4 modulated miR-15a in NPC. Moreover, we also explored the role of miR-15a and the effect on DDP in NPC. Finally, we analyzed the correlation of miR-15a and CDK4 expression in NPC tissues. In addition to let-7 family members, we observed that upregulated expression of miR-15a was significantly induced in CDK4-suppressed NPC cells. Further, we found that knocking down CDK4 suppressed c-Myc expression, and the latter directly suppressed the expression of miR-15a in NPC. Furthermore, miR-15a as a tumor suppressor antagonized CDK4 repressing cell cycle progression and cell growth in vitro and in vivo and induced the sensitivity of cells to DDP by regulating the c-Myc/CCND1/CDK4/E2F1 pathway in NPC. Finally, miR-15a was negatively weak correlated with the expression of CDK4 in NPC. Our studies demonstrate that CDK4 and miR-15a comprise an abnormal automodulatory feedback loop stimulating the pathogenesis and inducing chemotherapy resistance in NPC. The online version of this article (doi:10.1186/s12885-016-2277-2) contains supplementary material, which is available to authorized users

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

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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.

Inactivation of CDK/pRb pathway normalizes survival pattern of lymphoblasts expressing the FTLD-progranulin mutation c.709-1G>A.

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Carolina Alquezar

Full Text Available BACKGROUND: Mutations in the progranulin (PGRN gene, leading to haploinsufficiency, cause familial frontotemporal lobar degeneration (FTLD-TDP, although the pathogenic mechanism of PGRN deficit is largely unknown. Allelic loss of PGRN was previously shown to increase the activity of cyclin-dependent kinase (CDK CDK6/pRb pathway in lymphoblasts expressing the c.709-1G>A PGRN mutation. Since members of the CDK family appear to play a role in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults, we investigated the role of CDK6/pRb in cell survival/death mechanisms following serum deprivation. METHODOLOGY/PRINCIPAL FINDINGS: We performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carriers of c.709-1G>A PGRN mutation, asymptomatic and FTLD-TDP diagnosed individuals. Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A bearing lymphoblasts. Apparently, this feature allows PGRN-deficient cells to escape from serum withdrawal-induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the vulnerability of lymphoblasts from FTLD-TDP patients to trophic factor withdrawal. CONCLUSION/SIGNIFICANCE: The use of PGRN-deficient lymphoblasts from FTLD-TDP patients may be a useful model to investigate cell biochemical aspects of this disease. It is suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDP.

Machine Learning Methods for Prediction of CDK-Inhibitors

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Ramana, Jayashree; Gupta, Dinesh

2010-01-01

Progression through the cell cycle involves the coordinated activities of a suite of cyclin/cyclin-dependent kinase (CDK) complexes. The activities of the complexes are regulated by CDK inhibitors (CDKIs). Apart from its role as cell cycle regulators, CDKIs are involved in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. As the complexes perform crucial and diverse functions, these are important drug targets for tumour and stem cell therapeutic interventions. However, CDKIs are represented by proteins with considerable sequence heterogeneity and may fail to be identified by simple similarity search methods. In this work we have evaluated and developed machine learning methods for identification of CDKIs. We used different compositional features and evolutionary information in the form of PSSMs, from CDKIs and non-CDKIs for generating SVM and ANN classifiers. In the first stage, both the ANN and SVM models were evaluated using Leave-One-Out Cross-Validation and in the second stage these were tested on independent data sets. The PSSM-based SVM model emerged as the best classifier in both the stages and is publicly available through a user-friendly web interface at http://bioinfo.icgeb.res.in/cdkipred. PMID:20967128

Treating ER+ Breast Cancer with CDK4/6 Inhibitors.

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2017-08-01

Data from the MONARCH2, PALOMA-1, and TREnd trials strongly support using CDK4/6 inhibitors alongside standard endocrine therapy for advanced ER-positive breast cancer. Including these targeted agents not only improves progression-free survival but may reverse acquired resistance to hormone treatment. ©2017 American Association for Cancer Research.

Diverse models for the prediction of CDK4 inhibitory activity of ...

Indian Academy of Sciences (India)

employed for development of models for the prediction of CDK4 inhibitory activity using a dataset comprising of 52 analogues of ... index; molecular connectivity index; connective eccentricity topochemical index. 1. ... 80% of human cancers.

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+-induced neuronal death in cultured hippocampal neurons

International Nuclear Information System (INIS)

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

2008-01-01

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

Structure-based drug design of a highly potent CDK1,2,4,6 inhibitor with novel macrocyclic quinoxalin-2-one structure.

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Kawanishi, Nobuhiko; Sugimoto, Tetsuya; Shibata, Jun; Nakamura, Kaori; Masutani, Kouta; Ikuta, Mari; Hirai, Hiroshi

2006-10-01

The design of a novel series of cyclin-dependent kinase (CDK) inhibitors containing a macrocyclic quinoxaline-2-one is reported. Structure-based drug design and optimization from the starting point of diarylurea 2, which we previously reported as a moderate CDK1,2,4,6 inhibitor [J. Biol.Chem.2001, 276, 27548], led to the discovery of potent CDK1,2,4,6 inhibitor that were suitable for iv administration for in vivo study.

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

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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

Aminopurvalanol A, a Potent, Selective, and Cell Permeable Inhibitor of Cyclins/Cdk Complexes, Causes the Reduction of in Vitro Fertilizing Ability of Boar Spermatozoa, by Negatively Affecting the Capacitation-Dependent Actin Polymerization

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Nicola Bernabò

2017-12-01

Full Text Available The adoption of high-througput technologies demonstrated that in mature spermatozoa are present proteins that are thought to be not present or active in sperm cells, such as those involved in control of cell cycle. Here, by using an in silico approach based on the application of networks theory, we found that Cyclins/Cdk complexes could play a central role in signal transduction active during capacitation. Then, we tested this hypothesis in the vitro model. With this approach, spermatozoa were incubated under capacitating conditions in control conditions (CTRL or in the presence of Aminopurvalanol A a potent, selective and cell permeable inhibitor of Cyclins/Cdk complexes at different concentrations (2, 10, and 20 μM. We found that this treatment caused dose-dependent inhibition of sperm fertilizing ability. We attribute this event to the loss of acrosome integrity due to the inhibition of physiological capacitation-dependent actin polymerization, rather than to a detrimental effect on membrane lipid remodeling or on other signaling pathways such as tubulin reorganization or MAPKs activation. In our opinion, these data could revamp the knowledge on biochemistry of sperm capacitation and could suggest new perspectives in studying male infertility.

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

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

2005-11-01

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

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

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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

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

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Xiaohua Jin

2016-01-01

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

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

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Mariela C Marazita

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

CDK5 A Novel Role in Prostate Cancer Immunotherapy

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2016-10-01

Parallel: No scientific or budgetary overlap 90091646 (PI: Drake) Title: Enhancing Prostate Cancer Immunotherapy through Epigenetic Reprogramming for...Enhancing Prostate Cancer Immunotherapy through Epigenetic Reprogramming for Optimal Activation of Specific Effector T-Cells Time commitment: 1.2 calendar...AWARD NUMBER: W81XWH-15-1-0670 TITLE: CDK5-A Novel Role in Prostate Cancer Immunotherapy PRINCIPAL INVESTIGATOR: Dr. Barry Nelkin

Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 arrest and apoptosis

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Jada, S R; Matthews, C; Saad, M S; Hamzah, A S; Lajis, N H; Stevens, M F G; Stanslas, J

2008-01-01

Background and purpose: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death. Experimental approach: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry. Key results: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G1 arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis. Conclusion and implications: The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G1 phase cell cycle arrest, coupled with induction of apoptosis. PMID:18806812

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

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

2018-06-01

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

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

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Kar, Siddhartha; Wang, Meifang; Ham, Seung Wook; Carr, Brian I

2006-10-01

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.

CyclinD1, CDK4, and P21 expression by IEC-6 cells in response to NiTi alloy and polymeric biomaterials

International Nuclear Information System (INIS)

Wang, Zhanhui; Yan, Jun; Zheng, Qi; Wang, Zhigang

2012-01-01

In order to investigate how cells recognize biomaterials, mRNA that was expressed in attached Intestinal epithelial cells (IEC-6) on various suture substrates was evaluated. The expressed cell cycle regulators (cyclin D1, CDK4 and p21) mRNA were then isolated and detected using the real time- polymerase chain reaction (PCR) method. As a result, cyclin D1 gene expression was affected by cell-polymer adhesion and was associated with cell proliferation. In addition, CDK4 gene expression was affected by cell proliferation rather than by cell-biomaterial interaction. The p21 mRNA gene expression was higher in cells on more hydrophilic surfaces than on hydrophobic surfaces. Further, the cyclin D1, CDK4 and p21 gene expression were also influenced by the surface chemistry of suture materials. We concluded that the expression of cyclin D1, CDK4 and p21 mRNA was a powerful method for studying cell-biomaterial interactions or the evaluation of the carcinogenic activity of biomaterials. - Highlights: ►We evaluated the effects of biomaterials on the cyclin D1, CDK4 and p21 expression. ►Cell-polymer adhesion and cell proliferation affected cyclin D1 and CDK4 expression. ►The p21 expression was higher on more hydrophilic surfaces than on hydrophobic. ►They were also influenced by surface chemistry of biomaterials.

The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

Science.gov (United States)

Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

2015-01-15

Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

International Nuclear Information System (INIS)

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

2015-01-01

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

Energy Technology Data Exchange (ETDEWEB)

Zhao, Zhuo [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013 (China); Wang, Hao; Lin, Marina [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Groban, Leanne, E-mail: lgroban@wakehealth.edu [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Hypertension and Vascular Disease Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States); Office of Women in Medicine and Science, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States)

2015-03-27

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.

Structure–kinetic relationship study of CDK8/CycC specific compounds

Science.gov (United States)

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

Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro

International Nuclear Information System (INIS)

Gupta, Rupesh K.; Singh, Jeffery M.; Leslie, Tracie C.; Meachum, Sharon; Flaws, Jodi A.; Yao, Humphrey H-C

2010-01-01

Any insult that affects survival of ovarian antral follicles can cause abnormal estradiol production and fertility problems. Phthalate esters (PEs) are plasticizers used in a wide range of consumer and industrial products. Exposure to these chemicals has been linked to reduced fertility in humans and animal models. Di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) decrease serum estradiol levels and aromatase (Arom) expression, prolong estrous cycles, and cause anovulation in animal and culture models. These observations suggest PEs directly target antral follicles. We therefore tested the hypothesis that DEHP (1-100 μg/ml) and MEHP (0.1-10 μg/ml) directly inhibit antral follicular growth and estradiol production. Antral follicles from adult mice were cultured with DEHP or MEHP, and/or estradiol for 96 h. During culture, follicle size was measured every 24 h as a measurement of follicle growth. After culture, media were collected for measurement of estradiol levels and follicles were subjected to measurement of cylin-D-2 (Ccnd2), cyclin-dependant-kinase-4 (Cdk4), and Arom. We found that DEHP and MEHP inhibited growth of follicles and decreased estradiol production compared to controls at the highest doses. DEHP and MEHP also decreased mRNA expression of Ccnd2, Cdk4, and Arom at the highest dose. Addition of estradiol to the culture medium prevented the follicles from DEHP- and MEHP-induced inhibition of growth, reduction in estradiol levels, and decreased Ccnd2 and Cdk4 expression. Collectively, our results indicate that DEHP and MEHP may directly inhibit antral follicle growth via a mechanism that partially includes reduction in levels of estradiol production and decreased expression of cell cycle regulators.

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

DEFF Research Database (Denmark)

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

2007-01-01

) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV-cyclin D1-Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may...... sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGF beta and HGF, resulting in desmoplasia. The MMTV-DIK2 mice should provide a useful model system...

Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

International Nuclear Information System (INIS)

Tieszen, Chelsea R; Goyeneche, Alicia A; Brandhagen, BreeAnn N; Ortbahn, Casey T; Telleria, Carlos M

2011-01-01

Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased

Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

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Ortbahn Casey T

2011-05-01

Full Text Available Abstract Background Mifepristone (MF has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR. The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Methods Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. Results MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Conclusions Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and

CDK5-A Novel Role in Prostate Cancer Immunotherapy

Science.gov (United States)

2017-10-01

castration resistant prostate cancer (CRPC) Specific Aims: 1. Effect of dinaciclib on androgen receptor (AR) S81 phosphorylation and function. 2. Effect of...circulating tumor DNA (ctDNA) and T-cell receptor (TCR) repertoire profiling as biomarkers for men with oligometastatic prostate cancer treated with...AWARD NUMBER: W81XWH-15-1-0670 TITLE: CDK5-A Novel Role in Prostate Cancer Immunotherapy PRINCIPAL INVESTIGATOR: Dr. Barry Nelkin

The BDNF/TrkB Signaling Pathway Is Involved in Heat Hyperalgesia Mediated by Cdk5 in Rats

OpenAIRE

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

Background 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 spin...

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

DEFF Research Database (Denmark)

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

2007-01-01

Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMITV...

SKLB188 inhibits the growth of head and neck squamous cell carcinoma by suppressing EGFR signalling.

Science.gov (United States)

Barzegar, Mansoureh; Ma, Shuang; Zhang, Chao; Chen, Xin; Gu, Ying; Shang, Chaowei; Jiang, Xiaojuan; Yang, Jiao; Nathan, Cherie-Ann; Yang, Shengyong; Huang, Shile

2017-10-10

Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. Several small molecule EGFR inhibitors have been tested in clinical trials for treatment of HNSCC, but none of them are more effective than the current chemotherapeutic drugs. Thus, it is urgently needed to develop novel EGFR inhibitors for HNSCC treatment. By screening an in-house focused library containing approximately 650 000 known kinase inhibitors and kinase inhibitor-like compounds containing common kinase inhibitor core scaffolds, we identified SKLB188 as a lead compound for inhibition of EGFR. The anticancer effects of SKLB188 on HNSCC cells were investigated by in vitro cell growth, cell cycle and apoptosis assays, as well as in vivo FaDu xenograft mouse model. Molecular docking, in vitro kinase profiling and western blotting were performed to characterise EGFR as the molecular target. SKLB188 inhibited HNSCC cell proliferation by inducing G 1 cell cycle arrest, which was associated with downregulating the expression of Cdc25A, cyclins D1/A and cyclin-dependent kinases (CDK2/4), and upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (p21 Cip1 and p27 Kip1 ), leading to decreased phosphorylation of Rb. SKLB188 also induced caspase-dependent apoptosis of HNSCC cells by downregulating the expression of Mcl-1 and survivin. Molecular docking revealed that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay showed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC 50 =5 nM). Western blot analysis demonstrated that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose

Antitumor activity of novel chimeric peptides derived from cyclinD/CDK4 and the protein transduction domain 4.

Science.gov (United States)

Wang, Haili; Chen, Xi; Chen, Yanping; Sun, Lei; Li, Guodong; Zhai, Mingxia; Zhai, Wenjie; Kang, Qiaozhen; Gao, Yanfeng; Qi, Yuanming

2013-02-01

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.

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

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Ade Kallas

2014-01-01

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

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

Energy Technology Data Exchange (ETDEWEB)

Lee, Kyung-Mi [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Yun, Ji Ho [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Lee, Dong Hwa [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Park, Young Gyun [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Son, Kun Ho [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Kim, Yeong Shik, E-mail: kims@snu.ac.kr [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of)

2015-04-17

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

International Nuclear Information System (INIS)

Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa; Park, Young Gyun; Son, Kun Ho; Nho, Chu Won; Kim, Yeong Shik

2015-01-01

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus

Fascaplysin Exerts Anti-Cancer Effects through the Downregulation of Survivin and HIF-1α and Inhibition of VEGFR2 and TRKA

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Taek-In Oh

2017-09-01

Full Text Available Fascaplysin has been reported to exert anti-cancer effects by inhibiting cyclin-dependent kinase 4 (CDK4; however, the precise mode of action by which fascaplysin suppresses tumor growth is not clear. Here, we found that fascaplysin has stronger anti-cancer effects than other CDK4 inhibitors, including PD0332991 and LY2835219, on lung cancer cells that are wild-type or null for retinoblastoma (RB, indicating that unknown target molecules might be involved in the inhibition of tumor growth by fascaplysin. Fascaplysin treatment significantly decreased tumor angiogenesis and increased cleaved-caspase-3 in xenografted tumor tissues. In addition, survivin and HIF-1α were downregulated in vitro and in vivo by suppressing 4EBP1-p70S6K1 axis-mediated de novo protein synthesis. Kinase screening assays and drug-protein docking simulation studies demonstrated that fascaplysin strongly inhibited vascular endothelial growth factor receptor 2 (VEGFR2 and tropomyosin-related kinase A (TRKA via DFG-out non-competitive inhibition. Overall, these results suggest that fascaplysin inhibits TRKA and VEGFR2 and downregulates survivin and HIF-1α, resulting in suppression of tumor growth. Fascaplysin, therefore, represents a potential therapeutic approach for the treatment of multiple types of solid cancer.

Evolution of cyclin-dependent kinases (CDKs) and CDK-activating kinases (CAKs): differential conservation of CAKs in yeast and metazoa.

Science.gov (United States)

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

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity.

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Yang Shu

Full Text Available Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2 gene with Parkinson's disease (PD. Among the mutations, LRRK2 c.4883G>C (R1628P variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn't alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5. Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+ phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age‐related declines in voluntary physical activity

Science.gov (United States)

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�

Opiate exposure state controls dopamine D3 receptor and cdk5/calcineurin signaling in the basolateral amygdala during reward and withdrawal aversion memory formation.

Science.gov (United States)

Rosen, Laura G; Rushlow, Walter J; Laviolette, Steven R

2017-10-03

The dopamine (DA) D3 receptor (D3R) is highly expressed in the basolateral nucleus of the amygdala (BLA), a neural region critical for processing opiate-related reward and withdrawal aversion-related memories. Functionally, D3R transmission is linked to downstream Cdk5 and calcineurin signaling, both of which regulate D3R activity states and play critical roles in memory-related synaptic plasticity. Previous evidence links D3R transmission to opiate-related memory processing, however little is known regarding how chronic opiate exposure may alter D3R-dependent memory mechanisms. Using conditioned place preference (CPP) and withdrawal aversion (conditioned place aversion; CPA) procedures in rats, combined with molecular analyses of BLA protein expression, we examined the effects of chronic opiate exposure on the functional role of intra-BLA D3R transmission during the acquisition of opiate reward or withdrawal aversion memories. Remarkably, we report that the state of opiate exposure during behavioural conditioning (opiate-naïve/non-dependent vs. chronically exposed and in withdrawal) controlled the functional role of intra-BLA D3R transmission during the acquisition of both opiate reward memories and withdrawal-aversion associative memories. Thus, whereas intra-BLA D3R blockade had no effect on opiate reward memory formation in the non-dependent state, blockade of intra-BLA D3R transmission prevented the formation of opiate reward and withdrawal aversion memory in the chronically exposed state. This switch in the functional role of D3R transmission corresponded to significant increases in Cdk5 phosphorylation and total expression levels of calcineurin, and a corresponding decrease in intra-BLA D3R expression. Inhibition of either intra-BLA Cdk5 or calcineurin reversed these effects, switching intra-BLA associative memory formation back to a D3R-independent mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis.

Science.gov (United States)

Lipavská, Helena; Masková, Petra; Vojvodová, Petra

2011-05-01

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied

Synthesis, biological evaluation and molecular modeling of a novel series of 7-azaindole based tri-heterocyclic compounds as potent CDK2/Cyclin E inhibitors.

Science.gov (United States)

Baltus, Christine B; Jorda, Radek; Marot, Christophe; Berka, Karel; Bazgier, Václav; Kryštof, Vladimír; Prié, Gildas; Viaud-Massuard, Marie-Claude

2016-01-27

From four molecules, inspired by the structural features of fascaplysin, with an interesting potential to inhibit cyclin-dependent kinases (CDKs), we designed a new series of tri-heterocyclic derivatives based on 1H-pyrrolo[2,3-b]pyridine (7-azaindole) and triazole heterocycles. Using a Huisgen type [3 + 2] cycloaddition as the convergent key step, 24 derivatives were synthesized and their biological activities were evaluated. Comparative molecular field analysis (CoMFA), based on three-dimensional quantitative structure-activity relationship (3D-QSAR) studies, was conducted on a series of 30 compounds from the literature with high to low known inhibitory activity towards CDK2/cyclin E and was validated by a test set of 5 compounds giving satisfactory predictive r(2) value of 0.92. Remarkably, it also gave a good prediction of pIC50 for our tri-heterocyclic series which reinforce the validation of this model for the pIC50 prediction of external set compounds. The most promising compound, 43, showed a micro-molar range inhibitory activity against CDK2/cyclin E and also an antiproliferative and proapoptotic activity against a panel of cancer cell lines. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Park, Eun-Seok [Department of Applied Biochemistry, Division of Life Science, College of Health and Biomedical Science, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Kang, Shin-il [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of); Yoo, Kyu-dong [Hazardous Substances Analysis Division, Gwangju Regional Food and Drug Administration, Gwangju (Korea, Republic of); Lee, Mi-Yea [Department of Nursing Kyungbok University, Pocheon (Korea, Republic of); Yoo, Hwan-Soo; Hong, Jin-Tae [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of); Shin, Hwa-Sup [Department of Applied Biochemistry, Division of Life Science, College of Health and Biomedical Science, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Yun, Yeo-Pyo, E-mail: ypyun@chungbuk.ac.kr [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of)

2013-04-15

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway. - Highlights: ► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

International Nuclear Information System (INIS)

Park, Eun-Seok; Kang, Shin-il; Yoo, Kyu-dong; Lee, Mi-Yea; Yoo, Hwan-Soo; Hong, Jin-Tae; Shin, Hwa-Sup; Kim, Bokyung; Yun, Yeo-Pyo

2013-01-01

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway. - Highlights: ► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway

MEK inhibition potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells.

Science.gov (United States)

Zhang, Tao; Li, Yanyan; Zhu, Zhenkun; Gu, Mancang; Newman, Bryan; Sun, Duxin

2010-10-04

The Ras/Raf/MEK/ERK signaling has been implicated in uncontrolled cell proliferation and tumor progression in pancreatic cancer. The purpose of this study is to evaluate the antitumor activity of MEK inhibitor U0126 in combination with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in pancreatic cancer cells. Western blotting showed that 17-AAG caused a 2- to 3-fold transient activation of MEK/ERK signaling in pancreatic cancer cells. The activation sustained for 6 h before phospho-ERK (p-ERK) destabilization. The selective MEK inhibitor U0126 completely abolished 17-AAG induced ERK1/2 activation and resulted in more than 80% of phospho-ERK degradation after only 15 min treatment. Moreover, U0126 had complementary effect on 17-AAG regulated oncogenic and cell cycle related proteins. Although 17-AAG downregulated cyclin D1, cyclin E, CDK4 and CDK6, it led to cyclin A and CDK2 accumulation, which was reversed by the addition of U0126. Antiproliferation assay showed that combination of U0126 and 17-AAG resulted in synergistic cytotoxic effect. More importantly, 17-AAG alone only exhibited moderate inhibition of cell migration in vitro, while addition of U0126 dramatically enhanced the inhibitory effect by 2- to 5-fold. Taken together, these data demonstrate that MEK inhibitor U0126 potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. The combination of Hsp90 and MEK inhibition could provide a promising avenue for the treatment of pancreatic cancer.

Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

Energy Technology Data Exchange (ETDEWEB)

Sávio, André Luiz Ventura, E-mail: savio.alv@gmail.com [UNESP – Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, Departamento de Patologia, Botucatu, SP (Brazil); Nicioli da Silva, Glenda [UFOP – Universidade Federal de Ouro Preto, Escola de Farmácia, Departamento de Análises Clínicas, Ouro Preto, MG (Brazil); Salvadori, Daisy Maria Fávero [UNESP – Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, Departamento de Patologia, Botucatu, SP (Brazil)

2015-01-15

Highlights: • AITC inhibits mutant and wild-type TP53 cell proliferation. • Morphological changes and cells debris were observed after AITC treatment in both cells. • BAX and BCL2 expression modulation was observed in wild-type TP53 cells. • BCL2, BAX and ANLN increased and S100P decreased expression was detected in mutated TP53 cells. • AITC effects in gene modulation are dependent TP53 gene status. - Abstract: Natural compounds hold great promise for combating antibiotic resistance, the failure to control some diseases, the emergence of new diseases and the toxicity of some contemporary medical products. Allyl isothiocyanate (AITC), which is abundant in cruciferous vegetables and mustard seeds and is commonly referred to as mustard essential oil, exhibits promising antineoplastic activity against bladder cancer, although its mechanism of action is not fully understood. Therefore, the aim of this study was to investigate the effects of AITC activity on bladder cancer cell lines carrying a wild type (wt; RT4) or mutated (T24) TP53 gene. Morphological changes, cell cycle kinetics and CDK1, SMAD4, BAX, BCL2, ANLN and S100P gene expression were evaluated. In both cell lines, treatment with AITC inhibited cell proliferation (at 62.5, 72.5, 82.5 and 92.5 μM AITC) and induced morphological changes, including scattered and elongated cells and cellular debris. Gene expression profiles revealed increased S100P and BAX and decreased BCL2 expression in RT4 cells following AITC treatment. T24 cells displayed increased BCL2, BAX and ANLN and decreased S100P expression. No changes in SMAD4 and CDK1 expression were observed in either cell line. In conclusion, AITC inhibits cell proliferation independent of TP53 status. However, the mechanism of action of AITC differed in the two cell lines; in RT4 cells, it mainly acted via the classical BAX/BCL2 pathway, while in T24 cells, AITC modulated the activities of ANLN (related to cytokinesis) and S100P. These data confirm

Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

International Nuclear Information System (INIS)

Sávio, André Luiz Ventura; Nicioli da Silva, Glenda; Salvadori, Daisy Maria Fávero

2015-01-01

Highlights: • AITC inhibits mutant and wild-type TP53 cell proliferation. • Morphological changes and cells debris were observed after AITC treatment in both cells. • BAX and BCL2 expression modulation was observed in wild-type TP53 cells. • BCL2, BAX and ANLN increased and S100P decreased expression was detected in mutated TP53 cells. • AITC effects in gene modulation are dependent TP53 gene status. - Abstract: Natural compounds hold great promise for combating antibiotic resistance, the failure to control some diseases, the emergence of new diseases and the toxicity of some contemporary medical products. Allyl isothiocyanate (AITC), which is abundant in cruciferous vegetables and mustard seeds and is commonly referred to as mustard essential oil, exhibits promising antineoplastic activity against bladder cancer, although its mechanism of action is not fully understood. Therefore, the aim of this study was to investigate the effects of AITC activity on bladder cancer cell lines carrying a wild type (wt; RT4) or mutated (T24) TP53 gene. Morphological changes, cell cycle kinetics and CDK1, SMAD4, BAX, BCL2, ANLN and S100P gene expression were evaluated. In both cell lines, treatment with AITC inhibited cell proliferation (at 62.5, 72.5, 82.5 and 92.5 μM AITC) and induced morphological changes, including scattered and elongated cells and cellular debris. Gene expression profiles revealed increased S100P and BAX and decreased BCL2 expression in RT4 cells following AITC treatment. T24 cells displayed increased BCL2, BAX and ANLN and decreased S100P expression. No changes in SMAD4 and CDK1 expression were observed in either cell line. In conclusion, AITC inhibits cell proliferation independent of TP53 status. However, the mechanism of action of AITC differed in the two cell lines; in RT4 cells, it mainly acted via the classical BAX/BCL2 pathway, while in T24 cells, AITC modulated the activities of ANLN (related to cytokinesis) and S100P. These data confirm

Interaction with CCNH/CDK7 facilitates CtBP2 promoting esophageal squamous cell carcinoma (ESCC) metastasis via upregulating epithelial-mesenchymal transition (EMT) progression.

Science.gov (United States)

Zhang, Jianguo; Zhu, Junya; Yang, Lei; Guan, Chengqi; Ni, Runzhou; Wang, Yuchan; Ji, Lili; Tian, Ye

2015-09-01

CtBP2, as a transcriptional corepressor of epithelial-specific genes, has been reported to promote tumor due to upregulating epithelial-mesenchymal transition (EMT) in cancer cells. CtBP2 was also demonstrated to contribute to the proliferation of esophageal squamous cell carcinoma (ESCC) cells through a negative transcriptional regulation of p16(INK4A). In this study, for the first time, we reported that CtBP2 expression, along with CCNH/CDK7, was higher in ESCC tissues with lymph node metastases than in those without lymph node metastases. Moreover, both CtBP2 and CCNH/CDK7 were positively correlated with E-cadherin, tumor grade, and tumor metastasis. However, the concrete mechanism of CtBP2's role in enhancing ESCC migration remains incompletely understood. We confirmed that CCNH/CDK7 could directly interact with CtBP2 in ESCC cells in vivo and in vitro. Furthermore, our data demonstrate for the first time that CtBP2 enhanced the migration of ESCC cells in a CCNH/CDK7-dependent manner. Our results indicated that CCNH/CDK7-CtBP2 axis may augment ESCC cell migration, and targeting the interaction of both may provide a novel therapeutic target of ESCC.

Inhibition of human prostate cancer cells proliferation by a selective alpha1-adrenoceptor antagonist labedipinedilol-A involves cell cycle arrest and apoptosis

International Nuclear Information System (INIS)

Liou, S.-F.; Lin, H.-H.; Liang, J.-C.; Chen, I.-J.; Yeh, J.-L.

2009-01-01

In this research, we conducted an in vitro analysis to evaluate the prostate cancer cells response to labedipinedilol-A in order to determine the effect of this selective α 1 -adrenoceptor antagonist to suppress prostate cancer cell growth by affecting cell proliferation and apoptosis. Here, we report that treatment of androgen-sensitive (LNCaP) and androgen-insensitive (PC-3) prostate cancer cells with labedipinedilol-A inhibited cell proliferation in concentration-dependent and time-dependent manners. Moreover, norepinephrine-stimulated proliferation of both cell lines are markedly inhibited by labedipinedilol-A. The probable involvement of α 1 -adrenoceptors in this cellular response is suggested. Labedipinedilol-A-induced growth inhibition was associated with G 0 /G 1 arrest, and G 2 /M arrest depending upon concentrations. Cell cycle blockade was associated with reduced amounts of cyclin D1/2, cyclin E, Cdk2, Cdk4, and Cdk6 and increased levels of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27). In addition, labedipinedilol-A also induced apoptosis in PC-3 cells, as determined by using Hoechst 33342 staining, DNA fragmentation, and Annexin V staining assay. Furthermore, labedipinedilol-A triggered the mitochondrial apoptotic pathway, as indicated by increasing the expression of Bax, but decreasing the level of Bcl-2, resulting in mitochondrial membrane potential loss, cytochrome c release, and activation of caspase-9 and -3. We further investigated the role of MAPK cascades in the anti-proliferative and apoptosis effects of labedipinedilol-A, and confirmed that labedipinedilol-A could activate JNK1/2 but not p38 in both cell lines. Unlike JNK1/2, however, labedipinedilol-A treatment resulted in down-regulation of phospho-ERK1/2 expression. We concluded that labedipinedilol-A possessed the growth-suppressive and apoptotic effects on LNCaP and PC-3 cells by its α 1 -adrenoceptor blockade, and the apoptotic effects of labedipinedilol-A primarily through

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

Science.gov (United States)

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

2016-10-01

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

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.

Science.gov (United States)

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.

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

Science.gov (United States)

Tsai, Kuang-Lei; Sato, Shigeo; Tomomori-Sato, Chieri; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2013-01-01

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism. PMID:23563140

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

Science.gov (United States)

Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

2017-07-14

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Synapsin III Acts Downstream of Semaphorin 3A/CDK5 Signaling to Regulate Radial Migration and Orientation of Pyramidal Neurons In Vivo

Directory of Open Access Journals (Sweden)

Laura E. Perlini

2015-04-01

Full Text Available Synapsin III (SynIII is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5 phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation.

Regulation of the G1/S Transition in Hepatocytes: Involvement of the Cyclin-Dependent Kinase Cdk1 in the DNA Replication

Directory of Open Access Journals (Sweden)

Anne Corlu

2012-01-01

Full Text Available A singular feature of adult differentiated hepatocytes is their capacity to proliferate allowing liver regeneration. This review emphasizes the literature published over the last 20 years that established the most important pathways regulating the hepatocyte cell cycle. Our article also aimed at illustrating that many discoveries in this field benefited from the combined use of in vivo models of liver regeneration and in vitro models of primary cultures of human and rodent hepatocytes. Using these models, our laboratory has contributed to decipher the different steps of the progression into the G1 phase and the commitment to S phase of proliferating hepatocytes. We identified the mitogen dependent restriction point located at the two-thirds of the G1 phase and the concomitant expression and activation of both Cdk1 and Cdk2 at the G1/S transition. Furthermore, we demonstrated that these two Cdks contribute to the DNA replication. Finally, we provided strong evidences that Cdk1 expression and activation is correlated to extracellular matrix degradation upon stimulation by the pro-inflammatory cytokine TNFα leading to the identification of a new signaling pathway regulating Cdk1 expression at the G1/S transition. It also further confirms the well-orchestrated regulation of liver regeneration via multiple extracellular signals and pathways.

Dibenzocyclooctadiene lignans, gomisins J and N inhibit the Wnt/{beta}-catenin signaling pathway in HCT116 cells

Energy Technology Data Exchange (ETDEWEB)

Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); Kim, Chul Young [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); College of Pharmacy, Hanyang University, Ansan 426-791 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of)

2012-11-16

Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract: Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.

Dibenzocyclooctadiene lignans, gomisins J and N inhibit the Wnt/β-catenin signaling pathway in HCT116 cells

International Nuclear Information System (INIS)

Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju; Kim, Chul Young; Nho, Chu Won

2012-01-01

Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/β-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: ► Gomisins J and N inhibited Wnt/β-catenin signaling pathway in HCT116 cells. ► Gomisins J and N disrupted the binding of β-catenin to specific DNA sequences, TBE. ► Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. ► Gomisins J and N inhibited the expression of Cyc D1, a Wnt/β-catenin target gene. -- Abstract: Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/β-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/β-catenin signaling by disrupting the interaction between β-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the β-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/β-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/β-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity.

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Heijink, Anne Margriet; Blomen, Vincent A; Bisteau, Xavier; Degener, Fabian; Matsushita, Felipe Yu; Kaldis, Philipp; Foijer, Floris; van Vugt, Marcel A T M

2015-12-08

The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.

Combined Inhibition of CDK4/6 and PI3K/AKT/mTOR Pathways Induces a Synergistic Anti-Tumor Effect in Malignant Pleural Mesothelioma Cells

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Mara A. Bonelli

2017-08-01

Full Text Available Malignant pleural mesothelioma (MPM is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF, encoding for the cell cycle inhibitors p16INK4a and p14ARF, deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991, a highly selective inhibitor of cyclin-dependent kinase (CDK 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM.

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

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Jeong, Jin Boo; Jeong, Hyung Jin

2010-01-01

Research highlights: → 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. → 2M4VP inhibited hyper-phosphorylation of Rb protein. → 2M4VP induced cell cycle arrest from G1 to S. → 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. → 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

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Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

2010-10-01

Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

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Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

2011-01-01

Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

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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.

CDK4/6 inhibitor PD0332991 in glioblastoma treatment: does it have a future?

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Lisette eSchroder

2015-11-01

Full Text Available Glioblastoma is aggressive, highly infiltrating, and the most frequent malignant form of brain cancer. With a median survival time of only 14.6 months, when treated with the standard of care, it is essential to find new therapeutic options. A specific CDK4/6 inhibitor, PD0332991, obtained accelerated approval from the Food and Drug Administration for the treatment of patients with advanced estrogen receptor-positive and HER2-negative breast cancer. Common alterations in the cyclin D1-Cyclin Dependent Kinase 4/6-Retinoblastoma 1 pathway in glioblastoma make PD0332991 also an interesting drug for the treatment of glioblastoma. Promising results in in vitro studies, where patient derived glioblastoma cell lines showed sensitivity to PD0332991, gave motive to start in vivo studies. Outcomes of these studies have been contrasting in terms of PD0332991 efficacy within the brain: more research is necessary to conclude whether CDK4/6 inhibitor can be beneficial in the treatment of glioblastoma.

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

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Gu, Jun [Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu (China); Department of Orthopaedics, Xishan People' s Hospital, Wuxi, Jiangsu (China); Ni, Yingjie; Xu, Lin; Xu, Hongliang [Department of Orthopaedics, Xishan People' s Hospital, Wuxi, Jiangsu (China); Cai, Zhengdong, E-mail: caizhengdongsh@163.com [Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu (China)

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 the 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.

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

International Nuclear Information System (INIS)

Gu, Jun; Ni, Yingjie; Xu, Lin; Xu, Hongliang; Cai, Zhengdong

2016-01-01

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 the 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.

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

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Young-Chae Kim

Full Text Available 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.

CDK4/6 dual inhibitor abemaciclib demonstrates compelling preclinical activity against esophageal adenocarcinoma: a novel therapeutic option for a deadly disease.

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Kosovec, Juliann E; Zaidi, Ali H; Omstead, Ashten N; Matsui, Daisuke; Biedka, Mark J; Cox, Erin J; Campbell, Patrick T; Biederman, Robert W W; Kelly, Ronan J; Jobe, Blair A

2017-11-21

Esophageal adenocarcinoma (EAC) is a deadly disease with limited therapeutic options. In the present study, we determined the preclinical efficacy of CDK4/6 inhibitor abemaciclib for treatment of EAC. In vitro , apoptosis, proliferation, and pathway regulation were evaluated in OE19, OE33, and FLO1 EAC cell lines. In vivo , esophagojejunostomy was performed on rats to induce EAC. At 36 weeks post-surgery, MRI and endoscopic biopsy established baseline tumor volume and molecular correlates, respectively. Next, the study animals were randomized to 26mg/kg intraperitoneal abemaciclib treatment or vehicle control for 28 days. Pre and post treatment MRIs, histopathology, and qRT-PCR were utilized to determine response. Our results demonstrated treatment with abemaciclib lead to increased apoptosis, and decreased proliferation in OE19 (p=0.185), OE33 (p=0.048), and FLO1 (p=0.043) with anticipated downstream molecular inhibition. In vivo , 78.9% of treatment animals demonstrated >20% tumor volume decrease (placebo 0%). Mean tumor volume changed in the treatment arm by -65.5% (placebo +133.5%) (p<0.01), and prevalence changed by -37.5% (placebo +16.7%) (p<0.01). Pre vs post treatment qRT-PCR demonstrated significant inhibition of all downstream molecular correlates. Overall our findings suggest potent antitumor efficacy of abemaciclib against EAC with evident molecular pathway inhibition and reasonable safety, establishing the rationale for future clinical development.

CDB-4124, a progesterone receptor modulator, inhibits mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis.

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Wiehle, Ronald; Lantvit, Daniel; Yamada, Tohru; Christov, Konstantin

2011-03-01

CDB-4124 (Proellex or telapristone acetate) is a modulator of progesterone receptor (PR) signaling, which is currently employed in preclinical studies for prevention and treatment of breast cancer and has been used in clinical studies for treatment of uterine fibroids and endometriosis. Here we provide evidence for its action on steroid hormone-signaling, cell cycle-regulated genes and in vivo on mammary carcinogenesis. When CDB-4124 is given to rats at 200 mg/kg for 24 months, it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. Also, CDB-4124 given as subcutaneous pellets at two different doses suppressed, dose dependently, N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. The high dose (30 mg, over 84 days) increased tumor latency from 66 ± 24 days to 87 ± 20 days (P CDB-4124 inhibited cell proliferation and induced apoptosis in MNU-induced mammary tumors, which correlated with a decreased proportion of PR(+) tumor cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. In a mechanistic study employing T47D cells we found that CDB-4124 suppressed G(1)/G(0)-S transition by inhibiting CDK2 and CDK4 expressions, which correlated with inhibition of estrogen receptor (ER) expression. Taken together, these data indicate that CDB-4124 can suppress the development of precancerous lesions and carcinogen-induced ER(+) mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer.

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).

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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

TFP5/TP5 peptide provides neuroprotection in the MPTP model of Parkinson′s disease

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B K Binukumar

2016-01-01

Full Text Available Cyclin-dependent kinase 5 (Cdk5 is a member of the serine-threonine kinase family of cyclin-dependent kinases. Cdk5 is critical to normal mammalian nervous system development and plays important regulatory roles in multiple cellular functions. Recent evidence indicates that Cdk5 is inappropriately activated in several neurodegenerative conditions, including Parkinson′s disease (PD. PD is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. During neurotoxicity, p35 is cleaved to form p25. Binding of p25 with Cdk5 leads deregulation of Cdk5 resulting in number of neurodegenerative pathologies. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Here we show that inhibition of p25/Cdk5 hyperactivation through TFP5/TP5, truncated 24-aa peptide derived from the Cdk5 activator p35 rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP + in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show inhibition of Cdk5/p25-hyperactivation by TFP5/TP5 peptide, which identifies Cdk5/p25 as a potential therapeutic target to reduce neurodegeneration in PD.

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

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Agarwala Usha

2011-06-01

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

Inhibition of STAT3 activity delays obesity-induced thyroid carcinogenesis in a mouse model

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Park, Jeong Won; Han, Cho Rong; Zhao, Li; Willingham, Mark C.; Cheng, Sheue-yann

2015-01-01

Compelling epidemiologic studies indicate that obesity is a risk factor for many human cancers, including thyroid cancer. In recent decades, the incidence of thyroid cancer has dramatically increased along with a marked rise in obesity prevalence. We previously demonstrated that a high fat diet (HFD) effectively induced the obese phenotype in a mouse model of thyroid cancer (ThrbPV/PVPten+/− mice). Moreover, HFD activates the STAT3 signal pathway to promote more aggressive tumor phenotypes. The aim of the present study was to evaluate the effect of S3I-201, a specific inhibitor of STAT3 activity, on HFD-induced aggressive cancer progression in the mouse model of thyroid cancer. Wild type and ThrbPV/PVPten+/− mice were treated with HFD together with S3I-201 or vehicle-only as controls. We assessed the effects of S3I-201 on HFD-induced thyroid cancer progression, the leptin-JAK2-STAT3 signaling pathway, and key regulators of epithelial-mesenchymal transition. S3I-201 effectively inhibited HFD-induced aberrant activation of STAT3 and its downstream targets to markedly inhibit thyroid tumor growth and to prolong survival. Decreased protein levels of cyclins D1 and B1, cyclin dependent kinase (CDK) 4, CDK 6, and phosphorylated retinoblastoma protein led to the inhibition of tumor cell proliferation in S3I-201-treated ThrbPV/PVPten+/− mice. Reduced occurrence of vascular invasion and blocking of anaplasia and lung metastasis in thyroid tumors of S3I-201-treated ThrbPV/PVPten+/− mice were mediated via decreased expression of vimentin and matrix metalloproteinases, two key effectors of epithelial-mesenchymal transition. The present findings suggest that inhibition of the STAT3 activity would be a novel treatment strategy for obesity-induced thyroid cancer. PMID:26552408

Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption

DEFF Research Database (Denmark)

Beck, Halfdan; Nähse-Kumpf, Viola; Larsen, Marie Sofie Yoo

2012-01-01

Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation of replic......Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation...... of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted...

CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

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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

Rca1 inhibits APC-Cdh1(Fzr) and is required to prevent cyclin degradation in G2.

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Grosskortenhaus, Ruth; Sprenger, Frank

2002-01-01

We demonstrate that Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome (APC) in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy (Cdc20(Fzy)) and Cdh1-Fizzy-related (Cdh1(Fzr)), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1(Fzr)-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1(Fzr) mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1(Fzr) function. We show that Rca1 and Cdh1(Fzr) are in a complex that also includes the APC component Cdc27. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. Our data reveal a different mode of APC regulation by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1(Fzr) interaction.

Loss of γ-tubulin, GCP-WD/NEDD1 and CDK5RAP2 from the Centrosome of Neurons in Developing Mouse Cerebral and Cerebellar Cortex

International Nuclear Information System (INIS)

Yonezawa, Satoshi; Shigematsu, Momoko; Hirata, Kazuto; Hayashi, Kensuke

2015-01-01

It has been recently reported that the centrosome of neurons does not have microtubule nucleating activity. Microtubule nucleation requires γ-tubulin as well as its recruiting proteins, GCP-WD/NEDD1 and CDK5RAP2 that anchor γ-tubulin to the centrosome. Change in the localization of these proteins during in vivo development of brain, however, has not been well examined. In this study we investigate the localization of γ-tubulin, GCP-WD and CDK5RAP2 in developing cerebral and cerebellar cortex with immunofluorescence. We found that γ-tubulin and its recruiting proteins were localized at centrosomes of immature neurons, while they were lost at centrosomes in mature neurons. This indicated that the loss of microtubule nucleating activity at the centrosome of neurons is due to the loss of γ-tubulin-recruiting proteins from the centrosome. RT-PCR analysis revealed that these proteins are still expressed after birth, suggesting that they have a role in microtubule generation in cell body and dendrites of mature neurons. Microtubule regrowth experiments on cultured mature neurons showed that microtubules are nucleated not at the centrosome but within dendrites. These data indicated the translocation of microtubule-organizing activity from the centrosome to dendrites during maturation of neurons, which would explain the mixed polarity of microtubules in dendrites

Therapeutic rationale to target highly expressed CDK7 conferring poor outcomes in triple-negative breast cancer

NARCIS (Netherlands)

Li, Bo; Chonghaile, Triona Ni; Fan, Yue; Madden, Stephen F.; Klinger, Rut; O'Connor, Aisling E.; Walsh, Louise; O'Hurley, Gillian; Udupi, Girish Mallya; Joseph, Jesuchristopher; Tarrant, Finbarr; Conroy, Emer; Gaber, Alexander; Chin, Suet-Feung; Bardwell, Helen A; Provenzano, Elena; Crown, John; Dubois, Thierry; Linn, Sabine; Jirstrom, Karin; Caldas, Carlos; O'Connor, Darran P; Gallagher, William M

2017-01-01

Triple-negative breast cancer (TNBC) patients commonly exhibit poor prognosis and high relapse after treatment, but there remains a lack of biomarkers and effective targeted therapies for this disease. Here, we report evidence highlighting the cell-cycle–related kinase CDK7 as a driver and candidate

The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

Directory of Open Access Journals (Sweden)

Tao Zhang

2013-12-01

Full Text Available Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4, E2F1 and an increase of p21waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK and suppressed mammalian target of rapamycin (mTOR, the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA, cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.

CDK-mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer

Science.gov (United States)

Cepeda, Diana; Ng, Hwee-Fang; Sharifi, Hamid Reza; Mahmoudi, Salah; Cerrato, Vanessa Soto; Fredlund, Erik; Magnusson, Kristina; Nilsson, Helén; Malyukova, Alena; Rantala, Juha; Klevebring, Daniel; Viñals, Francesc; Bhaskaran, Nimesh; Zakaria, Siti Mariam; Rahmanto, Aldwin Suryo; Grotegut, Stefan; Nielsen, Michael Lund; Szigyarto, Cristina Al-Khalili; Sun, Dahui; Lerner, Mikael; Navani, Sanjay; Widschwendter, Martin; Uhlén, Mathias; Jirström, Karin; Pontén, Fredrik; Wohlschlegel, James; Grandér, Dan; Spruck, Charles; Larsson, Lars-Gunnar; Sangfelt, Olle

2013-01-01

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer. PMID:23776131

Modelling the CDK-dependent transcription cycle in fission yeast.

Science.gov (United States)

Sansó, Miriam; Fisher, Robert P

2013-12-01

CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

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Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.; Yamamoto, Kazuki; Kaeser, Benjamin; Ichikawa, Satoshi; Lee, Seok-Yong

2018-02-19

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.

Theoretical studies on the selective mechanisms of GSK3β and CDK2 by molecular dynamics simulations and free energy calculations.

Science.gov (United States)

Zhao, Sufang; Zhu, Jingyu; Xu, Lei; Jin, Jian

2017-06-01

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase which is widely involved in cell signaling and controls a broad number of cellular functions. GSK3 contains α and β isoforms, and GSK3β has received more attention and becomes an attractive drug target for the treatment of several diseases. The binding pocket of cyclin-dependent kinase 2 (CDK2) shares high sequence identity to that of GSK3β, and therefore, the design of highly selective inhibitors toward GSK3β remains a big challenge. In this study, a computational strategy, which combines molecular docking, molecular dynamics simulations, free energy calculations, and umbrella sampling simulations, was employed to explore the binding mechanisms of two selective inhibitors to GSK3β and CDK2. The simulation results highlighted the key residues critical for GSK3β selectivity. It was observed that although GSK3β and CDK2 share the conserved ATP-binding pockets, some different residues have significant contributions to protein selectivity. This study provides valuable information for understanding the GSK3β-selective binding mechanisms and the rational design of selective GSK3β inhibitors. © 2016 John Wiley & Sons A/S.

Sequencing Analysis of Mutant Allele $cdc$28-$srm$ of Protein Kinase CDC28 and Molecular Dynamics Study of Glycine-Rich Loop in Wild-Type and Mutant Allele G16S of CDK2 as Model

CERN Document Server

Koltovaya, N A; Kholmurodov, Kh T; Kretov, D A

2005-01-01

The central role that cyclin-dependent kinases play in the timing of cell division and the high incidence of genetic alteration of CDKs or deregulation of CDK inhibitors in a number of cancers make CDC28 of the yeast \\textit{Saccharomyces cerevisiae }very attractive model for studies of mechanisms of CDK regulation. Earlier it was found that certain gene mutations including \\textit{cdc28-srm} affect cell cycle progression, maintenance of different genetic structures and increase cell sensitivity to ionizing radiation. A~\\textit{cdc28-srm} mutation is not temperature-sensitive mutation and differs from the known \\textit{cdc28-ts }mutations because it has the evident phenotypic manifestations at 30 $^{\\circ}$C. Sequencing analysis of \\textit{cdc28-srm} revealed a single nucleotide substitution G20S. This is a third glycine in a conserved sequence GxGxxG in the G-rich loop positioned opposite the activation T-loop. Despite its demonstrated importance, the role of the G-loop has remained unclear. The crystal stru...

RNA interference targeting raptor inhibits proliferation of gastric cancer cells

International Nuclear Information System (INIS)

Wu, William Ka Kei; Lee, Chung Wa; Cho, Chi Hin; Chan, Francis Ka Leung; Yu, Jun; Sung, Joseph Jao Yiu

2011-01-01

Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G 0 /G 1 -phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D 3 and p21 Waf1 , which stabilizes cyclin D/cdk4 complex for G 1 -S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition.

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Maria J Aristizabal

2015-10-01

Full Text Available RNA polymerase II (RNAPII contains a unique C-terminal domain that is composed of heptapeptide repeats and which plays important regulatory roles during gene expression. RNAPII is responsible for the transcription of most protein-coding genes, a subset of non-coding genes, and retrotransposons. Retrotransposon transcription is the first step in their multiplication cycle, given that the RNA intermediate is required for the synthesis of cDNA, the material that is ultimately incorporated into a new genomic location. Retrotransposition can have grave consequences to genome integrity, as integration events can change the gene expression landscape or lead to alteration or loss of genetic information. Given that RNAPII transcribes retrotransposons, we sought to investigate if the RNAPII-CTD played a role in the regulation of retrotransposon gene expression. Importantly, we found that the RNAPII-CTD functioned to maintaining genome integrity through inhibition of retrotransposon gene expression, as reducing CTD length significantly increased expression and transposition rates of Ty1 elements. Mechanistically, the increased Ty1 mRNA levels in the rpb1-CTD11 mutant were partly due to Cdk8-dependent alterations to the RNAPII-CTD phosphorylation status. In addition, Cdk8 alone contributed to Ty1 gene expression regulation by altering the occupancy of the gene-specific transcription factor Ste12. Loss of STE12 and TEC1 suppressed growth phenotypes of the RNAPII-CTD truncation mutant. Collectively, our results implicate Ste12 and Tec1 as general and important contributors to the Cdk8, RNAPII-CTD regulatory circuitry as it relates to the maintenance of genome integrity.

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

Science.gov (United States)

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.

Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis.

Science.gov (United States)

Annibali, Daniela; Whitfield, Jonathan R; Favuzzi, Emilia; Jauset, Toni; Serrano, Erika; Cuartas, Isabel; Redondo-Campos, Sara; Folch, Gerard; Gonzàlez-Juncà, Alba; Sodir, Nicole M; Massó-Vallés, Daniel; Beaulieu, Marie-Eve; Swigart, Lamorna B; Mc Gee, Margaret M; Somma, Maria Patrizia; Nasi, Sergio; Seoane, Joan; Evan, Gerard I; Soucek, Laura

2014-08-18

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

TFIIH and P-TEFb coordinate transcription with capping enzyme recruitment at specific genes in fission yeast.

Science.gov (United States)

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

2009-03-27

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 inhibition by 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.

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

NARCIS (Netherlands)

Linke, C.; Chasapi, A.; González-Novo, A.; Al Sawad, I.; Tognetti, S.; Klipp, E.; Loog, M.; Krobitsch, S.; Posas, F.; Xenarios, I.; Barberis, M.

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

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.

Science.gov (United States)

Hu, G; Lee, H; Price, S M; Shen, M M; Abate-Shen, C

2001-06-01

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

Science.gov (United States)

Cepeda, Diana; Ng, Hwee-Fang; Sharifi, Hamid Reza; Mahmoudi, Salah; Cerrato, Vanessa Soto; Fredlund, Erik; Magnusson, Kristina; Nilsson, Helén; Malyukova, Alena; Rantala, Juha; Klevebring, Daniel; Viñals, Francesc; Bhaskaran, Nimesh; Zakaria, Siti Mariam; Rahmanto, Aldwin Suryo; Grotegut, Stefan; Nielsen, Michael Lund; Szigyarto, Cristina Al-Khalili; Sun, Dahui; Lerner, Mikael; Navani, Sanjay; Widschwendter, Martin; Uhlén, Mathias; Jirström, Karin; Pontén, Fredrik; Wohlschlegel, James; Grandér, Dan; Spruck, Charles; Larsson, Lars-Gunnar; Sangfelt, Olle

2013-07-01

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

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.

Science.gov (United States)

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.

Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina.

Science.gov (United States)

Wang, Tzu-Ming; Holzhausen, Lars C; Kramer, Richard H

2014-02-01

The reciprocal synapse between photoreceptors and horizontal cells underlies lateral inhibition and establishes the antagonistic center-surround receptive fields of retinal neurons to enhance visual contrast. Despite decades of study, the signal mediating the negative feedback from horizontal cells to cones has remained under debate because the small, invaginated synaptic cleft has precluded measurement. Using zebrafish retinas, we show that light elicits a change in synaptic proton concentration with the correct magnitude, kinetics and spatial dependence to account for lateral inhibition. Light, which hyperpolarizes horizontal cells, causes synaptic alkalinization, whereas activating an exogenously expressed ligand-gated Na(+) channel, which depolarizes horizontal cells, causes synaptic acidification. Whereas acidification was prevented by blocking a proton pump, re-alkalinization was prevented by blocking proton-permeant ion channels, suggesting that distinct mechanisms underlie proton efflux and influx. These findings reveal that protons mediate lateral inhibition in the retina, raising the possibility that protons are unrecognized retrograde messengers elsewhere in the nervous system.

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.

Science.gov (United States)

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.

Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer

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Jeff C. Liu

2018-04-01

Full Text Available Summary: CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC, RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs. : Liu et al. report that inhibition of the protein phosphatase CDC25 kills diverse triple-negative breast cancer (TNBC cells. Moreover, CDC25 antagonists cooperate with other drugs, such as PI3K inhibitors, to efficiently suppress growth of human TNBC engrafted into mice. Keywords: triple negative breast cancer, basal-like breast cancer, therapy, RB1, PTEN, TP53, CDC25, WEE1, CHK1, checkpoint control

Cdk1 Phosphorylates Drosophila Sas-4 to Recruit Polo to Daughter Centrioles and Convert Them to Centrosomes.

Science.gov (United States)

Novak, Zsofia A; Wainman, Alan; Gartenmann, Lisa; Raff, Jordan W

2016-06-20

Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic "centriole conversion" is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

Science.gov (United States)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; Zhong, Liyun

2017-04-01

Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

Licoricidin inhibits the growth of SW480 human colorectal adenocarcinoma cells in vitro and in vivo by inducing cycle arrest, apoptosis and autophagy

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Ji, Shuai [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004 (China); Tang, Shunan; Li, Kai; Li, Ziwei; Liang, Wenfei; Qiao, Xue; Wang, Qi [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Yu, Siwang, E-mail: swang_yu@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Ye, Min, E-mail: yemin@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China)

2017-07-01

Licorice (Glycyrrhiza uralensis Fisch.) possesses significant anti-cancer activities, but the active ingredients and underlying mechanisms have not been revealed. By screening the cytotoxic activities of 122 licorice compounds against SW480 human colorectal adenocarcinoma cells, we found that licoricidin (LCD) inhibited SW480 cell viability with an IC{sub 50} value of 7.2 μM. Further studies indicated that LCD significantly induced G1/S cell cycle arrest and apoptosis in SW480 cells, accompanied by inhibition of cyclins/CDK1 expression and activation of caspase-dependent pro-apoptotic signaling. Meanwhile, LCD promoted autophagy in SW480 cells, and activated AMPK signaling and inhibited Akt/mTOR pathway. Overexpression of a dominant-negative AMPKα2 abolished LCD-induced inhibition of Akt/mTOR, autophagic and pro-apoptotic signaling pathways, and significantly reversed loss of cell viability, suggesting activation of AMPK is essential for the anti-cancer activity of LCD. In vivo anti-tumor experiments indicated that LCD (20 mg/kg, i.p.) significantly inhibited the growth of SW480 xenografts in nude mice with an inhibitory rate of 43.5%. In addition, we obtained the glycosylated product LCDG by microbial transformation, and found that glycosylation slightly enhanced the in vivo anti-cancer activities of LCD. This study indicates that LCD could inhibit SW480 cells by inducing cycle arrest, apoptosis and autophagy, and is a potential chemopreventive or chemotherapeutic agent against colorectal cancer. - Highlights: • Molecular mechanisms for cytotoxic activity of licoricidin (LCD) were investigated. • LCD promoted autophagy of SW480 cells through AMPK and Akt/mTOR signaling pathways. • Both LCD and its glucoside showed in vivo anti-colorectal cancer activities.

Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

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Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

2015-01-01

Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

Oxidative stress accumulates in adipose tissue during aging and inhibits adipogenesis.

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Findeisen, Hannes M; Pearson, Kevin J; Gizard, Florence; Zhao, Yue; Qing, Hua; Jones, Karrie L; Cohn, Dianne; Heywood, Elizabeth B; de Cabo, Rafael; Bruemmer, Dennis

2011-04-14

Aging constitutes a major independent risk factor for the development of type 2 diabetes and is accompanied by insulin resistance and adipose tissue dysfunction. One of the most important factors implicitly linked to aging and age-related chronic diseases is the accumulation of oxidative stress. However, the effect of increased oxidative stress on adipose tissue biology remains elusive. In this study, we demonstrate that aging in mice results in a loss of fat mass and the accumulation of oxidative stress in adipose tissue. In vitro, increased oxidative stress through glutathione depletion inhibits preadipocyte differentiation. This inhibition of adipogenesis is at least in part the result of reduced cell proliferation and an inhibition of G(1)→S-phase transition during the initial mitotic clonal expansion of the adipocyte differentiation process. While phosphorylation of the retinoblastoma protein (Rb) by cyclin/cdk complexes remains unaffected, oxidative stress decreases the expression of S-phase genes downstream of Rb. This silencing of S phase gene expression by increased oxidative stress is mediated through a transcriptional mechanism involving the inhibition of E2F recruitment and transactivation of its target promoters. Collectively, these data demonstrate a previously unrecognized role of oxidative stress in the regulation of adipogenesis which may contribute to age-associated adipose tissue dysfunction.

A novel muscarinic antagonist R2HBJJ inhibits non-small cell lung cancer cell growth and arrests the cell cycle in G0/G1.

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Nan Hua

Full Text Available Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs. In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.

Embelin suppresses growth of human pancreatic cancer xenografts, and pancreatic cancer cells isolated from KrasG12D mice by inhibiting Akt and Sonic hedgehog pathways.

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Minzhao Huang

Full Text Available Pancreatic cancer is a deadly disease, and therefore effective treatment and/or prevention strategies are urgently needed. The objectives of this study were to examine the molecular mechanisms by which embelin inhibited human pancreatic cancer cell growth in vitro, and xenografts in Balb C nude mice, and pancreatic cancer cell growth isolated from KrasG12D transgenic mice. XTT assays were performed to measure cell viability. AsPC-1 cells were injected subcutaneously into Balb c nude mice and treated with embelin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of Akt, and Sonic Hedgehog (Shh and their target gene products were measured by the immunohistochemistry, and Western blot analysis. The effects of embelin on pancreatic cancer cells isolated from 10-months old KrasG12D mice were also examined. Embelin inhibited cell viability in pancreatic cancer AsPC-1, PANC-1, MIA PaCa-2 and Hs 766T cell lines, and these inhibitory effects were blocked either by constitutively active Akt or Shh protein. Embelin-treated mice showed significant inhibition in tumor growth which was associated with reduced expression of markers of cell proliferation (Ki67, PCNA and Bcl-2 and cell cycle (cyclin D1, CDK2, and CDK6, and induction of apoptosis (activation of caspase-3 and cleavage of PARP, and increased expression of Bax. In addition, embelin inhibited the expression of markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8, and metastasis (MMP-2 and MMP-9 in tumor tissues. Antitumor activity of embelin was associated with inhibition of Akt and Shh pathways in xenografts, and pancreatic cancer cells isolated from KrasG12D mice. Furthermore, embelin also inhibited epithelial-to-mesenchymal transition (EMT by up-regulating E-cadherin and inhibiting the expression of Snail, Slug, and ZEB1. These data suggest that embelin can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt and

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer

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Cepeda, Diana; Ng, Hwee-Fang; Sharifi, Hamid Reza

2013-01-01

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28...... results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role...... in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer....

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

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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

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

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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.

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

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

2015-01-01

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

Calcium dysregulation and Cdk5-ATM pathway involved in a mouse model of fragile X-associated tremor/ataxia syndrome.

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Robin, Gaëlle; López, José R; Espinal, Glenda M; Hulsizer, Susan; Hagerman, Paul J; Pessah, Isaac N

2017-07-15

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurological disorder that affects premutation carriers with 55-200 CGG-expansion repeats (preCGG) in FMR1, presenting with early alterations in neuronal network formation and function that precede neurodegeneration. Whether intranuclear inclusions containing DNA damage response (DDR) proteins are causally linked to abnormal synaptic function, neuronal growth and survival are unknown. In a mouse that harbors a premutation CGG expansion (preCGG), cortical and hippocampal FMRP expression is moderately reduced from birth through adulthood, with greater FMRP reductions in the soma than in the neurite, despite several-fold elevation of Fmr1 mRNA levels. Resting cytoplasmic calcium concentration ([Ca2+]i) in cultured preCGG hippocampal neurons is chronically elevated, 3-fold compared to Wt; elevated ROS and abnormal glutamatergic responses are detected at 14 DIV. Elevated µ-calpain activity and a higher p25/p35 ratio in the cortex of preCGG young adult mice indicate abnormal Cdk5 regulation. In support, the Cdk5 substrate, ATM, is upregulated by 1.5- to 2-fold at P0 and 6 months in preCGG brain, as is p-Ser1981-ATM. Bax:Bcl-2 is 30% higher in preCGG brain, indicating a greater vulnerability to apoptotic activation. Elevated [Ca2+]i, ROS, and DDR signals are normalized with dantrolene. Chronic [Ca2+]i dysregulation amplifies Cdk5-ATM signaling, possibly linking impaired glutamatergic signaling and DDR to neurodegeneration in preCGG brain. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

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Yang Xu

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

CDK5-mediated phosphorylation of p19INK4d avoids DNA damage-induced neurodegeneration in mouse hippocampus and prevents loss of cognitive functions.

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Ogara, María Florencia; Belluscio, Laura M; de la Fuente, Verónica; Berardino, Bruno G; Sonzogni, Silvina V; Byk, Laura; Marazita, Mariela; Cánepa, Eduardo T

2014-07-01

DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions. Copyright © 2014 Elsevier B.V. All rights reserved.

Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen

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Cimica, Velasco; Smith, Melissa E; Zhang, Zhikai; Mathur, Deepti; Mani, Sridhar; Kalpana, Ganjam V

2010-01-01

Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs. The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined. We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G 2 arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G 2 arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G 2 arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities. Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further

Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen

Energy Technology Data Exchange (ETDEWEB)

Cimica, Velasco; Smith, Melissa E; Zhang, Zhikai; Mathur, Deepti [Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 (United States); Mani, Sridhar [Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 (United States); Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 (United States); Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 (United States); Kalpana, Ganjam V [Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 (United States); Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 (United States)

2010-11-19

Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs. The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined. We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G{sub 2} arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G{sub 2} arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G{sub 2} arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities. Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results

Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen

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Mani Sridhar

2010-11-01

Full Text Available Abstract Background Rhabdoid Tumors (RTs are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam in order to reduce the concentration of flavopiridol needed for inhibition of RTs. Methods The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i cell survival assays, ii FACS analysis, iii caspase activity assays, and iv immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined. Results We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G2 arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G2 arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G2 arrest, but enhanced flavopiridol- (but not 4OH-Tam- mediated apoptosis, by enhancing caspase 2 and 3 activities. Conclusions Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was

Methylferulate from Tamarix aucheriana inhibits growth and enhances chemosensitivity of human colorectal cancer cells: possible mechanism of action.

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Abaza, Mohamed Salah I; Afzal, Mohammad; Al-Attiyah, Raja'a J; Guleri, Radhika

2016-10-01

Natural products are valuable sources for anticancer agents. In the present study, methylferulate (MF) was identified for the first time from Tamarix aucheriana. Spectral data were used for identification of MF. The potential of MF to control cell growth, cell cycle, apoptosis, generation of reactive oxygen species (ROS), cancer cell invasion, nuclear factor kappa B (NFkB) DNA-binding activity and proteasomal activities, as well as the enhancement of chemosensitivity in human colorectal cancer cells, were evaluated. The possible molecular mechanism of MF's therapeutic efficacy was also assessed. Column chromatography and spectral data were used for isolation and identification of MF. MTT, immunofluorescence, flow cytometry, in vitro invasion, fluoremetry, EIA and Real time qPCR were used to measure antiproliferative, chemo-sensitizing effects and other biochemical parameters. MF showed a dose-dependent anti-proliferative effect on colorectal cancer cells (IC 50  = 1.73 - 1.9 mM) with a nonsignificant cytotoxicity toward normal human fibroblast. Colony formation inhibition (P ≤ 0.001, 0.0001) confirmed the growth inhibition by MF. MF arrested cell cycle progression in the S and G2/M phases; induced apoptosis and ROS generation; and inhibited NF-kB DNA-binding activity, proteasomal activities and cell invasion in colorectal cancer cells. MF up-regulated cyclin-dependent kinase inhibitors (p19 INK4D , p21 WAF1/CIP1 , p27 KIP1 ), pro-apoptotic gene expression (Bax, Bad, Apaf1, Bid, Bim, Smac) and caspases (caspase 2, 3, 6, 7, 8, 9). Moreover, MF down-regulated cyclin-dependent kinases (Cdk1, Cdk2) and anti-apoptotic gene expression (c-IAP-1, c-IAP-2, Bcl2,FLIP). In addition, MF differentially potentiated the sensitivity of colorectal cancer cells to standard chemotherapeutic drugs. MF showed a multifaceted anti-proliferative and chemosensitizing effects. These results suggest the chemotherapeutic and co-adjuvant potential of MF.

3,3'Diindolylmethane suppresses vascular smooth muscle cell phenotypic modulation and inhibits neointima formation after carotid injury.

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Hongjing Guan

Full Text Available 3,3'Diindolylmethane (DIM, a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.DIM dose-dependently inhibited the platelet-derived growth factor (PDGF-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK 4/6 as well as an increase in p27(Kip1 levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptorβ (PDGF-Rβ and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK3β, extracellular signal-regulated kinase1/2 (ERK1/2, and signal transducers and activators of transcription 3 (STAT3. Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.These results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-Rβ and the

Association of germline variation in CCNE1 and CDK2 with breast cancer risk, progression and survival among Chinese Han women.

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Ji-Yuan Han

Full Text Available BACKGROUND: Somatic alterations of cyclin-dependent kinase 2 (CDK2-cyclin E complex have been shown to contribute to breast cancer (BC development and progression. This study aimed to explore the effects of single nucleotide polymorphisms (SNPs in CDK2 and CCNE1 (a gene encoding G1/S specific cyclin E1 protein, formerly called cyclin E on BC risk, progression and survival in a Chinese Han population. METHODOLOGY/PRINCIPAL FINDINGS: We herein genotyped 6 haplotype-tagging SNPs (htSNPs of CCNE1 and 2 htSNPs of CDK2 in 1207 BC cases and 1207 age-matched controls among Chinese Han women, and then reconstructed haplotype blocks according to our genotyping data and linkage disequilibrium status of these htSNPs. For CCNE1, the minor allele homozygotes of three htSNPs were associated with BC risk (rs3218035: adjusted odds ratio [aOR] = 3.35, 95% confidence interval [CI] = 1.69-6.67; rs3218038: aOR = 1.81, 95% CI = 1.22-2.70; rs3218042: aOR = 2.64, 95% CI = 1.31-5.34, and these three loci showed a dose-dependent manner in increasing BC risk (P(trend = 0.0001. Moreover, the 5-SNP haplotype CCGTC, which carried none of minor alleles of the 3 at-risk SNPs, was associated with a favorable event-free survival (hazard ratio [HR] = 0.53, 95% CI = 0.32-0.90. Stratified analysis suggested that the minor-allele homozygote carriers of rs3218038 had a worse event-free survival among patients with aggressive tumours (in tumour size>2 cm group: HR = 2.06, 95% CI = 1.06-3.99; in positive lymph node metastasis group: HR = 2.41, 95% CI = 1.15-5.03; in stage II-IV group: HR = 2.03, 95% CI = 1.09-3.79. For CDK2, no significant association was found. CONCLUSIONS/SIGNIFICANCE: This study indicates that genetic variants in CCNE1 may contribute to BC risk and survival in Chinese Han population. They may become molecular markers for individual evaluation of BC susceptibility and prognosis. Nevertheless, further validation studies are needed.

Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B

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Venkatesan, Balachandar; Ghosh-Choudhury, Nandini; Das, Falguni; Mahimainathan, Lenin; Kamat, Amrita; Kasinath, Balakuntalam S.; Abboud, Hanna E.; Choudhury, Goutam Ghosh

2008-01-01

Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF-induced DNA synthesis in mesangial cells with an IC50 of 10 μM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF-stimulated c-Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin-dependent kinase-2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb-2 binding sites tyrosine-751 and tyrosine-716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF-stimulated phosphorylation at tyrosine-751 and tyrosine-716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF-induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh-Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. PMID:18567737

Two Bistable Switches Govern M Phase Entry.

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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.

Prenatal alcohol exposure alters p35, CDK5 and GSK3β in the medial frontal cortex and hippocampus of adolescent mice

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Samantha L. Goggin

2014-01-01

Full Text Available Fetal alcohol spectrum disorders (FASDs are the number one cause of preventable mental retardation. An estimated 2–5% of children are diagnosed as having a FASD. While it is known that children prenatally exposed to alcohol experience cognitive deficits and a higher incidence of psychiatric illness later in life, the pathways underlying these abnormalities remain uncertain. GSK3β and CDK5 are protein kinases that are converging points for a vast number of signaling cascades, including those controlling cellular processes critical to learning and memory. We investigated whether levels of GSK3β and CDK5 are affected by moderate prenatal alcohol exposure (PAE, specifically in the hippocampus and medial frontal cortex of the adolescent mouse. In the present work we utilized immunoblotting techniques to demonstrate that moderate PAE increased hippocampal p35 and β-catenin, and decreased total levels of GSK3β, while increasing GSK3β Ser9 and Tyr216 phosphorylation. Interestingly, different alterations were seen in the medial frontal cortex where p35 and CDK5 were decreased and increased total GSK3β was accompanied by reduced Tyr216 of the enzyme. These results suggest that kinase dysregulation during adolescence might be an important contributing factor to the effects of PAE on hippocampal and medial frontal cortical functioning; and by extension, that global modulation of these kinases may produce differing effects depending on brain region.

BDE-47 and BDE-209 inhibit proliferation of Neuro-2a cells via inducing G1-phase arrest.

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Chen, Hongmei; Tang, Xuexi; Zhou, Bin; Xu, Ningning; Zhou, Zhongyuan; Fang, Kuan; Wang, You

2017-03-01

Cell proliferation is closely related to cell cycle which is strictly regulated by genes and regulatory proteins. In the present study, we comparatively analyzed the toxic effects of BDE-47 and BDE-209 on cell proliferation of Neuro-2a cells, and the possible mechanism was discussed. The results indicated that BDE-47 significantly inhibited the cell proliferation and the cell cycle were arrest at G1 phase, while BDE-209 had little effects on either cell proliferation or cell cycle. qRT-PCR and Western blot assay presented that BDE-47 up-regulated the gene expressions of p53 and p21, which down-regulated the expresseion of cyclinD1 and CDK2, and inhibited retinoblastoma protein (pRb) phosphorylation. This process could effectively arrest the cell cycle at G1 phase, which finally caused the inhibition on Neuro-2a cell proliferation. However, BDE-209 was only up-regulated the gene expressions of p53, also suggested to be involved in the inhibition on Neuro-2a cell proliferation. Copyright © 2016. Published by Elsevier B.V.

Exploring the physicochemical profile and the binding patterns of selected novel anticancer Himalayan plant derived active compounds with macromolecular targets

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Arun Bahadur Gurung

Full Text Available Plants are vital source of compounds offering plethora of therapeutic effects against various ailments without much side effects. Due to wide spread prevalence and drug resistance in cancer; there is an urgent need for discovery of new anti-cancer drugs. In the present study, selected novel anti-cancer plants derived compounds (cmpd1 to cmpd15 from Himalayan region were docked with defined molecular targets that regulate cell proliferation and apoptosis. The binding energies of best docked compounds ranged between −8.0 kcal/mol and −11.71 kcal/mol. Further analysis revealed critical hydrogen bonds and hydrophobic interactions between compounds and targets. The best docked compounds viz., cmpd15 against cyclin-dependent kinase-2 (CDK-2, cmpd8 against CDK-6 and cmpd9 against Topoisomerase I and II showed higher binding affinities than the native co-crystal ligands. The root mean square deviation (RMSD and potential energy plot clearly indicates the stability of the complexes during 20 ns molecular dynamics (MD simulation. The Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA binding energy analysis revealed Van der Waals energy component which is the principal stabilizing energy for their interactions except CDK-2/cmpd15 complex. The polar solvation energy did not have favorable contribution to their stabilization. The binding energy decomposition analysis revealed per residue contribution for each docked complexes. Physicochemical profile studies showed that majority of the compounds conform to Lipinski's rule of five (ROF having low to high blood brain barrier (BBB penetration, human intestinal absorption, plasma binding protein inhibition and P glycoprotein inhibition. Keywords: ADMET, Anticancer, MM/PBSA, Molecular docking, Molecular dynamics simulation and plant derived compounds

Inhibition of G1-phase arrest induced by ionizing radiation in hematopoietic cells by overexpression of genes involved in the G1/S-phase transition

International Nuclear Information System (INIS)

Epperly, M.; Berry, L.; Halloran, A.; Greenberger, J.S.

1995-01-01

D-type cyclins and cyclin-dependent kinase (cdk-4) are likely involved in regulating passage of cells through the G 1 phase of the cell cycle. A decrease in the proportion of cells in G 1 , a relatively radiation-sensitive phase of the cell cycle, should result in increased resistance to ionizing radiation; however, the effect of such overexpression on X-ray-induced G 1 -phase arrest is not known. Radiation survival curves were obtained at a dose rate of either 8 cGy/min or 1 Gy/min for subclones of the IL-3-dependent hematopoietic progenitor cell line 32D cl 3 expressing transgenes for either cyclin-D1, D2 or D3 or cdk-4. We compared the results to those with overexpression of the transgene for Bcl-2, whose expression enhances radiation survival and delays apoptosis. Cells overexpressing transgenes for each D-type cyclin or Bcl-2 had an increased number of cells in S phase compared to parent line 32D cl 3; however, overexpression of cdk-4 had no effect on cell cycle distribution. Cell death resulting from withdrawal of IL-3 was not affected by overexpression of D2, cdk-4 or Bcl-2. Flow cytometry 24 h after 5 Gy irradiation demonstrated that overexpression of each G 1 -phase regulatory transgene decreased the proportion of cells at the G 1 /S-phase border. Western analysis revealed induction of cyclin-D protein levels by irradiation, but no change in the D O , but a significant increase in the rvec n for cyclin-D or cdk-4 transgene-overexpressing clones at 1 Gy/min (P 1 /S-phase arrest. 31 refs., 4 figs., 4 tabs

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

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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

Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways

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Wu Weilin

2008-06-01

Full Text Available Abstract Human T-cell leukemia virus type-1 (HTLV-1 induces adult T-cell leukemia/lymphoma (ATL/L, a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP, a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-κB and the cell cycle pathways. The observation that NF-κB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-κB and CDK inhibitors (total of 35 compounds to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKβ kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A

Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways.

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Agbottah, Emmanuel; Yeh, Wen-I; Berro, Reem; Klase, Zachary; Pedati, Caitlin; Kehn-Hall, Kyleen; Wu, Weilin; Kashanchi, Fatah

2008-06-10

Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-kappaB and the cell cycle pathways. The observation that NF-kappaB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-kappaB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKbeta kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

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Candida Vannini

Full Text Available Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

miR Profiling Identifies Cyclin-Dependent Kinase 6 Downregulation as a Potential Mechanism of Acquired Cisplatin Resistance in Non-Small-Cell Lung Carcinoma.

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Bar, Jair; Gorn-Hondermann, Ivan; Moretto, Patricia; Perkins, Theodore J; Niknejad, Nima; Stewart, David J; Goss, Glenwood D; Dimitroulakos, Jim

2015-11-01

To identify the mechanisms of cisplatin resistance, global microRNA (miR) expression was tested. The expression of miR-145 was consistently higher in resistant cells. The expression of cyclin-dependent kinase 6 (CDK6), a potential target of miR-145, was lower in resistant cells, and inhibition of CDK4/6 protected cells from cisplatin. Cell cycle inhibition, currently being tested in clinical trials, might be antagonistic to cisplatin and other cytotoxic drugs. Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Platinum-based chemotherapeutic drugs are the most active agents in treating advanced disease. Resistance to these drugs is common and multifactorial; insight into the molecular mechanisms involved will likely enhance efficacy. A set of NSCLC platinum-resistant sublines was created from the Calu6 cell line. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentially expressed microRNAs (miRs) in these lines were identified using Affymetrix miR arrays. The potential genes targeted by these miRs were searched using the TargetScan algorithm. The expression levels of miRs and mRNA were tested using real-time polymerase chain reaction. miR-145 was reproducibly elevated in all the resistant sublines tested; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin-dependent kinase 6 (CDK6), an important regulator of cell proliferation. The mRNA and protein levels of CDK6 were both downregulated in the resistant sublines. An inhibitor of CDK4/6 (PD0332991) protected parental NSCLC cells from cisplatin cytotoxicity. In the present study, we identified miRs differentially expressed in cisplatin-resistant cell lines, including miR-145. A predicted target of miR-145 is CDK6, and its expression was found to be downregulated in the resistant sublines, although not directly by miR-145. Inhibition

New castanospermine glycoside analogues inhibit breast cancer cell proliferation and induce apoptosis without affecting normal cells.

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Ghada Allan

Full Text Available sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231 cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4, cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21(Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.

SKLB70326, a novel small-molecule inhibitor of cell-cycle progression, induces G{sub 0}/G{sub 1} phase arrest and apoptosis in human hepatic carcinoma cells

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Han, Yuanyuan; He, Haiyun [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Peng, Feng [Department of Thoracic Oncology of the Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Liu, Jiyan; Dai, Xiaoyun; Lin, Hongjun; Xu, Youzhi; Zhou, Tian; Mao, Yongqiu; Xie, Gang; Yang, Shengyong; Yu, Luoting; Yang, Li [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Zhao, Yinglan, E-mail: alancenxb@sina.com [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China)

2012-05-18

Highlights: Black-Right-Pointing-Pointer SKLB70326 is a novel compound and has activity of anti-HCC. Black-Right-Pointing-Pointer SKLB70326 induces cell cycle arrest and apoptosis in HepG2 cells. Black-Right-Pointing-Pointer SKLB70326 induces G{sub 0}/G{sub 1} phase arrest via inhibiting the activity of CDK2, CDK4 and CDK6. Black-Right-Pointing-Pointer SKLB70326 induces apoptosis through the intrinsic pathway. -- Abstract: We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G{sub 0}/G{sub 1} phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G{sub 0}/G{sub 1} phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

Cyclin-Dependent Kinase Inhibitor AT7519 as a Potential Drug for MYCN-Dependent Neuroblastoma.

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Dolman, M Emmy M; Poon, Evon; Ebus, Marli E; den Hartog, Ilona J M; van Noesel, Carel J M; Jamin, Yann; Hallsworth, Albert; Robinson, Simon P; Petrie, Kevin; Sparidans, Rolf W; Kok, Robbert J; Versteeg, Rogier; Caron, Huib N; Chesler, Louis; Molenaar, Jan J

2015-11-15

MYCN-dependent neuroblastomas have low cure rates with current multimodal treatment regimens and novel therapeutic drugs are therefore urgently needed. In previous preclinical studies, we have shown that targeted inhibition of cyclin-dependent kinase 2 (CDK2) resulted in specific killing of MYCN-amplified neuroblastoma cells. This study describes the in vivo preclinical evaluation of the CDK inhibitor AT7519. Preclinical drug testing was performed using a panel of MYCN-amplified and MYCN single copy neuroblastoma cell lines and different MYCN-dependent mouse models of neuroblastoma. AT7519 killed MYCN-amplified neuroblastoma cell lines more potently than MYCN single copy cell lines with a median LC50 value of 1.7 compared to 8.1 μmol/L (P = 0.0053) and a significantly stronger induction of apoptosis. Preclinical studies in female NMRI homozygous (nu/nu) mice with neuroblastoma patient-derived MYCN-amplified AMC711T xenografts revealed dose-dependent growth inhibition, which correlated with intratumoral AT7519 levels. CDK2 target inhibition by AT7519 was confirmed by significant reductions in levels of phosphorylated retinoblastoma (p-Rb) and nucleophosmin (p-NPM). AT7519 treatment of Th-MYCN transgenic mice resulted in improved survival and clinically significant tumor regression (average tumor size reduction of 86% at day 7 after treatment initiation). The improved efficacy of AT7519 observed in Th-MYCN mice correlated with higher tumor exposure to the drug. This study strongly suggests that AT7519 is a promising drug for the treatment of high-risk neuroblastoma patients with MYCN amplification. ©2015 American Association for Cancer Research.

Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

DEFF Research Database (Denmark)

Ballabeni, Andrea; Melixetian, Marina; Zamponi, Raffaella

2004-01-01

-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle....... Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate...

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

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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.

Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

International Nuclear Information System (INIS)

Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na; Guo, Qinglong

2013-01-01

Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function. �

Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

Energy Technology Data Exchange (ETDEWEB)

Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na, E-mail: luna555@163.com; Guo, Qinglong, E-mail: anticancer_drug@yahoo.com.cn

2013-09-01

Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function. �

Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

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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).

Effective molecular targeting of CDK4/6 and IGF-1R in a rare FUS-ERG fusion CDKN2A-deletion doxorubicin-resistant Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) nude-mouse model.

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Murakami, Takashi; Singh, Arun S; Kiyuna, Tasuku; Dry, Sarah M; Li, Yunfeng; James, Aaron W; Igarashi, Kentaro; Kawaguchi, Kei; DeLong, Jonathan C; Zhang, Yong; Hiroshima, Yukihiko; Russell, Tara; Eckardt, Mark A; Yanagawa, Jane; Federman, Noah; Matsuyama, Ryusei; Chishima, Takashi; Tanaka, Kuniya; Bouvet, Michael; Endo, Itaru; Eilber, Fritz C; Hoffman, Robert M

2016-07-26

Ewing's sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewing's sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewing's sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewing's sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer.

MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

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Hui Xu

2010-11-01

Full Text Available MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

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Xu, Hui; Yan, Yaping; Williams, Mark S; Carey, Gregory B; Yang, Jingxian; Li, Hongmei; Zhang, Guang-Xian; Rostami, Abdolmohamad

2010-11-01

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

Cyclin-Dependent Kinase 5/p35/p39: A Novel and Imminent Therapeutic Target for Diabetes Mellitus

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Danish Ahmed

2011-01-01

Full Text Available Present therapies to minify hyperglycaemia and insulin resistance mainly target ATP-sensitive K+ channels (KATP of pancreatic cells and PPAR-γ to enhance the insulin secretion and potential for GLUT expression, respectively. These current approaches are frequently associated with the various side effects such as hypoglycaemia and cardiovascular adverse events. CDK5 is a serine/threonine protein kinase, which forms active complexes with p35 or p39 found principally in neurons and in pancreatic β cells. Pieces of evidence from recent studies recommend the vital role of CDK5 in physiological functions in nonneuronal cells such as glucose-stimulated insulin secretion in pancreatic cells. Inhibition of CDK5 averts the decrease of insulin gene expression through the inhibition of nuclear translocation of PDX-1 which is a transcription factor for the insulin gene. The present pieces of evidence designate that CDK5 might be a potential drug target for the regulation of glucose-stimulated insulin secretion in the treatment of diabetes mellitus.

Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization

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Memin, Elisabeth, E-mail: molinac@mail.montclair.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Genzale, Megan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Crow, Marni; Molina, Carlos A. [Department of Biology and Molecular Biology, Montclair State University, Montclair, NJ, 07043 (United States)

2011-10-15

In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors.

High CDK6 protects cells from fulvestrant-mediated apoptosis and is a predictor of resistance to fulvestrant in estrogen receptor-positive metastatic breast cancer

DEFF Research Database (Denmark)

Alves, Carla Maria Lourenco; Elias, Daniel; Lyng, Maria B

2016-01-01

expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib re-sensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer...

Combination of HDAC inhibitor TSA and silibinin induces cell cycle arrest and apoptosis by targeting survivin and cyclinB1/Cdk1 in pancreatic cancer cells.

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Feng, Wan; Cai, Dawei; Zhang, Bin; Lou, Guochun; Zou, Xiaoping

2015-08-01

Histone deacetylases (HDAC) are involved in diverse biological processes and therefore emerge as potential targets for pancreatic cancer. Silibinin, an active component of silymarin, is known to inhibit growth of pancreatic cancer in vivo and in vitro. Herein, we examined the cytotoxic effects of TSA in combination with silibinin and investigated the possible mechanism in two pancreatic cancer cell lines (Panc1 and Capan2). Our study found that combination treatment of HDAC inhibitor and silibinin exerted additive growth inhibitory effect on pancreatic cancer cell. Annexin V-FITC/PI staining and flow cytometry analysis demonstrated that combination therapy induced G2/M cell cycle arrest and apoptosis in Panc1and Capan2 cells. The induction of apoptosis was further confirmed by evaluating the activation of caspases. Moreover, treatment with TSA and silibinin resulted in a profound reduction in the expression of cyclinA2, cyclinB1/Cdk1 and survivin. Taken together, our study might indicate that the novel combination of HDAC inhibitor and silibinin could offer therapeutic potential against pancreatic cancer. Copyright © 2015. Published by Elsevier Masson SAS.

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

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Benech Philippe

2009-08-01

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

Efficacy of cyclin dependent kinase 4 inhibitors as potent neuroprotective agents against insults relevant to Alzheimer's disease.

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Priyankar Sanphui

Full Text Available Alzheimer's disease (AD is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4 is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF deprivation and oligomeric beta amyloid (Aβ that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.

Kaempferol inhibits gastric cancer tumor growth: An in vitro and in vivo study.

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Song, Haibin; Bao, Junjie; Wei, Yuzhe; Chen, Yang; Mao, Xiaoguang; Li, Jianguo; Yang, Zhiwei; Xue, Yingwei

2015-02-01

Kaempferol, which is one of the general flavonoids, has recently been reported to suppress proliferation, induce cell cycle arrest and promote apoptosis in various human cancer cell lines. In the present study, the effect and mechanism of kaempferol on gastric cancer (GC) was examined. The results showed that kaempferol significantly inhibited the proliferation of MKN28 and SGC7901 cell lines. However, no significant inhibition in the GSE-1 normal gastric epithelial cell line in our experimental dose was detected. Additionally, significant apoptosis and G2/M phase cell cycle arrest were identified following the treatment of kaempferol. More importantly, we observed that kaempferol inhibited the growth of the tumor xenografts although no marked effects on liver, spleen or body weight were induced. The expression levels of G2/M cell cycle‑regulating factors, cyclin B1, Cdk1 and Cdc25C, were significantly reduced. In addition, kaempferol treatment markedly decreased the level of Bcl-2 concomitant with an increase in Bax expression, resulting in the upregulation of cleaved caspase-3 and -9, which promoted PARP cleavage. Kaempferol-treated cells also led to a decrease in p-Akt, p-ERK and COX-2 expression levels. The present study therefore provided evidence that kaempferol may be a therapeutic agent for GC.

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

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Butz, Henriett; Németh, Kinga; Czenke, Dóra; Likó, István; Czirják, Sándor; Zivkovic, Vladimir; Baghy, Kornélia; Korbonits, Márta; Kovalszky, Ilona; Igaz, Péter; Rácz, Károly; Patócs, Attila

2017-07-01

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially

Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

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Gao, Jingjing; Yao, Licheng; Xia, Tingting; Liao, Xuebin; Zhu, Deyu; Xiang, Ye

2018-04-01

The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ∼110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-α-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

Tob1 induces apoptosis and inhibits proliferation, migration and invasion of gastric cancer cells by activating Smad4 and inhibiting β‑catenin signaling.

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Kundu, Juthika; Wahab, S M Riajul; Kundu, Joydeb Kumar; Choi, Yoon-La; Erkin, Ozgur Cem; Lee, Hun Seok; Park, Sang Gyu; Shin, Young Kee

2012-09-01

Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of β‑catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3β, and increased the expression of β‑catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting β‑catenin-mediated signaling pathways.

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

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Suzuki, Kanayo [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Tanaka, Satoshi [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Yoshimoto, Tadashi [Department of Life Science, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka 572-8508 (Japan); Takaoka, Masanori [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan)

2014-01-03

Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDK inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

Imidazo[1,2-c]pyrimidin-5(6H)-one as a novel core of cyclin-dependent kinase 2 inhibitors: Synthesis, activity measurement, docking, and quantum mechanical scoring.

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Ajani, Haresh; Jansa, Josef; Köprülüoğlu, Cemal; Hobza, Pavel; Kryštof, Vladimír; Lyčka, Antonín; Lepsik, Martin

2018-04-23

We report on the synthesis, activity testing, docking, and quantum mechanical scoring of novel imidazo[1,2-c]pyrimidin-5(6H)-one scaffold for cyclin-dependent kinase 2 (CDK2) inhibition. A series of 26 compounds substituted with aromatic moieties at position 8 has been tested in in vitro enzyme assays and shown to inhibit CDK2. 2D structure-activity relationships have ascertained that small substituents at position 8 (up to the size of naphtyl or methoxyphenyl) generally lead to single-digit micromolar IC 50 values, whereas bigger substituents (substituted biphenyls) decreased the compounds' activities. The binding modes of the compounds obtained using Glide docking have exhibited up to 2 hinge-region hydrogen bonds to CDK2 and differed in the orientation of the inhibitor core and the placement of the 8-substituents. Semiempirical quantum mechanics-based scoring identified probable favourable binding modes, which will serve for future structure-based design and synthetic optimization of substituents of the heterocyclic core. In summary, we have identified a novel core for CDK2 inhibition and will explore it further to increase the potencies of the compounds and also monitor selectivities against other protein kinases. Copyright © 2018 John Wiley & Sons, Ltd.

Optogenetic Inhibition Reveals Distinct Roles for Basolateral Amygdala Activity at Discrete Time Points during Risky Decision Making.

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Orsini, Caitlin A; Hernandez, Caesar M; Singhal, Sarthak; Kelly, Kyle B; Frazier, Charles J; Bizon, Jennifer L; Setlow, Barry

2017-11-29

in which inhibition occurs. BLA inhibition during a period of deliberation between small, safe and large, risky outcomes decreased risky choice. In contrast, BLA inhibition during receipt of the large, punished outcome increased risky choice. These findings highlight the importance of temporally targeted approaches to understand neural substrates underlying complex cognitive processes. More importantly, they reveal novel information about dynamic BLA modulation of risky choice. Copyright © 2017 the authors 0270-6474/17/3711537-12$15.00/0.

Preparation of coffee oil-algae oil-based nanoemulsions and the study of their inhibition effect on UVA-induced skin damage in mice and melanoma cell growth.

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Yang, Chu-Ching; Hung, Chi-Feng; Chen, Bing-Huei

2017-01-01

Coffee grounds, a waste by-product generated after making coffee, contains approximately 15% coffee oil which can be used as a raw material in cosmetics. Algae oil rich in docosahexaenoic acid (DHA) has been demonstrated to possess anticancer and anti-inflammation functions. The objectives of this study were to develop a gas chromatography-mass spectrometry (GC-MS) method for the determination of fatty acids in coffee oil and algae oil and prepare a nanoemulsion for studying its inhibition effect on ultraviolet A-induced skin damage in mice and growth of melanoma cells B16-F10. A total of 8 and 5 fatty acids were separated and quantified in coffee oil and algae oil by GC-MS, respectively, with linoleic acid (39.8%) dominating in the former and DHA (33.9%) in the latter. A nanoemulsion with a particle size of 30 nm, zeta potential -72.72 mV, and DHA encapsulation efficiency 100% was prepared by using coffee oil, algae oil, surfactant (20% Span 80 and 80% Tween 80), and deionized water. Differential scanning calorimetry (DSC) analysis revealed a high stability of nanoemulsion when heated up to 110°C at a pH 6, whereas no significant changes in particle size distribution and pH occurred over a 90-day storage period at 4°C. Animal experiments showed that a dose of 0.1% coffee oil-algae oil nanoemulsion was effective in mitigating trans-epidermal water loss, skin erythema, melanin formation, and subcutaneous blood flow. Cytotoxicity test implied effective inhibition of melanoma cell growth by nanoemulsion with an IC 50 value of 26.5 µg/mL and the cell cycle arrested at G2/M phase. A dose-dependent upregulation of p53, p21, cyclin B, and cyclin A expressions and downregulation of CDK1 and CDK2 occurred. Also, both Bax and cytochrome c expressions were upregulated and bcl-2 expression downregulated, accompanied by a rise in caspase-3, caspase-8, and caspase-9 activities for apoptosis execution. Collectively, the apoptosis pathway of melanoma cells B16-F10 may involve

Ganoderma lucidum suppresses growth of breast cancer cells through the inhibition of Akt/NF-kappaB signaling.

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Jiang, Jiahua; Slivova, Veronika; Harvey, Kevin; Valachovicova, Tatiana; Sliva, Daniel

2004-01-01

Ganoderma lucidum (Reishi, Lingzhi) is a popular Asian mushroom that has been used for more than 2 millennia for the general promotion of health and was therefore called the "Mushroom of Immortality." Ganoderma lucidum was also used in traditional Chinese medicine to prevent or treat a variety of diseases, including cancer. We previously demonstrated that Ganoderma lucidum suppresses the invasive behavior of breast cancer cells by inhibiting the transcription factor NF-kappaB. However, the molecular mechanisms responsible for the inhibitory effects of Ganoderma lucidum on the growth of highly invasive and metastatic breast cancer cells has not been fully elucidated. Here, we show that Ganoderma lucidum inhibits proliferation of breast cancer MDA-MB-231 cells by downregulating Akt/NF-kappaB signaling. Ganoderma lucidum suppresses phosphorylation of Akt on Ser473 and downregulates the expression of Akt, which results in the inhibition of NF-kappaB activity in MDA-MB-231 cells. The biological effect of Ganoderma lucidum was demonstrated by cell cycle arrest at G0/G1, which was the result of the downregulation of expression of NF-kappaB-regulated cyclin D1, followed by the inhibition of cdk4. Our results suggest that Ganoderma lucidum inhibits the growth of MDA-MB-231 breast cancer cells by modulating Akt/NF-kappaB signaling and could have potential therapeutic use for the treatment of breast cancer.

Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

Science.gov (United States)

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).

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

Science.gov (United States)

Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

DEFF Research Database (Denmark)

Syljuåsen, Randi G; Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg

2005-01-01

by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose...

Transferable scoring function based on semiempirical quantum mechanical PM6-DH2 method: CDK2 with 15 structurally diverse inhibitors

Czech Academy of Sciences Publication Activity Database

Dobeš, Petr; Fanfrlík, Jindřich; Řezáč, Jan; Otyepka, M.; Hobza, Pavel

2011-01-01

Roč. 25, č. 3 (2011), s. 223-235 ISSN 0920-654X R&D Projects: GA MŠk LC512; GA ČR GAP208/11/0295 Grant - others:European Social Fund(XE) CZ.1.05/2.1.00/03.0058 Institutional research plan: CEZ:AV0Z40550506 Keywords : CDK2 * semiempirical quantum mechanical method PM6-DH2 * drug design Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.386, year: 2011

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

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Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

2016-07-11

mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

The prolyl isomerase Pin1 acts synergistically with CDK2 to regulate the basal activity of estrogen receptor α in breast cancer.

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Chiara Lucchetti

Full Text Available In hormone receptor-positive breast cancers, most tumors in the early stages of development depend on the activity of the estrogen receptor and its ligand, estradiol. Anti-estrogens, such as tamoxifen, have been used as the first line of therapy for over three decades due to the fact that they elicit cell cycle arrest. Unfortunately, after an initial period, most cells become resistant to hormonal therapy. Peptidylprolyl isomerase 1 (Pin1, a protein overexpressed in many tumor types including breast, has been demonstrated to modulate ERalpha activity and is involved in resistance to hormonal therapy. Here we show a new mechanism through which CDK2 drives an ERalpha-Pin1 interaction under hormone- and growth factor-free conditions. The PI3K/AKT pathway is necessary to activate CDK2, which phosphorylates ERalphaSer294, and mediates the binding between Pin1 and ERalpha. Site-directed mutagenesis demonstrated that ERalphaSer294 is essential for Pin1-ERalpha interaction and modulates ERalpha phosphorylation on Ser118 and Ser167, dimerization and activity. These results open up new drug treatment opportunities for breast cancer patients who are resistant to anti-estrogen therapy.

Hepatitis C virus core protein expression leads to biphasic regulation of the p21 cdk inhibitor and modulation of hepatocyte cell cycle

International Nuclear Information System (INIS)

Nguyen, Hau; Mudryj, Maria; Guadalupe, Moraima; Dandekar, Satya

2003-01-01

Hepatitis C virus (HCV) Core protein is implicated in viral pathogenesis by the modulation of hepatocyte gene expression and function. To determine the effect of Core protein on the cell-cycle control of hepatocytes, a HepG2 cell line containing a Flag-tagged Core under the control of an inducible promoter was generated. Initial Core protein expression included the presence of unprocessed (191 aa) and processed (173 aa) forms of the Core proteins with the processed form becoming dominant later. Expression of the 191 aa form of Core protein corresponded to an increase in the expression of the p21, a decrease in cdk2-dependent kinase activity, and a decrease in the percentage of cells in S-phase along with an accumulation of cells in the G 0 /G 1 phase of the cell cycle. As the processed form accumulated, the p21 levels started to decline, suggesting that Core protein regulates p21 expression in a biphasic manner. These findings implicate Core protein in potentially modulating hepatocyte cell cycle differentially in the early stages of infection through biphasic regulation of p21 cdk kinase inhibitor

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

International Nuclear Information System (INIS)

Carletti, Eugenie; Santoni, Gianluca; Colletier, Jacques-Philippe; Schopfer, Lawrence M.; Lockridge, Oksana; Masson, Patrick; Nachon, Florian; Weik, Martin

2013-01-01

Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aero-toxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bio-scavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 Angstroms snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H 2 16 O or H 2 18 O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP. (authors)

Theobromine inhibits differentiation of 3T3-L1 cells during the early stage of adipogenesis via AMPK and MAPK signaling pathways.

Science.gov (United States)

Jang, Yeon Jeong; Koo, Hyun Jung; Sohn, Eun-Hwa; Kang, Se Chan; Rhee, Dong-Kwon; Pyo, Suhkneung

2015-07-01

Obesity is characterized by hypertrophy and/or by the differentiation or adipogenesis of pre-existing adipocytes. In this study, we investigated the inhibitory effects of theobromine, a type of alkaloid in cocoa, on adipocyte differentiation of 3T3-L1 preadipocytes and its mechanisms of action. Theobromine inhibited the accumulation of lipid droplets, the expression of PPARγ and C/EBPα, and the mRNA expression of aP2 and leptin. The inhibition of adipogenic differentiation by theobromine occurred primarily in the early stages of differentiation. In addition, theobromine arrested the cell cycle at the G0/G1 phase and regulated the expressions of CDK2, p27, and p21. Theobromine treatment increased AMPK phosphorylation and knockdown of AMPKα1/α2 prevented the ability of theobromine to inhibit PPARγ expression in the differentiating 3T3-L1 cells. Theobromine reduced the phosphorylation of ERK and JNK. Moreover, the secretion and the mRNA level of TNF-α and IL-6 were inhibited by theobromine treatment. These data suggest that theobromine inhibits adipocyte differentiation during the early stages of adipogenesis by regulating the expression of PPARγ and C/EBPα through the AMPK and ERK/JNK signaling pathways in 3T3-L1 preadipocytes.

SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

International Nuclear Information System (INIS)

Cao, Rubo; Ding, Qian; Li, Pindong; Xue, Jun; Zou, Zhenwei; Huang, Jing; Peng, Gang

2013-01-01

Radioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1). A NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression. Compared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression. SHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance

Activation of Cyclin-Dependent Kinase 5 Is a Consequence of Cell Death

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Yixia Ye

2009-01-01

Full Text Available Cyclin-dependent kinase 5 (Cdk5 is similar to other Cdks but is activated during cell differentiation and cell death rather than cell division. Since activation of Cdk5 has been reported in many situations leading to cell death, we attempted to determine if it was required for any form of cell death. We found that Cdk5 is activated during apoptotic deaths and that the activation can be detected even when the cells continue to secondary necrosis. This activation can occur in the absence of Bim, calpain, or neutral cathepsins. The kinase is typically activated by p25, derived from p35 by calpain-mediated cleavage, but inhibition of calpain does not affect cell death or the activation of Cdk5. Likewise, RNAi-forced suppression of the synthesis of Cdk5 does not affect the incidence or kinetics of cell death. We conclude that Cdk5 is activated as a consequence of metabolic changes that are common to many forms of cell death. Thus its activation suggests processes during cell death that will be interesting or important to understand, but activation of Cdk5 is not necessary for cells to die.

Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

Energy Technology Data Exchange (ETDEWEB)

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

2010-01-01

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

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

Science.gov (United States)

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Elevated C1orf63 expression is correlated with CDK10 and predicts better outcome for advanced breast cancers: a retrospective study

International Nuclear Information System (INIS)

Hong, Chao-Qun; Zhang, Fan; You, Yan-Jie; Qiu, Wei-Li; Giuliano, Armando E.; Cui, Xiao-Jiang; Zhang, Guo-Jun; Cui, Yu-Kun

2015-01-01

Chromosome 1 open reading frame 63 (C1orf63) is located on the distal short arm of chromosome 1, whose allelic loss has been observed in several human cancers. C1orf63 has been reported to be up-regulated in IL-2-starved T lymphocytes, which suggests it might be involved in cell cycle control, a common mechanism for carcinogenesis. Here we investigated the expression and clinical implication of C1orf63 in breast cancer. Paraffin-embedded specimens, clinicopathological features and follow-up data of the breast cancer patients were collected. Publicly available microarray and RNA-seq datasets used in this study were downloaded from ArrayExpress of EBI and GEO of NCBI. KM plotter tool was also adopted. The expression of C1orf63 and CDK10, one known cell cycle-dependent tumor suppressor in breast cancer, was assessed by immunohistochemistry. Western blotting was performed to detect C1orf63 protein in human breast cancer cell lines, purchased from the Culture Collection of the Chinese Academy of Sciences, Shanghai. In a group of 12 human breast tumors and their matched adjacent non-cancerous tissues, C1orf63 expression was observed in 7 of the 12 breast tumors, but not in the 12 adjacent non-cancerous tissues (P < 0.001). Similar results were observed of C1orf63 mRNA expression both in breast cancer and several other cancers, including lung cancer, prostate cancer and hepatocellular carcinoma. In another group of 182 breast cancer patients, C1orf63 expression in tumors was not correlated with any clinicopathological features collected in this study. Survival analyses showed that there was no significant difference of overall survival (OS) rates between the C1orf63 (+) group and the C1orf63 (−) group (P = 0.145). However, the analyses of KM plotter displayed a valid relationship between C1orf63 and RFS (relapse free survival)/OS (P < 0.001; P = 0.007). Notablely, in breast cancers with advanced TNM stages (III ~ IV) among these 182 patients, C1orf63 expression was an

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins

International Nuclear Information System (INIS)

Deng, Lin; Lu, Yuanyuan; Zhao, Xiaodi; Sun, Yi; Shi, Yongquan; Fan, Hongwei; Liu, Changhao; Zhou, Jinfeng; Nie, Yongzhan; Wu, Kaichun; Fan, Daiming; Guo, Xuegang

2013-01-01

Highlights: •Overexpression of Ran in pancreatic cancer was correlated with histological grade. •Downregulation of Ran could induce cell apoptosis and inhibit cell proliferation. •The effects were mediated by cell cycle proteins, Survivin and cleaved Caspase-3. -- Abstract: Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3

GL-1196 Suppresses the Proliferation and Invasion of Gastric Cancer Cells via Targeting PAK4 and Inhibiting PAK4-Mediated Signaling Pathways

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Jian Zhang

2016-04-01

Full Text Available Gastric cancer, which is the most common malignant gastrointestinal tumor, has jumped to the third leading cause of cancer-related mortality worldwide. It is of great importance to identify novel and potent drugs for gastric cancer treatment. P21-activated kinase 4 (PAK4 has emerged as an attractive target for the development of anticancer drugs in consideration of its vital functions in tumorigenesis and progression. In this paper, we reported that GL-1196, as a small molecular compound, effectively suppressed the proliferation of human gastric cancer cells through downregulation of PAK4/c-Src/EGFR/cyclinD1 pathway and CDK4/6 expression. Moreover, GL-1196 prominently inhibited the invasion of human gastric cancer cells in parallel with blockage of the PAK4/LIMK1/cofilin pathway. Interestingly, GL-1196 also inhibited the formation of filopodia and induced cell elongation in SGC7901 and BGC823 cells. Taken together, these results provided novel insights into the potential therapeutic strategy for gastric cancer.

Profile of palbociclib in the treatment of metastatic breast cancer

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Ehab M

2016-05-01

Full Text Available Moataz Ehab,1 Mohamad Elbaz2,31Department of Pharmacy Practice, 2Department of Pharmacology, Pharmacy School, Helwan University, Egypt; 3Department of Pathology, The Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH, USAAbstract: Breast cancer is the most common cancer diagnosed in women. Each year, thousands die either because of disease progression or failure of treatment. Breast cancer is classified into different subtypes based on the molecular expression of estrogen receptor (ER, progesterone receptor, and/or human epidermal growth factor receptor 2 (HER2. These receptors represent important therapeutic targets either through monoclonal antibodies or through small-molecule inhibitors directed toward them. However, up to 40% of patients develop either a primary or a secondary resistance to the current treatments. Therefore, there is an urgent need for investigating new targets in order to overcome the resistance and/or enhance the current therapies. Cell cycle is altered in many human cancers, especially in breast cancer. Cyclin-dependent kinases (CDKs, especially CDK4 and CDK6, play a pivotal role in cell cycle progression that makes them potential targets for new promising therapies. CDK inhibition has shown strong antitumor activities, ranging from cytostatic antiproliferative effects to synergistic effects in combination with other antitumor drugs. In order to overcome the drawbacks of the first-generation CDK inhibitors, recently, new CDK inhibitors have emerged that are more selective to CDK4 and CDK6 such as palbociclib, which is the most advanced CDK4/6 inhibitor in trials. In preclinical studies, palbociclib has shown a very promising antitumor activity, especially against ERα+ breast cancer subtype. Palbociclib has gained world attention, and US the Food and Drug Administration has accelerated its approval for first-line treatment in combination with letrozole for the first-line systematic

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

Science.gov (United States)

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.

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

Science.gov (United States)

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

2015-01-01

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

In vitro toxicity of polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons to cetacean cells and tissues

Energy Technology Data Exchange (ETDEWEB)

Carvan, M.J. III.

1993-01-01

Cetaceans bioaccumulate high aromatic hydrocarbon tissue residues, and elevated levels of PCB residues in tissues are proposed to have occurred concurrently with recent epizootic deaths of dolphins. The objectives of this study were: (1) to develop and characterize an epithelial cell line derived from dolphin tissues, (2) to investigate the effects of hydrocarbon pollutants on those cells, and (3) to analyze the toxicity of hydrocarbon pollutants on cetacean tissues in vitro. An epithelial cell line, Carvan dolphin kidney (CDK), isolated from a spontaneously aborted female bottlenose dolphin, Tursiops truncatus, grew rapidly. These cells were neither transformed nor immortal. Velocity sedimentation analysis showed CDK cells contained nuclear aryl hydrocarbon receptor, suggestive of cytochrome P450 inducibility. BaP inhibited mitosis in CDK cells in a dose-dependent manner. Data indicate that CDK cells metabolize BaP, that BaP metabolites bind to cellular DNA initiating unscheduled DNA synthesis, and that the inhibition of cytochrome P450 metabolism decrease the BaP-associated inhibition of mitosis in dolphin cells. The data also suggest that TCDD acts synergistically to increase the levels of DNA damage by the procarcinogen BaP. Cetacean liver microsomes was isolated and evaluated for the presence of cytochrome P450 proteins by SDS-PAGE, apparent minimum molecular weight determination, and immunoblot analysis. P450 activity was induced in cetacean tissue samples and CDK cells by exposure in vitro to one of several cytochrome P450-inducing chemicals. The data suggest that cetacean tissues and cells can be utilized to study the in vitro induction of cytochrome P450, resultant metabolism of xenobiotic contaminants, and the subsequent cellular and molecular responses. However, the identity of specific P450 isozymes involved in this process will remain undetermined until monoclonal antibodies that recognize cetacean P450s can be generated.

Genome-Wide Association and Functional Follow-Up Reveals New Loci for Kidney Function

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Fuchsberger, Christian; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Taliun, Daniel; Li, Man; Gao, Xiaoyi; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C.; O'Seaghdha, Conall M.; Glazer, Nicole; Isaacs, Aaron; Liu, Ching-Ti; Smith, Albert V.; O'Connell, Jeffrey R.; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Johnson, Andrew D.; Gierman, Hinco J.; Feitosa, Mary; Hwang, Shih-Jen; Atkinson, Elizabeth J.; Lohman, Kurt; Cornelis, Marilyn C.; Johansson, Åsa; Tönjes, Anke; Dehghan, Abbas; Chouraki, Vincent; Holliday, Elizabeth G.; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y.; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B.; Launer, Lenore J.; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D.; Boerwinkle, Eric; Schmidt, Helena; Cavalieri, Margherita; Rao, Madhumathi; Hu, Frank B.; Demirkan, Ayse; Oostra, Ben A.; de Andrade, Mariza; Turner, Stephen T.; Ding, Jingzhong; Andrews, Jeanette S.; Freedman, Barry I.; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H.-Erich; Kolcic, Ivana; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E.; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H.; Wright, Alan F.; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Endlich, Karlhans; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K.; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G.; Rivadeneira, Fernando; Aulchenko, Yurii S.; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Ketkar, Shamika; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Giulianini, Franco; Krämer, Bernhard K.; Portas, Laura; Ford, Ian; Buckley, Brendan M.; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Metzger, Marie; Mitchell, Paul; Ciullo, Marina; Kim, Stuart K.; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J. Wouter; Probst-Hensch, Nicole M.; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R.; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; Siscovick, David S.; van Duijn, Cornelia M.; Borecki, Ingrid; Kardia, Sharon L. R.; Liu, Yongmei; Curhan, Gary C.; Rudan, Igor; Gyllensten, Ulf; Wilson, James F.; Franke, Andre; Pramstaller, Peter P.; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline C. M.; Hayward, Caroline; Ridker, Paul; Parsa, Afshin; Bochud, Murielle; Heid, Iris M.; Goessling, Wolfram; Chasman, Daniel I.; Kao, W. H. Linda; Fox, Caroline S.

2012-01-01

Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD. PMID:22479191

Genome-wide association and functional follow-up reveals new loci for kidney function.

Science.gov (United States)

Pattaro, Cristian; Köttgen, Anna; Teumer, Alexander; Garnaas, Maija; Böger, Carsten A; Fuchsberger, Christian; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Taliun, Daniel; Li, Man; Gao, Xiaoyi; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C; O'Seaghdha, Conall M; Glazer, Nicole; Isaacs, Aaron; Liu, Ching-Ti; Smith, Albert V; O'Connell, Jeffrey R; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Johnson, Andrew D; Gierman, Hinco J; Feitosa, Mary; Hwang, Shih-Jen; Atkinson, Elizabeth J; Lohman, Kurt; Cornelis, Marilyn C; Johansson, Åsa; Tönjes, Anke; Dehghan, Abbas; Chouraki, Vincent; Holliday, Elizabeth G; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B; Launer, Lenore J; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D; Boerwinkle, Eric; Schmidt, Helena; Cavalieri, Margherita; Rao, Madhumathi; Hu, Frank B; Demirkan, Ayse; Oostra, Ben A; de Andrade, Mariza; Turner, Stephen T; Ding, Jingzhong; Andrews, Jeanette S; Freedman, Barry I; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H-Erich; Kolcic, Ivana; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H; Wright, Alan F; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Endlich, Karlhans; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G; Rivadeneira, Fernando; Aulchenko, Yurii S; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Ketkar, Shamika; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Giulianini, Franco; Krämer, Bernhard K; Portas, Laura; Ford, Ian; Buckley, Brendan M; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Metzger, Marie; Mitchell, Paul; Ciullo, Marina; Kim, Stuart K; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J Wouter; Probst-Hensch, Nicole M; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; Siscovick, David S; van Duijn, Cornelia M; Borecki, Ingrid; Kardia, Sharon L R; Liu, Yongmei; Curhan, Gary C; Rudan, Igor; Gyllensten, Ulf; Wilson, James F; Franke, Andre; Pramstaller, Peter P; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline C M; Hayward, Caroline; Ridker, Paul; Parsa, Afshin; Bochud, Murielle; Heid, Iris M; Goessling, Wolfram; Chasman, Daniel I; Kao, W H Linda; Fox, Caroline S

2012-01-01

Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD.

Genome-wide association and functional follow-up reveals new loci for kidney function.

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Cristian Pattaro

Full Text Available Chronic kidney disease (CKD is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR, the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD.

Oxygen-Glucose Deprivation Induces G2/M Cell Cycle Arrest in Brain Pericytes Associated with ERK Inactivation.

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Wei, Wenjie; Yu, Zhiyuan; Xie, Minjie; Wang, Wei; Luo, Xiang

2017-01-01

Growing evidence has revealed that brain pericytes are multifunctional and contribute to the pathogenesis of a number of neurological disorders. However, the role of pericytes in cerebral ischemia, and especially the pathophysiological alterations in pericytes, remains unclear. In the present study, our aim was to determine whether the proliferation of pericytes is affected by cerebral ischemia and, if so, to identify the underlying mechanism(s). Cultured brain pericytes subjected to oxygen-glucose deprivation (OGD) were used as our model of cerebral ischemia; the protein expression levels of cyclin D1, cyclin E, cdk4, and cyclin B1 were determined by Western blot analysis, and cell cycle analysis was assessed by flow cytometry. The OGD treatment reduced the brain pericyte proliferation by causing G2/M phase arrest and downregulating the protein levels of cyclin D1, cyclin E, cdk4, and cyclin B1. Further studies demonstrated a simultaneous decrease in the activity of extracellular regulated protein kinases (ERK), suggesting a critical role of the ERK signaling cascade in the inhibition of OGD-induced pericyte proliferation. We suggest that OGD inhibition of the proliferation of brain pericytes is associated with the inactivation of the ERK signaling pathway, which arrests them in the G2/M phase.

A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

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Elgjo Kjell

2009-07-01

Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

NANOG Is Multiply Phosphorylated and Directly Modified by ERK2 and CDK1 In Vitro

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Justin Brumbaugh

2014-01-01

Full Text Available NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS. MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG.

Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells.

Science.gov (United States)

Cho, Sun-Mi; Lee, Eun-Ok; Kim, Sung-Hoon; Lee, Hyo-Jeong

2014-07-30

The essential oil of Pinus koraiensis (EOPK) is biologically active compound obtained from the leaves of P. koraiensis. The goal of this study was to investigate the anti-cancer mechanism of EOPK in HCT116 colorectal cancer cells. HCT116 cell proliferation was assessed by conducting crystal violet and BrdU assays. To assess the effects of EOPK on cell migration, we performed a wound-healing assay. Further, the contribution of PAK1 to EOPK-induced AKT and extracellular signal-regulated kinase (ERK) suppression was assessed by siRNA-mediated PAK1 knockdown. Changes to the expression and phosphorylation of PAK1 and its effectors were determined by western blotting, and changes to the actin cytoskeleton were determined by performing an immunofluorescence assay. EOPK significantly decreased HCT116 cell proliferation and migration, and induced G1 arrest without affecting normal cells. Additionally, EOPK suppressed the expression of PAK1, and decreased ERK and AKT phosphorylation in HCT116 cells. Finally, EOPK suppressed β-catenin, cyclin D1, and CDK4/6 expression. Our studies indicate that EOPK significantly reduced proliferation and migration of colorectal cancer cells. Furthermore, EOPK suppressed PAK1 expression in a dose-dependent manner, and this suppression of PAK1 led to inhibition of ERK, AKT, and β-catenin activities. Our findings suggest that EOPK exerts its anticancer activity via the inhibition of PAK1 expression, suggesting it may be a potent chemotherapeutic agent for colorectal cancer.

Net expression inhibits the growth of pancreatic ductal adenocarcinoma cell PL45 in vitro and in vivo.

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Baiwen Li

Full Text Available Pancreatic ductal adenocarcinoma has a poor prognosis due to late diagnosis and a lack of effective therapeutic options. Thus, it is important to better understand its molecular mechanisms and to develop more effective treatments for the disease. The ternary complex factor Net, which exerts its strong inhibitory function on transcription of proto-oncogene gene c-fos by forming ternary complexes with a second transcription factor, has been suspected of being involved in pancreatic cancer and other tumors biology. In this study, we found that the majority of pancreatic ductal adenocarcinoma tissues and cell lines had weak or no expression of Net, whereas significantly high level of Net expression occurred in paired adjacent normal tissues we studied. Furthermore, using in vitro and in vivo model systems, we found that overexpression of Net inhibited cell growth and survival and induced cell apoptosis in human pancreatic ductal adenocarcinoma cell PL45; the mechanisms by which Net inhibited the cell cycle progression were mainly through P21-Cyclin D1/CDK4 Pathway. Our data thus suggested that Net might play an important role in pancreatic carcinogenesis, possibly by acting as a tumor suppressor gene.

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

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Md Hafiz Uddin

Full Text Available 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.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.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.The present findings suggest that oncogenes, PSMD10 and CDK4, and tumor suppressors, p53 and RB, are involved in the

Regulation of the cell cycle by irradiation

International Nuclear Information System (INIS)

Akashi, Makoto

1995-01-01

The molecular mechanism of cell proliferation is extremely complex; deregulation results in neoplastic transformation. In eukaryotes, proliferation of cells is finely regulated through the cell cycle. Studies have shown that the cell cycle is regulated by s series of enzymes known as cyclin-dependent kinases (CDKs). The activities of CDKs are controlled by their association with regulatory subunits, cyclins; the expression of cyclins and the activation of the different cyclin-CDK complexes are required for the cell to cycle. Thus, the cell cycle is regulated by activating and inhibiting phosphorylation of the CDK subunits and this program has internal check points at different stages of the cell cycle. When cells are exposed to external insults such as DNA damaging agents, negative regulation of the cell cycle occurs; arrest in either G1 or G2 stage is induced to prevent the cells from prematurely entering into the next stage before DNA is repaired. Recently, a potent inhibitor of CDKs, which inhibits the phosphorylation of retinoblastoma susceptibility (Rb) gene product by cyclin A-CDK2, cyclin E-CDK2, cyclin D1-CDK4, and cyclin D2-CDK4 complexes has been identified. This protein named WAF1, Sdi1, Cip1, or p21 (a protein of Mr 21,000) contains a p53-binding site in its promoter and studies have reported that the expression of WAF1 was directly regulated by p53; cells with loss of p53 activity due to mutational alteration were unable to induce WAF1. This chapter will be focused on the mechanisms of the cell cycle including inhibitors of CDKs, and the induction of WAF1 by irradiation through a pathway independent of p53 will be also described. (author)

A comparative study of ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery.

Science.gov (United States)

Kim, Joonyup; Wilson, Rebecca L; Case, J Brett; Binder, Brad M

2012-11-01

Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa 'Nipponbare') seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics.

Cdk1-cyclin B1-mediated phosphorylation of tumor-associated microtubule-associated protein/cytoskeleton-associated protein 2 in mitosis.

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Hong, Kyung Uk; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-06-12

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis*

Science.gov (United States)

Uk Hong, Kyung; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-01-01

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis. PMID:19369249

Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway

International Nuclear Information System (INIS)

Agner, Jeppe; Falck, Jacob; Lukas, Jiri; Bartek, Jiri

2005-01-01

When exposed to DNA-damaging insults such as ionizing radiation (IR) or ultraviolet light (UV), mammalian cells activate checkpoint pathways to halt cell cycle progression or induce cell death. Here we examined the ability of five commonly used anticancer drugs with different mechanisms of action to activate the Chk1/Chk2-Cdc25A-CDK2/cyclin E cell cycle checkpoint pathway, previously shown to be induced by IR or UV. Whereas exposure of human cells to topoisomerase inhibitors camptothecin, etoposide, or adriamycin resulted in rapid (within 1 h) activation of the pathway including degradation of the Cdc25A phosphatase and inhibition of cyclin E/CDK2 kinase activity, taxol failed to activate this checkpoint even after a prolonged treatment. Unexpectedly, although the alkylating agent cisplatin also induced degradation of Cdc25A (albeit delayed, after 8-12 h), cyclin E/CDK2 activity was elevated and DNA synthesis continued, a phenomena that correlated with increased E2F1 protein levels and consequently enhanced expression of cyclin E. These results reveal a differential impact of various classes of anticancer chemotherapeutics on the Cdc25A-degradation pathway, and indicate that the kinetics of checkpoint induction, and the relative balance of key components within the DNA damage response network may dictate whether the treated cells arrest their cell cycle progression

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

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Jennifer L Bandura

Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

Science.gov (United States)

Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

2013-01-01

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II.

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Margaret A Schwarz

Full Text Available Endothelial-Monocyte Activating Polypeptide (EMAP II is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date.Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration.Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.

Direct effects of HIV-1 Tat on excitability and survival of primary dorsal root ganglion neurons: possible contribution to HIV-1-associated pain.

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Xianxun Chi

Full Text Available The vast majority of people living with human immunodeficiency virus type 1 (HIV-1 have pain syndrome, which has a significant impact on their quality of life. The underlying causes of HIV-1-associated pain are not likely attributable to direct viral infection of the nervous system due to the lack of evidence of neuronal infection by HIV-1. However, HIV-1 proteins are possibly involved as they have been implicated in neuronal damage and death. The current study assesses the direct effects of HIV-1 Tat, one of potent neurotoxic viral proteins released from HIV-1-infected cells, on the excitability and survival of rat primary dorsal root ganglion (DRG neurons. We demonstrated that HIV-1 Tat triggered rapid and sustained enhancement of the excitability of small-diameter rat primary DRG neurons, which was accompanied by marked reductions in the rheobase and resting membrane potential (RMP, and an increase in the resistance at threshold (R(Th. Such Tat-induced DRG hyperexcitability may be a consequence of the inhibition of cyclin-dependent kinase 5 (Cdk5 activity. Tat rapidly inhibited Cdk5 kinase activity and mRNA production, and roscovitine, a well-known Cdk5 inhibitor, induced a very similar pattern of DRG hyperexcitability. Indeed, pre-application of Tat prevented roscovitine from having additional effects on the RMP and action potentials (APs of DRGs. However, Tat-mediated actions on the rheobase and R(Th were accelerated by roscovitine. These results suggest that Tat-mediated changes in DRG excitability are partly facilitated by Cdk5 inhibition. In addition, Cdk5 is most abundant in DRG neurons and participates in the regulation of pain signaling. We also demonstrated that HIV-1 Tat markedly induced apoptosis of primary DRG neurons after exposure for longer than 48 h. Together, this work indicates that HIV-1 proteins are capable of producing pain signaling through direct actions on excitability and survival of sensory neurons.

An attempt to evaluate the effect of vitamin K3 using as an enhancer of anticancer agents.

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Matzno, Sumio; Yamaguchi, Yuka; Akiyoshi, Takeshi; Nakabayashi, Toshikatsu; Matsuyama, Kenji

2008-06-01

The possibility of vitamin K3 (VK3) as an anticancer agent was assessed. VK3 dose-dependently diminished the cell viability (measured as esterase activity) with IC50 of 13.7 microM and Hill coefficient of 3.1 in Hep G2 cells. It also decreased the population of S phase and arrested cell cycle in the G2/M phase in a dose-dependent manner. G2/M arrest was regulated by the increment of cyclin A/cdk1 and cyclin A/cdk2 complex, and contrasting cyclin B/cdk1 complex decrease. Finally, combined application demonstrated that VK3 significantly enhanced the cytotoxicity of etoposide, a G2 phase-dependent anticancer agent, whereas it reduced the cytotoxic activity of irinotecan, a S phase-dependent agent. These findings suggest that VK3 induces G2/M arrest by inhibition of cyclin B/cdk1 complex formation, and is thus useful as an enhancer of G2 phase-dependent drugs in hepatic cancer chemotherapy.

Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis

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Giaever Guri

2009-01-01

Full Text Available Abstract Background Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug. Results Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C. Conclusion Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells.

Compound K, a Ginsenoside Metabolite, Inhibits Colon Cancer Growth via Multiple Pathways Including p53-p21 Interactions

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Eugene B. Chang

2013-01-01

Full Text Available Compound K (20-O-beta-D-glucopyranosyl-20(S-protopanaxadiol, CK, an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC. A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied to investigate the anticancer effects of CK including antiproliferation, apoptosis and cell cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose- and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.

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

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Xiao-Su Zhao

Full Text Available 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.

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

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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.

Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells.

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Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

2011-10-01

Gastric cancer is a leading cause of cancer-related deaths, worldwide being second only to lung cancer as a cause of death. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms of arctigenin for anti-tumor effect on gastric cancer have not been examined. This study examined the biological effects of arctigenin on the human gastric cancer cell line SNU-1 and AGS. Cell proliferation was determined by MTT assay. In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by arctigenin in a time and dose dependent manner, as compared with SNU-1 and AGS cells cultured in the absence of arctigenin. Inhibition of cell proliferation by arctigenin was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bcl-2 to Bax by arctigenin. Also, arctigenin blocked cell cycle arrest from G(1) to S phase by regulating the expression of cell cycle regulatory proteins such as Rb, cyclin D1, cyclin E, CDK4, CDK2, p21Waf1/Cip1 and p15 INK4b. The antiproliferative effect of arctigenin on SNU-1 and AGS gastric cancer cells revealed in this study suggests that arctigenin has intriguing potential as a chemopreventive or chemotherapeutic agent. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

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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.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

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Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

2014-01-01

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

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

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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

Non-specific chemical inhibition of the Fanconi anemia pathway sensitizes cancer cells to cisplatin

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Jacquemont Céline

2012-04-01

Full Text Available Abstract Background Platinum compounds such as cisplatin and carboplatin are DNA crosslinking agents widely used for cancer chemotherapy. However, the effectiveness of platinum compounds is often tempered by the acquisition of cellular drug resistance. Until now, no pharmacological approach has successfully overcome cisplatin resistance in cancer treatment. Since the Fanconi anemia (FA pathway is a DNA damage response pathway required for cellular resistance to DNA interstrand crosslinking agents, identification of small molecules that inhibit the FA pathway may reveal classes of chemicals that sensitize cancer cells to cisplatin. Results Through a cell-based screening assay of over 16,000 chemicals, we identified 26 small molecules that inhibit ionizing radiation and cisplatin-induced FANCD2 foci formation, a marker of FA pathway activity, in multiple human cell lines. Most of these small molecules also compromised ionizing radiation-induced RAD51 foci formation and homologous recombination repair, indicating that they are not selective toward the regulation of FANCD2. These compounds include known inhibitors of the proteasome, cathepsin B, lysosome, CHK1, HSP90, CDK and PKC, and several uncharacterized chemicals including a novel proteasome inhibitor (Chembridge compound 5929407. Isobologram analyses demonstrated that half of the identified molecules sensitized ovarian cancer cells to cisplatin. Among them, 9 demonstrated increased efficiency toward FA pathway-proficient, cisplatin-resistant ovarian cancer cells. Six small molecules, including bortezomib (proteasome inhibitor, CA-074-Me (cathepsin B inhibitor and 17-AAG (HSP90 inhibitor, synergized with cisplatin specifically in FA-proficient ovarian cancer cells (2008 + FANCF, but not in FA-deficient isogenic cells (2008. In addition, geldanamycin (HSP90 inhibitor and two CHK1 inhibitors (UCN-01 and SB218078 exhibited a significantly stronger synergism with cisplatin in FA

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade

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David A. Schaer

2018-03-01

Full Text Available Summary: Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6, has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity. : Schaer, Beckmann et al. describe unique immune-modulating properties of abemaciclib that include upregulation of antigen presentation on tumor cells and increased T cell activation. These activities synergize with anti-PD-L1 therapy to further enhance immune activation, including macrophage and DC antigen presentation, and also lead to a reciprocal increase in abemaciclib-dependent cell cycle gene regulation. Keywords: CDK4/6, abemaciclib, PD-1, PD-L1, combination immunotherapy, cancer

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation.

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Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

2016-09-15

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0-15μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol

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Androic, Ilija; Krämer, Andrea; Yan, Ruilan; Rödel, Franz; Gätje, Regine; Kaufmann, Manfred; Strebhardt, Klaus; Yuan, Juping

2008-01-01

Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers. In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models. Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo. Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy

The ibrutinib B-cell proliferation inhibition is potentiated in vitro by dexamethasone: Application to chronic lymphocytic leukemia.

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Manzoni, Delphine; Catallo, Régine; Chebel, Amel; Baseggio, Lucile; Michallet, Anne-Sophie; Roualdes, Olivier; Magaud, Jean-Pierre; Salles, Gilles; Ffrench, Martine

2016-08-01

New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Salinity-induced inhibition of growth in the aquatic pteridophyte Azolla microphylla primarily involves inhibition of photosynthetic components and signaling molecules as revealed by proteome analysis.

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Thagela, Preeti; Yadav, Ravindra Kumar; Mishra, Vagish; Dahuja, Anil; Ahmad, Altaf; Singh, Pawan Kumar; Tiwari, Budhi Sagar; Abraham, Gerard

2017-01-01

Salinity stress causes adverse physiological and biochemical changes in the growth and productivity of a plant. Azolla, a symbiotic pteridophyte and potent candidate for biofertilizer due to its nitrogen fixation ability, shows reduced growth and nitrogen fixation during saline stress. To better understand regulatory components involved in salinity-induced physiological changes, in the present study, Azolla microphylla plants were exposed to NaCl (6.74 and 8.61 ds/m) and growth, photochemical reactions of photosynthesis, ion accumulation, and changes in cellular proteome were studied. Maximum dry weight was accumulated in control and untreated plant while a substantial decrease in dry weight was observed in the plants exposed to salinity. Exposure of the organism to different concentrations of salt in hydroponic conditions resulted in differential level of Na + and K + ion accumulation. Comparative analysis of salinity-induced proteome changes in A. microphylla revealed 58 salt responsive proteins which were differentially expressed during the salt exposure. Moreover, 42 % spots among differentially expressed proteins were involved in different signaling events. The identified proteins are involved in photosynthesis, energy metabolism, amino acid biosynthesis, protein synthesis, and defense. Downregulation of these key metabolic proteins appears to inhibit the growth of A. microphylla in response to salinity. Altogether, the study revealed that in Azolla, increased salinity primarily affected signaling and photosynthesis that in turn leads to reduced biomass.

Echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTH1

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Dong L

2015-12-01

Full Text Available Liwei Dong,1 Hongge Wang,1 Jiajing Niu,1 Mingwei Zou,2 Nuoting Wu,1 Debin Yu,1 Ye Wang,1 Zhihua Zou11Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China; 2Department of Psychology, College of Liberal Arts and Social Sciences, University of Houston, Houston, TX, USA Abstract: Inhibition of the nucleotide pool sanitizing enzyme MTH1 causes extensive oxidative DNA damages and apoptosis in cancer cells and hence may be used as an anticancer strategy. As natural products have been a rich source of medicinal chemicals, in the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in vitro screening assay to search for natural compounds capable of inhibiting MTH1. Echinacoside, a compound derived from the medicinal plants Cistanche and Echinacea, effectively inhibited the catalytic activity of MTH1 in an in vitro assay. Treatment of various human cancer cell lines with Echinacoside resulted in a significant increase in the cellular level of oxidized guanine (8-oxoguanine, while cellular reactive oxygen species level remained unchanged, indicating that Echinacoside also inhibited the activity of cellular MTH1. Consequently, Echinacoside treatment induced an immediate and dramatic increase in DNA damage markers and upregulation of the G1/S-CDK inhibitor p21, which were followed by marked apoptotic cell death and cell cycle arrest in cancer but not in noncancer cells. Taken together, these studies identified a natural compound as an MTH1 inhibitor and suggest that natural products can be an important source of anticancer agents. Keywords: Echinacoside, MTH1, 8-oxoG, DNA damage, apoptosis, cell cycle arrest

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi*

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Klayman, Lauren M.; Wedegaertner, Philip B.

2017-01-01

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. PMID:27994056

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

Science.gov (United States)

Klayman, Lauren M; Wedegaertner, Philip B

2017-02-03

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Paris polyphylla Suppresses Proliferation and Vasculogenic Mimicry of Human Osteosarcoma Cells and Inhibits Tumor Growth In Vivo.

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Yao, Nan; Ren, Ke; Wang, Yimin; Jin, Qiaomei; Lu, Xiao; Lu, Yan; Jiang, Cuihua; Zhang, Dongjian; Lu, Jun; Wang, Chen; Huo, Jiege; Chen, Yong; Zhang, Jian

2017-01-01

Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE. PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

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

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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.

Inhibition and Adsorption impact of Leave Extracts of Cnidoscolus ...

African Journals Online (AJOL)

Corrosion inhibition in the presence of alokaloid and non alkaloid extracts of Cnidoscolus aconitifolius in 1M HCl was studied using the weight loss and hydrogen evolution techniques at 303, 313 and 333 K. The results obtained revealed that the inhibition efficiency decreased with increase in temperature. Inhibition ...

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

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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.

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

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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.

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

International Nuclear Information System (INIS)

Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

2016-01-01

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0–15 μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. - Highlights: • Andrographolide is a diterpenoid phytochemical. �

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

Energy Technology Data Exchange (ETDEWEB)

Chen, Ching-Chu [Division of Endocrinology and Metabolism, Department of Medicine, China Medical University Hospital, Taichung, Taiwan (China); Division of Endocrinology and Metabolism, Department of Chinese Medicine, China Medical University, China Medical University, Taichung, Taiwan (China); Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Huang, Chin-Shiu [Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan (China); Department of Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Chen, Yun-Ting [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Chen, Haw-Wen, E-mail: chenhw@mail.cmu.edu.tw [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Lii, Chong-Kuei, E-mail: cklii@mail.cmu.edu.tw [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan (China)

2016-09-15

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0–15 μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. - Highlights: • Andrographolide is a diterpenoid phytochemical. �

RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.

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Tang, Lian; Dai, Fan; Liu, Yan; Yu, Xiaoqiang; Huang, Chao; Wang, Yuqin; Yao, Wenjuan

2018-05-20

The RhoA/ROCK signaling pathway regulates cell morphology, adhesion, proliferation, and migration. In this study, we investigated the regulatory role of RhoA/ROCK signaling on PDGF-BB-mediated smooth muscle phenotypic modulation and vascular remodeling and clarified the molecular mechanisms behind these effects. PDGF-BB treatment induced the activation of RhoA, ROCK, PDGF-Rβ, and the expression of PDGF-Rβ in HA-VSMCs (human aortic vascular smooth muscle cells). PDGF-Rβ inhibition and RhoA suppression blocked PDGF-BB-induced RhoA activation and ROCK induction. In addition, PDGF-BB-mediated cell proliferation and migration were suppressed by PDGF-Rβ inhibition, RhoA suppression, and ROCK inhibition, suggesting that PDGF-BB promotes phenotypic modulation of HA-VSMCs by activating the RhoA/ROCK pathway via the PDGF receptor. Moreover, suppressing both ROCK1 and ROCK2 blocked cell cycle progression from G0/G1 to S phase by decreasing the transcription and protein expression of cyclin D1, CDK2, and CDK4 via JNK/c-Jun pathway, thus reducing cell proliferation in PDGF-BB-treated HA-VSMCs. ROCK1 deletion, rather than ROCK2 suppression, significantly inhibited PDGF-BB-induced migration by reducing the expression of vimentin and preventing the remodeling of vimentin and phospho-vimentin. Furthermore, ROCK1 deletion suppressed vimentin by inhibiting the phosphorylation of Smad2/3 and the nuclear translocation of Smad4. These findings suggested that ROCK1 and ROCK2 might play different roles in PDGF-BB-mediated cell proliferation and migration in HA-VSMCs. In addition, PDGF-BB and its receptor participated in neointima formation and vascular remodeling by promoting cell cycle protein expression via the JNK pathway and enhancing vimentin expression in a rat balloon injury model; effects that were inhibited by treatment with fasudil. Together, the results of this study reveal a novel mechanism through which RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and

A Comparative Study of Ethylene Growth Response Kinetics in Eudicots and Monocots Reveals a Role for Gibberellin in Growth Inhibition and Recovery1[W][OA

Science.gov (United States)

Kim, Joonyup; Wilson, Rebecca L.; Case, J. Brett; Binder, Brad M.

2012-01-01

Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa ‘Nipponbare’) seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics. PMID:22977279

ROCK inhibition with Y27632 promotes the proliferation and cell cycle progression of cultured astrocyte from spinal cord.

Science.gov (United States)

Yu, Zhiyuan; Liu, Miao; Fu, Peicai; Xie, Minjie; Wang, Wei; Luo, Xiang

2012-12-01

Rho-associated Kinase (ROCK) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. Although its effects on astrocyte proliferation have not been well characterized, ROCK has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. In the present study, our aim was to investigate the effect of ROCK inhibition by [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (Y27632) on proliferation and DNA synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. Western blots showed that treatment of astrocytes with Y27632 increased their expression of cyclin D1, CDK4, and cyclin E, thereby causing cell cycle progression. Furthermore, Y27632-induced astrocyte proliferation was mediated through the extracellular-signal-regulated kinase signaling cascade. These results indicate the importance of ROCK in astrocyte proliferation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol

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Strebhardt Klaus

2008-12-01

Full Text Available Abstract Background Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1, is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers. Methods In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models. Results Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo. Conclusion Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.

Inhibition of thrombin by functionalized C60 nanoparticles revealed via in vitro assays and in silico studies.

Science.gov (United States)

Liu, Yanyan; Fu, Jianjie; Pan, Wenxiao; Xue, Qiao; Liu, Xian; Zhang, Aiqian

2018-01-01

The studies on the human toxicity of nanoparticles (NPs) are far behind the rapid development of engineered functionalized NPs. Fullerene has been widely used as drug carrier skeleton due to its reported low risk. However, different from other kinds of NPs, fullerene-based NPs (C 60 NPs) have been found to have an anticoagulation effect, although the potential target is still unknown. In the study, both experimental and computational methods were adopted to gain mechanistic insight into the modulation of thrombin activity by nine kinds of C 60 NPs with diverse surface chemistry properties. In vitro enzyme activity assays showed that all tested surface-modified C 60 NPs exhibited thrombin inhibition ability. Kinetic studies coupled with competitive testing using 3 known inhibitors indicated that six of the C 60 NPs, of greater hydrophobicity and hydrogen bond (HB) donor acidity or acceptor basicity, acted as competitive inhibitors of thrombin by directly interacting with the active site of thrombin. A simple quantitative nanostructure-activity relationship model relating the surface substituent properties to the inhibition potential was then established for the six competitive inhibitors. Molecular docking analysis revealed that the intermolecular HB interactions were important for the specific binding of C 60 NPs to the active site canyon, while the additional stability provided by the surface groups through van der Waals interaction also play a key role in the thrombin binding affinity of the NPs. Our results suggest that thrombin is a possible target of the surface-functionalized C 60 NPs relevant to their anticoagulation effect. Copyright © 2017. Published by Elsevier B.V.

Cyclin C influences the timing of mitosis in fission yeast.

Science.gov (United States)

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

2017-07-01

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

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

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Jingxiao Zhang

Full Text Available 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.

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

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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.

Cucurbitacin B inhibits proliferation, induces G2/M cycle arrest and autophagy without affecting apoptosis but enhances MTT reduction in PC12 cells

Directory of Open Access Journals (Sweden)

Chuanhong Wu

2016-03-01

Full Text Available In the present study, the effect of cucurbitacin B (a natural product with anti-cancer effect was studied on PC12 cells. It significantly reduced the cell number, changed cell morphology and inhibited colony formation while MTT results showed increased cell viability. Cucurbitacin B treatment increased activity of succinode hydrogenase. No alteration in the integrity of mem-brane, the release of lactic dehydrogenase, the mitochondrial membrane potential, and the expression of apoptotic proteins suggested that cucurbitacin B did not induce apoptosis. The cell cycle was remarkably arrested at G2/M phase. Furthermore, cucurbitacin B induced autophagy as evidence by accumulation of autophagic vacuoles and the increase of LC3II. In addition, cucurbitacin B up-regulated the expression of p-beclin-1, p-ULK1, p-Wee1, p21 and down-regulated p-mTOR, p-p70S6K, CDC25C, CDK1, Cyclin B1. In conclusion, cucurbitacin B inhibited PC12 proliferation but caused MTT pitfall. Cucurbitacin B induced G2/M cell cycle arrest, autophagy, but not the apoptosis in PC12 cells.

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

Science.gov (United States)

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

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Peng Xu

2017-03-01

Full Text Available Human parvovirus B19 (B19V infection of primary human erythroid progenitor cells (EPCs arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2 within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

fMRI reveals reciprocal inhibition between social and physical cognitive domains

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Jack, Anthony I.; Dawson, Abigail; Begany, Katelyn; Leckie, Regina L.; Barry, Kevin; Ciccia, Angela; Snyder, Abraham

2012-01-01

Two lines of evidence indicate that there exists a reciprocal inhibitory relationship between opposed brain networks. First, most attention-demanding cognitive tasks activate a stereotypical set of brain areas, known as the task-positive network and simultaneously deactivate a different set of brain regions, commonly referred to as the task negative or default mode network. Second, functional connectivity analyses show that these same opposed networks are anti-correlated in the resting state. We hypothesize that these reciprocally inhibitory effects reflect two incompatible cognitive modes, each of which is directed towards understanding the external world. Thus, engaging one mode activates one set of regions and suppresses activity in the other. We test this hypothesis by identifying two types of problem-solving task which, on the basis of prior work, have been consistently associated with the task positive and task negative regions: tasks requiring social cognition, i.e., reasoning about the mental states of other persons, and tasks requiring physical cognition, i.e., reasoning about the causal/mechanical properties of inanimate objects. Social and mechanical reasoning tasks were presented to neurologically normal participants during fMRI. Each task type was presented using both text and video clips. Regardless of presentation modality, we observed clear evidence of reciprocal suppression: social tasks deactivated regions associated with mechanical reasoning and mechanical tasks deactivated regions associated with social reasoning. These findings are not explained by self-referential processes, task engagement, mental simulation, mental time travel or external vs. internal attention, all factors previously hypothesized to explain default mode network activity. Analyses of resting state data revealed a close match between the regions our tasks identified as reciprocally inhibitory and regions of maximal anti-correlation in the resting state. These results indicate

fMRI reveals reciprocal inhibition between social and physical cognitive domains.

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Jack, Anthony I; Dawson, Abigail J; Begany, Katelyn L; Leckie, Regina L; Barry, Kevin P; Ciccia, Angela H; Snyder, Abraham Z

2013-02-01

Two lines of evidence indicate that there exists a reciprocal inhibitory relationship between opposed brain networks. First, most attention-demanding cognitive tasks activate a stereotypical set of brain areas, known as the task-positive network and simultaneously deactivate a different set of brain regions, commonly referred to as the task negative or default mode network. Second, functional connectivity analyses show that these same opposed networks are anti-correlated in the resting state. We hypothesize that these reciprocally inhibitory effects reflect two incompatible cognitive modes, each of which may be directed towards understanding the external world. Thus, engaging one mode activates one set of regions and suppresses activity in the other. We test this hypothesis by identifying two types of problem-solving task which, on the basis of prior work, have been consistently associated with the task positive and task negative regions: tasks requiring social cognition, i.e., reasoning about the mental states of other persons, and tasks requiring physical cognition, i.e., reasoning about the causal/mechanical properties of inanimate objects. Social and mechanical reasoning tasks were presented to neurologically normal participants during fMRI. Each task type was presented using both text and video clips. Regardless of presentation modality, we observed clear evidence of reciprocal suppression: social tasks deactivated regions associated with mechanical reasoning and mechanical tasks deactivated regions associated with social reasoning. These findings are not explained by self-referential processes, task engagement, mental simulation, mental time travel or external vs. internal attention, all factors previously hypothesized to explain default mode network activity. Analyses of resting state data revealed a close match between the regions our tasks identified as reciprocally inhibitory and regions of maximal anti-correlation in the resting state. These results

Downregulation of HDAC9 inhibits cell proliferation and tumor formation by inducing cell cycle arrest in retinoblastoma

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Zhang, Yiting; Wu, Dan; Xia, Fengjie; Xian, Hongyu; Zhu, Xinyue [Medical School of Nanjing University, Department of Ophthalmology, Jinling Hospital, Nanjing, 210002 (China); Cui, Hongjuan, E-mail: hcui@swu.edu.cn [State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, 400716 (China); Huang, Zhenping, E-mail: huangzhenping19633@163.com [Medical School of Nanjing University, Department of Ophthalmology, Jinling Hospital, Nanjing, 210002 (China)

2016-04-29

Histone deacetylase 9 (HDAC9) is a member of class II HDACs, which regulates a wide variety of normal and abnormal physiological functions. Recently, HDAC9 has been found to be overexpressed in some types of human cancers. However, the role of HDAC9 in retinoblastoma remains unclear. In this study, we found that HDAC9 was commonly expressed in retinoblastoma tissues and HDAC9 was overexpressed in prognostically poor retinoblastoma patients. Through knocking down HDAC9 in Y79 and WERI-Rb-1 cells, the expression level of HDAC9 was found to be positively related to cell proliferation in vitro. Further investigation indicated that knockdown HDAC9 could significantly induce cell cycle arrest at G1 phase in retinoblastoma cells. Western blot assay showed downregulation of HDAC9 could significantly decrease cyclin E2 and CDK2 expression. Lastly, xenograft study in nude mice showed that downregulation of HDAC9 inhibited tumor growth and development in vivo. Therefore, our results suggest that HDAC9 could serve as a novel potential therapeutic target in the treatment of retinoblastoma. - Highlights: • High expression of HDAC9 correlates with poor patient prognosis. • Downregulation of HDAC9 inhibits cell proliferation in retinoblastoma cells. • Downregulation of HDAC9 induces cell cycle arrest at G1 phase in retinoblastoma cells. • Downregulation of HDAC9 suppresses tumor growth in nude mice.

Hippuristanol Reduces the Viability of Primary Effusion Lymphoma Cells both in Vitro and in Vivo

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Masachika Senba

2013-09-01

Full Text Available Primary effusion lymphoma (PEL caused by Kaposi’s sarcoma-associated herpesvirus (also known as human herpesvirus-8 shows serious lymphomatous effusion in body cavities. PEL is difficult to treat and there is no standard treatment strategy. Hippuristanol is extracted from Okinawan coral Isis hippuris, and inhibits translational initiation by blocking eukaryotic initiation factor 4A, an ATP-dependent RNA helicase, binding to mRNA. Recently, there has been much interest in targeting translation initiation as an anticancer therapy. Here, we show that treatment of PEL cell lines with hippuristanol resulted in cell cycle arrest at G1 phase, and induced caspases activation and apoptosis. Hippuristanol also reduced the expression of cyclin D2, CDK2, CDK4, CDK6 and prosurvival XIAP and Mcl-1 proteins. Activation of activator protein-1, signal transducers and activators of transcription protein 3 and Akt pathways plays a critical role in the survival and growth of PEL cells. Hippuristanol suppressed the activities of these three pathways by inhibiting the expression of JunB, JunD, c-Fos, signal transducers and activators of transcription protein 3 and Akt proteins. In a xenograft mouse model that showed ascites and diffused organ invasion of PEL cells, treatment with hippuristanol significantly inhibited the growth and invasion of PEL cells compared with untreated mice. The results of the in vitro and in vivo experiments underline the potential usefulness of hippuristanol in the treatment of PEL.

Differential regulation of collapsin response mediator protein 2 (CRMP2 phosphorylation by GSK3ß and CDK5 following traumatic brain injury

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Sarah Marie Wilson

2014-05-01

Full Text Available Aberrant ion channel function has been heralded as a main underlying mechanism driving epilepsy and its symptoms. However, it has become increasingly clear that treatment strategies targeting voltage-gated sodium or calcium channels merely mask the symptoms of epilepsy without providing disease-modifying benefits. Ion channel function is likely only one important cog in a highly complex machine. Gross morphological changes, such as reactive sprouting and outgrowth, may also play a role in epileptogenesis. Mechanisms responsible for these changes are not well understood. Here we investigate the potential involvement of the neurite outgrowth-promoting molecule collapsin response mediator protein 2 (CRMP2. CRMP2 activity, in this respect, is regulated by phosphorylation state, where phosphorylation by a variety of kinases, including glycogen synthase kinase 3 β (GSK3β renders it inactive. Phosphorylation (inactivation of CRMP2 was decreased at two distinct phases following traumatic brain injury (TBI. While reduced CRMP2 phosphorylation during the early phase was attributed to the inactivation of GSK3β, the sustained decrease in CRMP2 phosphorylation in the late phase appeared to be independent of GSK3β activity. Instead, the reduction in GSK3β-phosphorylated CRMP2 was attributed to a loss of priming by cyclin-dependent kinase 5 (CDK5, which allows for subsequent phosphorylation by GSK3β. Based on the observation that the proportion of active CRMP2 is increased for up to 4 weeks following TBI, it was hypothesized that it may drive neurite outgrowth, and therefore, circuit reorganization during this time. Therefore, a novel small-molecule tool was used to target CRMP2 in an attempt to determine its importance in mossy fiber sprouting following TBI. In this report, we demonstrate novel differential regulation of CRMP2 phosphorylation by GSK3β and CDK5 following TBI.

Polysulfonate suramin inhibits Zika virus infection.

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Tan, Chee Wah; Sam, I-Ching; Chong, Wei Lim; Lee, Vannajan Sanghiran; Chan, Yoke Fun

2017-07-01

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log 10  PFU viral reduction with IC 50 value of ∼2.5-5 μg/ml (1.93 μM-3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

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Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

2009-01-01

Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

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

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Cheng, Ya-Hsin, E-mail: yhcheng@mail.cmu.edu.tw [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China); Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China); Hung, Chein-Hui [Graduate Institute of Clinical Medicine Sciences, Chang Gung University, Puizi City, Chiayi 613, Taiwan, ROC (China); Chang, Nai Wen [Department of Biochemistry, School of Medicine, China Medical University, Taichung, Taiwan, ROC (China); Lin, Chingju [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China)

2012-09-15

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

Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes

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Kool, Jaap; Uren, Anthony G; Martins, Carla P

2010-01-01

-throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific...... revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association...

Cooperation for Better Inhibiting.

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Novoa, Eva Maria; Ribas de Pouplana, Lluís

2015-06-18

Cladosporin is an antimalarial drug that acts as an ATP-mimetic to selectively inhibit Plasmodium lysyl-tRNA synthetase. Using multiple crystal structures, Fang et al. (2015) reveal in this issue of Chemistry & Biology the fascinating mechanism responsible for cladosporin selectivity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

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Zan, Xinyi; Cui, Fengjie; Li, Yunhong; Yang, Yan; Wu, Di; Sun, Wenjing; Ping, Lifeng

2015-05-01

HEG-5 is a novel polysaccharide-protein purified from the fermented mycelia of Hericium erinaceus CZ-2. The present study aims to investigate the effects of HEG-5 on proliferation, cell cycle and apoptosis of human gastric cancer cells SGC-7901. Here, we first uncover that HEG-5 significantly inhibited the proliferation and colony formation of SGC-7901 cells by promoting apoptosis and cell cycle arrest at S phase. RT-PCR and Western blot analysis suggested that HEG-5 could decrease the expressions of Bcl2, PI3K and AKT1, while increase the expressions of Caspase-8, Caspase-3, p53, CDK4, Bax and Bad. These findings indicated that the Caspase-8/-3-dependent, p53-dependent mitochondrial-mediated and PI3k/Akt signaling pathways involved in the molecular events of HEG-5 induced apoptosis and cell cycle arrest. Thus, our study provides in vitro evidence that HEG-5 may be taken as a potential candidate for treating gastric cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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Sørensen, Per Soelberg

2010-01-01

and the same direction of effect observed in the discovery phase. Three loci exceeded genome-wide significance in the joint analysis: RGS1 (P value=3.55 x 10(-9)), IL12A (P=3.08 x 10(-8)) and MPHOSPH9/CDK2AP1 (P=3.96 x 10(-8)). The RGS1 risk allele is shared with celiac disease (CD), and the IL12A risk allele......A recent meta-analysis identified seven single-nucleotide polymorphisms (SNPs) with suggestive evidence of association with multiple sclerosis (MS). We report an analysis of these polymorphisms in a replication study that includes 8,085 cases and 7,777 controls. A meta-analysis across...... the replication collections and a joint analysis with the discovery data set were performed. The possible functional consequences of the validated susceptibility loci were explored using RNA expression data. For all of the tested SNPs, the effect observed in the replication phase involved the same allele...

A mechanism for the coordination of proliferation and differentiation by spatial regulation of Fus2p in budding yeast

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Kim, Junwon; Rose, Mark D.

2012-01-01

Yeast cells induce the genes required for mating prior to the completion of mitosis. To ensure proper cell cycle progression prior to mating differentiation, a key cytoplasmic regulator of cell fusion, Fus2p, is sequestered in the nucleus by cyclin-dependent kinase (Cdk). In response to pheromone signaling, the mitogen-activated protein kinase Fus3p phosphorylates Ser 84 in Fus2p to drive nuclear export. We found that Fus3p becomes active and phosphorylates S84 as early as S phase, raising the question of how Cdk prevents inappropriate activation of Fus2p. Countering Fus3p, Cdk and a p21-activated kinase, Cla4p, maintain Fus2p's nuclear localization by phosphorylating Ser 67, which drives nuclear import and inhibits nuclear export. When Cdk and Cla4p activities drop after cell division, Fus3p promotes Fus2p export both via S84 phosphorylation and by down-regulating S67 phosphorylation. Thus, potential premature activation of Fus2p in mitosis is prevented by cell cycle-dependent phosphorylation that overrides the mating pheromone-induced phosphorylation that drives nuclear export. PMID:22588722

GQ-16, a Novel Peroxisome Proliferator-activated Receptor gamma (PPAR gamma) Ligand, Promotes Insulin Sensitization without Weight Gain

NARCIS (Netherlands)

Amato, Angelica A.; Rajagopalan, Senapathy; Lin, Jean Z.; Carvalho, Bruno M.; Figueira, Ana C. M.; Lu, Jenny; Ayers, Stephen D.; Mottin, Melina; Silveira, Rodrigo L.; Telles de Souza, Paulo; Mourao, Rosa H. V.; Saad, Mario J. A.; Togashi, Marie; Simeoni, Luiz A.; Abdalla, Dulcineia S. P.; Skaf, Munir S.; Polikparpov, Igor; Lima, Maria C. A.; Galdino, Suely L.; Brennan, Richard G.; Baxter, John D.; Pitta, Ivan R.; Webb, Paul; Phillips, Kevin J.; Neves, Francisco A. R.

2012-01-01

The recent discovery that peroxisome proliferator-activated receptor gamma (PPAR gamma) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report

Phosphorylation of pRb by cyclin D kinase is necessary for development of cardiac hypertrophy

DEFF Research Database (Denmark)

Hinrichsen, Rebecca; Hansen, A.H.; Haunsø, S.

2008-01-01

/6-phosphorylated retinoblastoma protein (pRb) during hypertrophy and expression of an unphosphorylatable pRb mutant impaired hypertrophic growth in cardiomyocytes. Transcription factor E2F was activated by hypertrophic elicitors but activation was impaired by pharmacological inhibition of cyclin D-cdk4...

p115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine-induced vascular smooth muscle cell migration and proliferation.

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Singh, Nikhlesh K; Janjanam, Jagadeesh; Rao, Gadiparthi N

2017-08-25

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-G i/o -Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased synaptophysin is involved in inflammation-induced heat hyperalgesia mediated by cyclin-dependent kinase 5 in rats.

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Hong-Hai Zhang

levels were involved in heat hyperalgesia mediated by Cdk5 in spinal cord dorsal horns of CFA-treated rats, suggesting that inhibiting abnormal activation of Cdk5-synaptophysin may present a novel target for diminishing inflammatory pain.

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

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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 (

The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion.

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Ramsubramaniam, Nikhil; Harris, Steven D; Marten, Mark R

2014-11-01

We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra-long reverse phase gradient, and identified using a "high-high" strategy (high mass accuracy on the parent and fragment ions) with higher-energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: "sites of polar growth," "vesicle mediated transport," and "cytoskeleton organization." In these same GO categories, kinase-substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase-substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An-Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 (http://proteomecentral.proteomexchange.org/dataset/PXD000715). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative modeling and docking studies of p16ink4/Cyclin D1/Rb pathway genes in lung cancer revealed functionally interactive residue of RB1 and its functional partner E2F1

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e Zahra Syeda Naqsh

2013-01-01

Full Text Available Abstract Background Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1 Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase (2 Growth inhibitory pathways (p53/Rb/P14ARF, STK11 (3 Apoptotic pathways (Bcl-2/Bax/Fas/FasL. Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies. Results YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of −0.132 and −0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. Conclusion This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of

Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

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Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden

2015-01-01

Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and

P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells

International Nuclear Information System (INIS)

Wei, Min; Gu, Qinlong; Wang, Zhiwei; Yao, Hongliang; Yang, Zhongyin; Zhang, Qing; Liu, Bingya; Yu, Yingyan; Su, Liping; Zhu, Zhenggang

2011-01-01

Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer. Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA. HMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition. The results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial

RNA glycosidase and other agents target Tat to inhibit HIV-1 transcription.

Science.gov (United States)

Harrich, David; Jin, Hongping

2018-03-20

The HIV-1 tat gene encodes a small 86-104 amino acid protein depending on the HIV-1 strain. Tat is essential for HIV-1 replication through interactions with numerous cellular transcription factors. The interaction between Tat and P-TEFb, which is a cellular protein complex composed of cyclin T1 and CDK9, delivers P-TEFb to the newly transcribed viral mRNAs where phosphorylation of RNA polymerase II by CDK9 leads to highly efficient mRNA transcription. It has long been recognized that Tat is a potential anti-HIV-1 target and possibly a viral Achilles' heel. However, specifically targeting Tat without affecting normal host cell functions has been challenging. Means to inactivate Tat have been reported that includes small compounds, transdominant negative Tat proteins, and by plant-derived antivirals. Investigations of these agents have reported encouraging outcomes that inform and may hopefully affect strategies for a functional HIV-1 cure. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Carvacrol suppresses proliferation and invasion in human oral squamous cell carcinoma

Directory of Open Access Journals (Sweden)

Dai W

2016-04-01

Full Text Available Wei Dai,1,2 Changfu Sun,1,2 Shaohui Huang,1,2 Qing Zhou1,21Department of Oromaxillofacial-Head and Neck Surgery, 2Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University, Shenyang, Liaoning, People’s Republic of ChinaAbstract: Carvacrol, a component of thyme oil, as a novel antitumor agent, has been implicated in several types of cancer cells. However, the mechanisms underlying the effect of carvacrol in human oral squamous cell carcinoma (OSCC remain unclear. Here, we report that carvacrol significantly inhibits tumor cell proliferation, metastasis and invasion, and induces apoptosis in OSCC. Our results demonstrated that the molecular mechanisms of the effect of carvacrol in Tca-8113 induces G1/S cell cycle arrest through downregulation of CDK regulator CCND1 and CDK4, and upregulation of CDK inhibitor P21. Further analysis demonstrated that carvacrol also inhibited Tca-8113 cells’ clone formation in clonogenic cell survival assay. Student’s t-test (two-tailed was used to compare differences between groups, and the significance level was P<0.01. Then, treatment of Tca-8113 cells with carvacrol resulted in downregulation of Bcl-2, Cox2, and upregulation of Bax. Carvacrol significantly inhibited the migration and invasion of human OSCC cells by blocking the phosphorylation of FAK and MMP-9 and MMP-2, transcription factor ZEB1, and β-catenin proteins’ expression. Taken together, these results provide novel insights into the mechanism of carvacrol and suggest potential therapeutic strategies for human OSCC.Keywords: carvacrol, proliferation, metastasis and invasion, oral squamous cell carcinoma

Kindergarteners' self-reported social inhibition and observed social reticence: moderation by adult-reported social inhibition and social anxiety disorder symptoms.

Science.gov (United States)

Kiel, Elizabeth J; Buss, Kristin A; Molitor, Joseph G

2015-04-01

Prevention of later anxiety problems would best be accomplished by identifying at-risk children early in development. For example, children who develop Social Anxiety Disorder (SAD) may show social withdrawal in the form of social inhibition (i.e., shyness with unfamiliar adults and peers) at school entry. Although the use of children's perceptions of their own social inhibition would provide insight into early risk, the utility of young children's self-reports remains unclear. The current study examined whether children deemed more extreme on social inhibition or social anxiety by adult report provided self-report of social inhibition that related to observed social reticence in the laboratory. Participants included 85 kindergarten children (36 female, 49 male), their parents, and their teachers. Moderation analyses revealed that children's self-reported social inhibition related significantly to observed social reticence under the conditions of high parent-reported social inhibition, high teacher-reported social inhibition, and high SAD symptoms. These results suggest that the most inhibited children are aware of their behavior and can report it in a meaningfully way as young as kindergarten age.

Digital expression profiling identifies RUNX2, CDC5L, MDM2, RECQL4, and CDK4 as potential predictive biomarkers for neo-adjuvant chemotherapy response in paediatric osteosarcoma.

Directory of Open Access Journals (Sweden)

Jeffrey W Martin

Full Text Available Osteosarcoma is the most common malignancy of bone, and occurs most frequently in children and adolescents. Currently, the most reliable technique for determining a patients' prognosis is measurement of histopathologic tumor necrosis following pre-operative neo-adjuvant chemotherapy. Unfavourable prognosis is indicated by less than 90% estimated necrosis of the tumor. Neither genetic testing nor molecular biomarkers for diagnosis and prognosis have been described for osteosarcomas. We used the novel nanoString mRNA digital expression analysis system to analyse gene expression in 32 patients with sporadic paediatric osteosarcoma. This system used specific molecular barcodes to quantify expression of a set of 17 genes associated with osteosarcoma tumorigenesis. Five genes, from this panel, which encoded the bone differentiation regulator RUNX2, the cell cycle regulator CDC5L, the TP53 transcriptional inactivator MDM2, the DNA helicase RECQL4, and the cyclin-dependent kinase gene CDK4, were differentially expressed in tumors that responded poorly to neo-adjuvant chemotherapy. Analysis of the signalling relationships of these genes, as well as other expression markers of osteosarcoma, indicated that gene networks linked to RB1, TP53, PI3K, PTEN/Akt, myc and RECQL4 are associated with osteosarcoma. The discovery of these networks provides a basis for further experimental studies of role of the five genes (RUNX2, CDC5L, MDM2, RECQL4, and CDK4 in differential response to chemotherapy.

Transducer of ERBB2.1 (TOB1) as a Tumor Suppressor: A Mechanistic Perspective.

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Lee, Hun Seok; Kundu, Juthika; Kim, Ryong Nam; Shin, Young Kee

2015-12-15

Transducer of ERBB2.1 (TOB1) is a tumor-suppressor protein, which functions as a negative regulator of the receptor tyrosine-kinase ERBB2. As most of the other tumor suppressor proteins, TOB1 is inactivated in many human cancers. Homozygous deletion of TOB1 in mice is reported to be responsible for cancer development in the lung, liver, and lymph node, whereas the ectopic overexpression of TOB1 shows anti-proliferation, and a decrease in the migration and invasion abilities on cancer cells. Biochemical studies revealed that the anti-proliferative activity of TOB1 involves mRNA deadenylation and is associated with the reduction of both cyclin D1 and cyclin-dependent kinase (CDK) expressions and the induction of CDK inhibitors. Moreover, TOB1 interacts with an oncogenic signaling mediator, β-catenin, and inhibits β-catenin-regulated gene transcription. TOB1 antagonizes the v-akt murine thymoma viral oncogene (AKT) signaling and induces cancer cell apoptosis by activating BCL2-associated X (BAX) protein and inhibiting the BCL-2 and BCL-XL expressions. The tumor-specific overexpression of TOB1 results in the activation of other tumor suppressor proteins, such as mothers against decapentaplegic homolog 4 (SMAD4) and phosphatase and tensin homolog-10 (PTEN), and blocks tumor progression. TOB1-overexpressing cancer cells have limited potential of growing as xenograft tumors in nude mice upon subcutaneous implantation. This review addresses the molecular basis of TOB1 tumor suppressor function with special emphasis on its regulation of intracellular signaling pathways.

Transducer of ERBB2.1 (TOB1 as a Tumor Suppressor: A Mechanistic Perspective

Directory of Open Access Journals (Sweden)

Hun Seok Lee

2015-12-01

Full Text Available Transducer of ERBB2.1 (TOB1 is a tumor-suppressor protein, which functions as a negative regulator of the receptor tyrosine-kinase ERBB2. As most of the other tumor suppressor proteins, TOB1 is inactivated in many human cancers. Homozygous deletion of TOB1 in mice is reported to be responsible for cancer development in the lung, liver, and lymph node, whereas the ectopic overexpression of TOB1 shows anti-proliferation, and a decrease in the migration and invasion abilities on cancer cells. Biochemical studies revealed that the anti-proliferative activity of TOB1 involves mRNA deadenylation and is associated with the reduction of both cyclin D1 and cyclin-dependent kinase (CDK expressions and the induction of CDK inhibitors. Moreover, TOB1 interacts with an oncogenic signaling mediator, β-catenin, and inhibits β-catenin-regulated gene transcription. TOB1 antagonizes the v-akt murine thymoma viral oncogene (AKT signaling and induces cancer cell apoptosis by activating BCL2-associated X (BAX protein and inhibiting the BCL-2 and BCL-XL expressions. The tumor-specific overexpression of TOB1 results in the activation of other tumor suppressor proteins, such as mothers against decapentaplegic homolog 4 (SMAD4 and phosphatase and tensin homolog-10 (PTEN, and blocks tumor progression. TOB1-overexpressing cancer cells have limited potential of growing as xenograft tumors in nude mice upon subcutaneous implantation. This review addresses the molecular basis of TOB1 tumor suppressor function with special emphasis on its regulation of intracellular signaling pathways.

Establishment of functioning human corneal endothelial cell line with high growth potential.

Directory of Open Access Journals (Sweden)

Tadashi Yokoi

Full Text Available Hexagonal-shaped human corneal endothelial cells (HCEC form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+- and K(+-dependent ATPase (Na(+/K(+-ATPase. Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs in the Rb pathway (p16-CDK4/CyclinD1-pRb. In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7 and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin. Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7, THCEH (Cyclin and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7 and THCEH (Cyclin. THCEH (Cyclin expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+/K(+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7. This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.

miR-24 inhibits cell proliferation by suppressing expression of E2F2, MYC and other cell cycle regulatory genes by binding to “seedless” 3′UTR microRNA recognition elements

Science.gov (United States)

Lal, Ashish; Navarro, Francisco; Maher, Christopher; Maliszewski, Laura E.; Yan, Nan; O'Day, Elizabeth; Chowdhury, Dipanjan; Dykxhoorn, Derek M.; Tsai, Perry; Hofman, Oliver; Becker, Kevin G.; Gorospe, Myriam; Hide, Winston; Lieberman, Judy

2009-01-01

Summary miR-24, up-regulated during terminal differentiation of multiple lineages, inhibits cell cycle progression. Antagonizing miR-24 restores post-mitotic cell proliferation and enhances fibroblast proliferation, while over-expressing miR-24 increases the G1 compartment. The 248 mRNAs down-regulated upon miR-24 over-expression are highly enriched for DNA repair and cell cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, CDC2) or inhibit (p27Kip1, VHL) cell cycle progression. miR-24 directly regulates MYC and E2F2 and some genes they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 over-expression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4 and FEN1 by recognizing seedless, but highly complementary, sequences. PMID:19748357

A nontranscriptional role for Oct4 in the regulation of mitotic entry

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Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

Geranylated flavanone tomentodiplacone B inhibits proliferation of human monocytic leukaemia (THP-1) cells

Czech Academy of Sciences Publication Activity Database

Kollár, P.; Bárta, Tomáš; Závalová, V.; Šmejkal, K.; Hampl, Aleš

2011-01-01

Roč. 162, č. 7 (2011), s. 1534-1541 ISSN 0007-1188 Grant - others:GA MŠk.(CZ) LC06077 Program:LC Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50390512 Keywords : flavonoids * CDK2 * antiproliferative effect Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.409, year: 2011

Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway.

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Rei, Damien; Mason, Xenos; Seo, Jinsoo; Gräff, Johannes; Rudenko, Andrii; Wang, Jun; Rueda, Richard; Siegert, Sandra; Cho, Sukhee; Canter, Rebecca G; Mungenast, Alison E; Deisseroth, Karl; Tsai, Li-Huei

2015-06-09

Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25, up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memory-related genes in the hippocampus. A combination of optogenetic and pharmacosynthetic approaches shows that BLA activation is both necessary and sufficient for stress-associated molecular changes and memory impairments. Furthermore, we show that this effect relies on direct glutamatergic projections from the BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction. Taken together, our data provide a neural circuit model for stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation.

Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway

Science.gov (United States)

Rei, Damien; Mason, Xenos; Seo, Jinsoo; Gräff, Johannes; Rudenko, Andrii; Wang, Jun; Rueda, Richard; Siegert, Sandra; Cho, Sukhee; Canter, Rebecca G.; Mungenast, Alison E.; Deisseroth, Karl; Tsai, Li-Huei

2015-01-01

Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25, up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memory-related genes in the hippocampus. A combination of optogenetic and pharmacosynthetic approaches shows that BLA activation is both necessary and sufficient for stress-associated molecular changes and memory impairments. Furthermore, we show that this effect relies on direct glutamatergic projections from the BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction. Taken together, our data provide a neural circuit model for stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation. PMID:25995364

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

Science.gov (United States)

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

Terbinafine inhibits gap junctional intercellular communication

International Nuclear Information System (INIS)

Lee, Ju Yeun; Yoon, Sei Mee; Choi, Eun Ju; Lee, Jinu

2016-01-01

Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca 2+ concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca 2+ concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.

Terbinafine inhibits gap junctional intercellular communication.

Science.gov (United States)

Lee, Ju Yeun; Yoon, Sei Mee; Choi, Eun Ju; Lee, Jinu

2016-09-15

Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca(2+) concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. Copyright © 2016 Elsevier Inc. All rights reserved.

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

Science.gov (United States)

Latham, Antony M; Kankanala, Jayakanth; Fearnley, Gareth W; Gage, Matthew C; Kearney, Mark T; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B; Fishwick, Colin W G; Ponnambalam, Sreenivasan

2014-01-01

Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Graphene: corrosion-inhibiting coating.

Science.gov (United States)

Prasai, Dhiraj; Tuberquia, Juan Carlos; Harl, Robert R; Jennings, G Kane; Rogers, Bridget R; Bolotin, Kirill I

2012-02-28

We report the use of atomically thin layers of graphene as a protective coating that inhibits corrosion of underlying metals. Here, we employ electrochemical methods to study the corrosion inhibition of copper and nickel by either growing graphene on these metals, or by mechanically transferring multilayer graphene onto them. Cyclic voltammetry measurements reveal that the graphene coating effectively suppresses metal oxidation and oxygen reduction. Electrochemical impedance spectroscopy measurements suggest that while graphene itself is not damaged, the metal under it is corroded at cracks in the graphene film. Finally, we use Tafel analysis to quantify the corrosion rates of samples with and without graphene coatings. These results indicate that copper films coated with graphene grown via chemical vapor deposition are corroded 7 times slower in an aerated Na(2)SO(4) solution as compared to the corrosion rate of bare copper. Tafel analysis reveals that nickel with a multilayer graphene film grown on it corrodes 20 times slower while nickel surfaces coated with four layers of mechanically transferred graphene corrode 4 times slower than bare nickel. These findings establish graphene as the thinnest known corrosion-protecting coating.

Kindergarteners’ Self-Reported Social Inhibition and Observed Social Reticence: Moderation by Adult-Reported Social Inhibition and Social Anxiety Disorder Symptoms

Science.gov (United States)

Kiel, Elizabeth J.; Buss, Kristin A.; Molitor, Joseph G.

2014-01-01

Prevention of later anxiety problems would best be accomplished by identifying at-risk children early in development. For example, children who develop Social Anxiety Disorder (SAD) may show social withdrawal in the form of social inhibition (i.e., shyness with unfamiliar adults and peers) at school entry. Although the use of children’s perceptions of their own social inhibition would provide insight into early risk, the utility of young children’s self-reports remains unclear. The current study examined whether children deemed more extreme on social inhibition or social anxiety by adult report provided self-report of social inhibition that related to observed social reticence in the laboratory. Participants included 85 kindergarten children (36 female, 49 male), their parents, and their teachers. Moderation analyses revealed that children’s self-reported social inhibition related significantly to observed social reticence under the conditions of high parent-reported social inhibition, high teacher-reported social inhibition, and high SAD symptoms. These results suggest that the most inhibited children are aware of their behavior and can report it in a meaningfully way as young as kindergarten age. PMID:25113397

Inhibition of AMPK catabolic action by GSK3

Science.gov (United States)

Suzuki, Tsukasa; Bridges, Dave; Nakada, Daisuke; Skiniotis, Georgios; Morrison, Sean J.; Lin, Jiandie; Saltiel, Alan R.; Inoki, Ken

2013-01-01

SUMMARY AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK. PMID:23623684

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.

Science.gov (United States)

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

Corrosion inhibition by lithium zinc phosphate pigment

International Nuclear Information System (INIS)

Alibakhshi, E.; Ghasemi, E.; Mahdavian, M.

2013-01-01

Highlights: •Synthesis of lithium zinc phosphate (LZP) by chemical co-precipitation method. •Corrosion inhibition activity of pigments compare with zinc phosphate (ZP). •LZP showed superior corrosion inhibition effect in EIS measurements. •Evaluation of adhesion strength and dispersion stability. -- Abstract: Lithium zinc phosphate (LZP) has been synthesized through a co-precipitation process and characterized by XRD and IR spectroscopy. The inhibitive performances of this pigment for corrosion of mild steel have been discussed in comparison with the zinc phosphate (ZP) in the pigment extract solution by means of EIS and in the epoxy coating by means of salt spray. The EIS and salt spray results revealed the superior corrosion inhibitive effect of LZP compared to ZP. Moreover, adhesion strength and dispersion stability of the pigmented epoxy coating showed the advantage of LZP compared to ZP

Transcriptome dynamics of the microRNA inhibition response

DEFF Research Database (Denmark)

Wen, Jiayu; Leucci, Elenora; Vendramin, Roberto

2015-01-01

We report a high-resolution time series study of transcriptome dynamics following antimiR-mediated inhibition of miR-9 in a Hodgkin lymphoma cell-line-the first such dynamic study of the microRNA inhibition response-revealing both general and specific aspects of the physiological response. We show...... validate the key observations with independent time series qPCR and we experimentally validate key predicted miR-9 targets. Methodologically, we developed sensitive functional data analytic predictive methods to analyse the weak response inherent in microRNA inhibition experiments. The methods...... of this study will be applicable to similar high-resolution time series transcriptome analyses and provides the context for more accurate experimental design and interpretation of future microRNA inhibition studies....

A current and comprehensive review of cyclin-dependent kinase inhibitors for the treatment of metastatic breast cancer.

Science.gov (United States)

Bilgin, Burak; Sendur, Mehmet A N; Şener Dede, Didem; Akıncı, Muhammed Bülent; Yalçın, Bülent

2017-09-01

Resistance to endocrine treatment generally occurs over time, especially in the metastatic stage. In this paper, we aimed to review the mechanisms of cyclin-dependent kinase (CDK) 4/6 inhibition and clinical usage of new agents in the light of recent literature updates. A literature search was carried out using PubMed, Medline and ASCO and ESMO annual-meeting abstracts by using the following search keywords; "palbociclib", "abemaciclib", "ribociclib", "cyclin-dependent kinase inhibitors" and "CDK 4/6" in metastatic breast cancer (MBC). The last search was on 10 June 2017. CDKs and cyclins are two molecules that have a key role in cell cycle progression. Today, there are three highly selective CDK4/6 inhibitors in clinical development - palbociclib, ribociclib and abemaciclib. Palbociclib and ribociclib were recently approved by the US FDA in combination with letrozole for the treatment of MBC in a first-line setting, as well as palbociclib in combination with fulvestrant for hormone-receptor (HR)-positive MBC that had progressed while on previous endocrine therapy according to the PALOMA-1, MONALEESA-2 and PALOMA-3 trials, respectively. In the recently published randomized phase III MONARCH 2 trial, abemaciclib plus letrozole had longer progression-free survival and higher objective response rates with less serious adverse events in advanced HR-positive breast cancer previously treated with hormonal treatment. CDK4/6 inhibition is a new and promising target for patients with hormone-receptor-positive MBC. Both palbociclib and ribociclib showed significant additive benefit for patients receiving first-line treatment for HR-positive, epidermal growth factor receptor-2-negative advanced breast cancer. Palbociclib and abemaciclib also had significant activity in combination with fulvestrant for patients with MBC that progressed on previous endocrine therapy.

Corrosion Inhibition by Sodium Gluconate-Zn2+-DTPMP System

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P. Manjula

2009-01-01

Full Text Available The inhibition efficiency of a phosphonic acid, Diethylene Triamine Pentamethylene Phosphonic acid (DTPMP in controlling corrosion of carbon steel immersed in an aqueous solution containing 60 ppm of Cl- has been evaluated by weight loss method in the absence and presence of Zn2+. The formulation consisting of DTPMP and Zn2+ has excellent inhibition efficiency (IE. A synergistic effect is noticed between Zn2+ and DTPMP. Addition of sodium gluconate (SG enhances the IE of Zn2+ and DTPMP system. The DTPMP-Zn2+-SG system function as a mixed inhibitor as revealed by polarization study. AC impedance spectrum, optical and atomic force micrographs reveal the formation of a protective film on the metal surface. FTIR spectra reveal that the protective film consists of Fe2+-DTPMP complex, Fe2+-SG complex and Zn(OH2.

Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

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Rami A Al-Horani

Full Text Available Factor XIIIa (FXIIIa is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa's active site by using sulfated glycosaminoglycans (GAGs or non-saccharide GAG mimetics (NSGMs would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%. Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71% and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants.

Nutrition controls mitochondrial biogenesis in the Drosophila adipose tissue through Delg and cyclin D/Cdk4.

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Claudia Baltzer

Full Text Available MITOCHONDRIA ARE CELLULAR ORGANELLES THAT PERFORM CRITICAL METABOLIC FUNCTIONS: they generate energy from nutrients but also provide metabolites for de novo synthesis of fatty acids and several amino acids. Thus mitochondrial mass and activity must be coordinated with nutrient availability, yet this remains poorly understood. Here, we demonstrate that Drosophila larvae grown in low yeast food have strong defects in mitochondrial abundance and respiration activity in the larval fat body. This correlates with reduced expression of genes encoding mitochondrial proteins, particularly genes involved in oxidative phosphorylation. Second, genes involved in glutamine metabolism are also expressed in a nutrient-dependent manner, suggesting a coordination of amino acid synthesis with mitochondrial abundance and activity. Moreover, we show that Delg (CG6338, the Drosophila homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, is required for proper expression of most genes encoding mitochondrial proteins. Our data demonstrate that Delg is critical to adjust mitochondrial abundance in respect to Cyclin D/Cdk4, a growth-promoting complex and glutamine metabolism according to nutrient availability. However, in contrast to nutrients, Delg is not involved in the regulation of mitochondrial activity in the fat body. These findings are the first genetic evidence that the regulation of mitochondrial mass can be uncoupled from mitochondrial activity.

Changes in cerebro-cerebellar interaction during response inhibition after performance improvement.

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Hirose, Satoshi; Jimura, Koji; Kunimatsu, Akira; Abe, Osamu; Ohtomo, Kuni; Miyashita, Yasushi; Konishi, Seiki

2014-10-01

It has been demonstrated that motor learning is supported by the cerebellum and the cerebro-cerebellar interaction. Response inhibition involves motor responses and the higher-order inhibition that controls the motor responses. In this functional MRI study, we measured the cerebro-cerebellar interaction during response inhibition in two separate days of task performance, and detected the changes in the interaction following performance improvement. Behaviorally, performance improved in the second day, compared to the first day. The psycho-physiological interaction (PPI) analysis revealed the interaction decrease from the right inferior frontal cortex (rIFC) to the cerebellum (lobule VII or VI). It was also revealed that the interaction increased from the same cerebellar region to the primary motor area. These results suggest the involvement of the cerebellum in response inhibition, and raise the possibility that the performance improvement was supported by the changes in the cerebro-cerebellar interaction. Copyright © 2014 Elsevier Inc. All rights reserved.

Terbinafine inhibits gap junctional intercellular communication

Energy Technology Data Exchange (ETDEWEB)

Lee, Ju Yeun, E-mail: whitewndus@naver.com [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of); Yoon, Sei Mee, E-mail: sei_mee@naver.com [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of); Department of Integrated OMICS for Biomedical Sciences, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Choi, Eun Ju, E-mail: yureas@naver.com [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of); Lee, Jinu, E-mail: jinulee@yonsei.ac.kr [College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983 (Korea, Republic of)

2016-09-15

Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca{sup 2+} concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca{sup 2+} concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.

Design principles of the yeast G1/S switch.

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Xiaojing Yang

2013-10-01

Full Text Available A hallmark of the G1/S transition in budding yeast cell cycle is the proteolytic degradation of the B-type cyclin-Cdk stoichiometric inhibitor Sic1. Deleting SIC1 or altering Sic1 degradation dynamics increases genomic instability. Certain key facts about the parts of the G1/S circuitry are established: phosphorylation of Sic1 on multiple sites is necessary for its destruction, and both the upstream kinase Cln1/2-Cdk1 and the downstream kinase Clb5/6-Cdk1 can phosphorylate Sic1 in vitro with varied specificity, cooperativity, and processivity. However, how the system works as a whole is still controversial due to discrepancies between in vitro, in vivo, and theoretical studies. Here, by monitoring Sic1 destruction in real time in individual cells under various perturbations to the system, we provide a clear picture of how the circuitry functions as a switch in vivo. We show that Cln1/2-Cdk1 sets the proper timing of Sic1 destruction, but does not contribute to its destruction speed; thus, it acts only as a trigger. Sic1's inhibition target Clb5/6-Cdk1 controls the speed of Sic1 destruction through a double-negative feedback loop, ensuring a robust all-or-none transition for Clb5/6-Cdk1 activity. Furthermore, we demonstrate that the degradation of a single-phosphosite mutant of Sic1 is rapid and switch-like, just as the wild-type form. Our mathematical model confirms our understanding of the circuit and demonstrates that the substrate sharing between the two kinases is not a redundancy but a part of the design to overcome the trade-off between the timing and sharpness of Sic1 degradation. Our study provides direct mechanistic insight into the design features underlying the yeast G1/S switch.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

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Rafael Zúñiga

2018-03-01

Full Text Available TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

Breast cancer stem-like cells are inhibited by a non-toxic aryl hydrocarbon receptor agonist.

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Gérald J Prud'homme

2010-11-01

Full Text Available Cancer stem cells (CSCs have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH. CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231 mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation. It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of

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

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Arase Sachiko

2012-03-01

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

Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: Possible Ca2+-channel inhibition

International Nuclear Information System (INIS)

Stauderman, K.A.; Gandhi, V.C.; Jones, D.J.

1992-01-01

Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K + -induced [ 3 H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+ . Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3 H]5-HT release induced by the Ca 2+ -ionophore A 23187 or Ca 2+ -independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K + -induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry

Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition

International Nuclear Information System (INIS)

Zhang, Ji-Gang; Zhang, Dan-Dan; Wu, Xin; Wang, Yu-Zhu; Gu, Sheng-Ying; Zhu, Guan-Hua; Li, Xiao-Yu; Li, Qin; Liu, Gao-Lin

2015-01-01

Studies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells. Cell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques. We explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence. Taken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC

Lauric acid and myristic acid from Allium sativum inhibit the growth of Mycobacterium tuberculosis H37Ra: in silico analysis reveals possible binding to protein kinase B.

Science.gov (United States)

Muniyan, Rajiniraja; Gurunathan, Jayaraman

2016-12-01

The bulb of Allium sativum Linn (Alliaceae) has numerous medicinal values. Though the petroleum ether extract of the bulb has shown to exhibit antimycobacterial activity, the phytochemical(s) responsible for this inhibitory activity is not known. To characterize the bioactive compounds in the petroleum ether extract of Allium sativum (garlic) that inhibit the growth of Mycobacterium tuberculosis H37Ra. Bioactivity-guided fractionation was employed to isolate the bioactive compounds. Antimycobacterial activity was evaluated by well-diffusion method and microplate alamar blue assay (MABA). Infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy were used to characterize the bioactive compounds. Autodock was used to obtain information on molecular recognition, and molecular dynamics simulation was performed using GROMACS. The bioactive compounds that inhibited the growth of M. tuberculosis H37Ra were found to be lauric acid (LA) and myristic acid (MA). The minimal inhibitory concentration of LA and MA was found to be 22.2 and 66.7 μg/mL, respectively. In silico analysis revealed that these fatty acids could bind at the cleft between the N-terminal and C-terminal lobes of the cytosolic domain of serine/threonine protein kinase B (PknB). The inhibition activity was dependent on the alkyl chain length of the fatty acid, and the amino acid residues involved in binding to fatty acid was found to be conserved across the Pkn family of proteins. The study indicates the possibility of using fatty acid derivatives, involving Pkn family of proteins, to inhibit the signal transduction processes in M. tuberculosis.

High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27^Kip Cdk-complexes

DEFF Research Database (Denmark)

Groth, Anja; Willumsen, Berthe Marie

2005-01-01

The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells...

Review and research analysis of computational target methods using BioRuby and in silico screening of herbal lead compounds against pancreatic cancer using R programming.

Science.gov (United States)

Jayadeepa, R M; Ray, Ankita; Naik, Dhaval; Sanyal, Debendra Nath; Shah, Disha

2014-01-01

Plants and their natural components sophisticated with the cornerstone of traditional conventional medicinal system throughout the globe for many years and extend to furnish mankind with latest remedies. Natural Products act as lead molecules for the synthesis of various potent drugs. In the current research a study is conducted on herbal small molecule and their potential binding chemical affinity to the effect or molecules of major diseases such as pancreatic cancer. Clinical studies demonstrate correlation between Cyclin- Dependent Kinase 4 (CDK4) and malignant progression of Pancreatic Cancer. Using Bioruby Gem's we were able to analyze better characteristics of the target protein. VegaZZ and NAMD were used to minimize the energy of the target protein. Therefore identification of effective, well- tolerated targets was analyzed. Further the target protein was subjected to docking with the anti cancer inhibitors which represents a rational chemo preventive strategy using AutoDock Vina. Later using the dock score top ranked phytochemicals were analyzed for Toxicity Analysis. Using the BioRuby gem we were able to measure the distance between the amino acid. Various R scripting libraries were used to hunt the best leads, as in this case the phytochemicals. Phytochemicals such as Wedelolactones and Catechin were analyzed computationally. This study has presented the various effects of naturally occurring anti pancreatic cancer compounds Catechin, Wedelolactones that inhibits Cyclin Dependent Kinase 4. The study results reveal that compounds use less binding energy to CDK4 and inhibit its activity. Future investigation of other various wet lab studies such as cell line studies will confirm results of these two herbal chemical formulations potential ones for treating Pancreatic Cancer.

The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

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Laura Graf

2013-12-01

Full Text Available 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.

Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90

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Xu Yuan-Ji

2011-08-01

Full Text Available Abstract Background Multiple myeloma (MM is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. Results In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. Conclusions Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.

Immunoregulatory effect of evodiamine in mice of various germlines.

Science.gov (United States)

Hu, Hai-Yan; Song, Zhao-Yang; Deng, Lan; Zhang, Mei-Xia

2008-08-01

The aim of this study was to investigate the effect of evodiamine on the proliferation and the immune function of thymocytes and splenocyte of mice from three germlines, which were 8 weeks old masculinity BALB/c, C57BL/6 and F1 hybridization mice. Cells of thymus and spleen were harvested and prepared as unicellular suspension. Cell proliferation was detected by MTT method, while the concentration of IL-2 was detected by ELISA, mRNA levels of bcl-2 and cdk2 in cells treated with evodiamine were detected by RT-PCR, the apoptosis rate and intracellular reactive oxygen species (ROS) concentration were analyzed by FCM, and the protein levels of BCL-2, CDK2 and BAX were determined by fluorescence microscope. The results indicated that at 0.5, 0.75 and 1 micromol/L, evodiamine inhibited the proliferation and externalization of thymocytes and splenocytes stimulated with ConA (p rate increased at a prolong period of time. After treatment with evodiamine for 24 and 48 hours, the cells were divided into two groups, one of which was negatively stained by 2 7-dichlorofluorescein (DCF), which indicated that ROS level decreased significantly in the dying cells. It is concluded that evodiamine inhibits proliferation and induces apoptosis of thymocytes and splenocytes from different germline mice, and at the same time decreases secretion of IL-2 through down-regulating bcl-2 and cdk2 levels.

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

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Antony M Latham

Full Text Available Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2 and cyclin-dependent kinase 1 (CDK1. This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Differential Cellular and Molecular Effects of Butyrate and Trichostatin A on Vascular Smooth Muscle Cells

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Kasturi Ranganna

2012-09-01

Full Text Available The histone deacetylase (HDAC inhibitors, butyrate and trichostatin A (TSA, are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.

Early circulating tumor DNA dynamics and clonal selection with palbociclib and fulvestrant for breast cancer.

Science.gov (United States)

O'Leary, Ben; Hrebien, Sarah; Morden, James P; Beaney, Matthew; Fribbens, Charlotte; Huang, Xin; Liu, Yuan; Bartlett, Cynthia Huang; Koehler, Maria; Cristofanilli, Massimo; Garcia-Murillas, Isaac; Bliss, Judith M; Turner, Nicholas C

2018-03-01

CDK4/6 inhibition substantially improves progression-free survival (PFS) for women with advanced estrogen receptor-positive breast cancer, although there are no predictive biomarkers. Early changes in circulating tumor DNA (ctDNA) level may provide early response prediction, but the impact of tumor heterogeneity is unknown. Here we use plasma samples from patients in the randomized phase III PALOMA-3 study of CDK4/6 inhibitor palbociclib and fulvestrant for women with advanced breast cancer and show that relative change in PIK3CA ctDNA level after 15 days treatment strongly predicts PFS on palbociclib and fulvestrant (hazard ratio 3.94, log-rank p = 0.0013). ESR1 mutations selected by prior hormone therapy are shown to be frequently sub clonal, with ESR1 ctDNA dynamics offering limited prediction of clinical outcome. These results suggest that early ctDNA dynamics may provide a robust biomarker for CDK4/6 inhibitors, with early ctDNA dynamics demonstrating divergent response of tumor sub clones to treatment.

Inhibition of urinary calculi -- a spectroscopic study

Science.gov (United States)

Manciu, Felicia; Govani, Jayesh; Durrer, William; Reza, Layra; Pinales, Luis

2008-10-01

Although a considerable number of investigations have already been undertaken and many causes such as life habits, metabolic disorders, and genetic factors have been noted as sources that accelerate calculi depositions and aggregations, there are still plenty of unanswered questions regarding efficient inhibition and treatment mechanisms. Thus, in an attempt to acquire more insights, we propose here a detailed scientific study of kidney stone formation and growth inhibition based on a traditional medicine approach with Rotula Aquatica Lour (RAL) herbal extracts. A simplified single diffusion gel growth technique was used for synthesizing the samples for the present study. The unexpected Zn presence in the sample with RAL inhibitor, as revealed by XPS measurements, explains the inhibition process and the dramatic reflectance of the incident light observed in the infrared transmission studies. Raman data demonstrate potential binding of the inhibitor with the oxygen of the kidney stone. Photoluminescence results corroborate to provide additional evidence of Zn-related inhibition.

Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling.

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Cajigas, Ivelisse; Leib, David E; Cochrane, Jesse; Luo, Hao; Swyter, Kelsey R; Chen, Sean; Clark, Brian S; Thompson, James; Yates, John R; Kingston, Robert E; Kohtz, Jhumku D

2015-08-01

Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains. © 2015. Published by The Company of Biologists Ltd.

The CDK inhibitor p21 is a novel target gene of ATF4 and contributes to cell survival under ER stress.

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Inoue, Yasumichi; Kawachi, Shiori; Ohkubo, Tsubasa; Nagasaka, Mai; Ito, Shogo; Fukuura, Keishi; Itoh, Yuka; Ohoka, Nobumichi; Morishita, Daisuke; Hayashi, Hidetoshi

2017-11-01

Activating transcription factor 4 (ATF4) is well known for its role in the endoplasmic reticulum (ER) stress response. ATF4 also transcriptionally induces multiple effectors that determine cell fate depending on cellular context. In addition, ATF4 can communicate both pro-apoptotic and pro-survival signals. How ATF4 mediates its prosurvival roles, however, requires further investigation. Here, we report that the CDK inhibitor p21 is a novel target gene of ATF4. We identified two ATF4-responsive elements, one of which directly binds ATF4, within the first intron of the p21 gene. Importantly, overexpression of p21 enhances cell survival following ER stress induction, while p21 knockdown increases cell death. These results suggest that p21 induction plays a vital role in the cellular response to ER stress and indicate that p21 is a prosurvival effector of ATF4. © 2017 Federation of European Biochemical Societies.

Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.

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Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

2016-04-12

Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines.

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Pentimalli, Francesca; Forte, Iris M; Esposito, Luca; Indovina, Paola; Iannuzzi, Carmelina A; Alfano, Luigi; Costa, Caterina; Barone, Daniela; Rocco, Gaetano; Giordano, Antonio

2018-04-02