Science.gov

Sample records for cdk inhibitor r-roscovitine

  1. R-roscovitine reduces lung inflammation induced by lipoteichoic acid and Streptococcus pneumoniae.

    PubMed

    Hoogendijk, Arie J; Roelofs, Joris J T H; Duitman, Janwillem; van Lieshout, Miriam H P; Blok, Dana C; van der Poll, Tom; Wieland, Catharina W

    2012-01-01

    Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense. PMID:22692577

  2. The cyclin-dependent kinase inhibitor seliciclib (R-roscovitine; CYC202) decreases the expression of mitotic control genes and prevents entry into mitosis.

    PubMed

    Whittaker, Steven R; Te Poele, Robert H; Chan, Florence; Linardopoulos, Spiros; Walton, Michael I; Garrett, Michelle D; Workman, Paul

    2007-12-15

    The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials. PMID:18075315

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

    PubMed

    Guendel, Irene; Agbottah, Emmanuel T; Kehn-Hall, Kylene; Kashanchi, Fatah

    2010-01-01

    Current therapy for human immunodeficiency virus (HIV-1) infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI) r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202) in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs. PMID:20334651

  4. The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors.

    PubMed

    Nellore, Anoma; Liu, Bianling; Patsoukis, Nikolaos; Boussiotis, Vassiliki A; Li, Lequn

    2014-01-01

    Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms' tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity. PMID:24631965

  5. The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors

    PubMed Central

    Nellore, Anoma; Liu, Bianling; Patsoukis, Nikolaos; Boussiotis, Vassiliki A.; Li, Lequn

    2014-01-01

    Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms’ tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity. PMID:24631965

  6. State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy

    PubMed Central

    Ganapathi, Sindura B.; Kester, Mark; Elmslie, Keith S.

    2009-01-01

    Human ether-a-go-go-related gene (HERG) potassium channel acts as a delayed rectifier in cardiac myocytes and is an important target for both pro- and antiarrhythmic drugs. Many drugs have been pulled from the market for unintended HERG block causing arrhythmias. Conversely, recent evidence has shown that HERG plays a role in cell proliferation and is overexpressed both in multiple tumor cell lines and in primary tumor cells, which makes HERG an attractive target for cancer treatment. Therefore, a drug that can block HERG but that does not induce cardiac arrhythmias would have great therapeutic potential. Roscovitine is a cyclin-dependent kinase (CDK) inhibitor that is in phase II clinical trials as an anticancer agent. In the present study we show that R-roscovitine blocks HERG potassium current (human embryonic kidney-293 cells stably expressing HERG) at clinically relevant concentrations. The block (IC50 = 27 μM) was rapid (τ = 20 ms) and reversible (τ = 25 ms) and increased with channel activation, which supports an open channel mechanism. Kinetic study of wild-type and inactivation mutant HERG channels supported block of activated channels by roscovitine with relatively little effect on either closed or inactivated channels. A HERG gating model reproduced all roscovitine effects. Our model of open channel block by roscovitine may offer an explanation of the lack of arrhythmias in clinical trials using roscovitine, which suggests the utility of a dual CDK/HERG channel block as an adjuvant cancer therapy. PMID:19244476

  7. Novel R-roscovitine NO-donor hybrid compounds as potential pro-resolution of inflammation agents

    PubMed Central

    Montanaro, Gabriele; Bertinaria, Massimo; Rolando, Barbara; Fruttero, Roberta; Lucas, Christopher D.; Dorward, David A.; Rossi, Adriano G.; Megson, Ian L.; Gasco, Alberto

    2013-01-01

    Neutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and possess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution derivatives, here we describe the design, synthesis and investigation of the biological potential of a small series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties (compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an increased pro-apoptotic activity when compared with either R-roscovitine or structurally related compounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apoptosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi–NO-donor hybrids may have additive pro-resolution of inflammation effects. PMID:23394865

  8. Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity

    PubMed Central

    Chen, Zhenghu; Wang, Zhenyu; Pang, Jonathan C.; Yu, Yang; Bieerkehazhi, Shayahati; Lu, Jiaxiong; Hu, Ting; Zhao, Yanling; Xu, Xin; Zhang, Hong; Yi, Joanna S.; Liu, Shangfeng; Yang, Jianhua

    2016-01-01

    Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients. PMID:27378523

  9. Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity.

    PubMed

    Chen, Zhenghu; Wang, Zhenyu; Pang, Jonathan C; Yu, Yang; Bieerkehazhi, Shayahati; Lu, Jiaxiong; Hu, Ting; Zhao, Yanling; Xu, Xin; Zhang, Hong; Yi, Joanna S; Liu, Shangfeng; Yang, Jianhua

    2016-01-01

    Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients. PMID:27378523

  10. Discovery and evaluation of dual CDK1 and CDK2 inhibitors.

    PubMed

    Payton, Marc; Chung, Grace; Yakowec, Peter; Wong, Andrew; Powers, Dave; Xiong, Ling; Zhang, Nancy; Leal, Juan; Bush, Tammy L; Santora, Vincent; Askew, Ben; Tasker, Andrew; Radinsky, Robert; Kendall, Richard; Coats, Steve

    2006-04-15

    In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective small-molecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G1 and G2, and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis. PMID:16618755

  11. Drug Delivery Strategies of Chemical CDK Inhibitors.

    PubMed

    Alvira, Daniel; Mondragón, Laura

    2016-01-01

    The pharmacological use of new therapeutics is often limited by a safe and effective drug-delivery system. In this sense, new chemical CDK inhibitors are not an exception. Nanotechnology may be able to solve some of the main problems limiting cancer treatments such as more specific delivery of therapeutics and reduction of toxic secondary effects. It provides new delivery systems able to specifically target cancer cells and release the active molecules in a controlled fashion. Specifically, silica mesoporous supports (SMPS) have emerged as an alternative for more classical drug delivery systems based on polymers. In this chapter, we describe the synthesis of a SMPS containing the CDK inhibitor roscovitine as cargo molecule and the protocols for confirmation of the proper cargo release of the nanoparticles in cell culture employing cell viability, cellular internalization, and cell death induction studies. PMID:26231714

  12. Cyclin dependent kinase (CDK) inhibitors as anticancer drugs.

    PubMed

    Sánchez-Martínez, Concepción; Gelbert, Lawrence M; Lallena, María José; de Dios, Alfonso

    2015-09-01

    Sustained proliferative capacity is a hallmark of cancer. In mammalian cells proliferation is controlled by the cell cycle, where cyclin-dependent kinases (CDKs) regulate critical checkpoints. CDK4 and CDK6 are considered highly validated anticancer drug targets due to their essential role regulating cell cycle progression at the G1 restriction point. This review provides an overview of recent advances on cyclin dependent kinase inhibitors in general with special emphasis on CDK4 and CDK6 inhibitors and compounds under clinical evaluation. Chemical structures, structure activity relationships, and relevant preclinical properties will be described. PMID:26115571

  13. The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes.

    PubMed

    Paternot, Sabine; Colleoni, Bianca; Bisteau, Xavier; Roger, Pierre P

    2014-01-01

    CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols. PMID:25486476

  14. Treating cancer with selective CDK4/6 inhibitors.

    PubMed

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

    2016-07-01

    Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process. Clinical implementation of first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, was originally hampered by the high risk of toxicity and lack of efficacy noted with these agents. The emergence of a new generation of selective CDK4/6 inhibitors, including ribociclib, abemaciclib and palbociclib, has enabled tumour types in which CDK4/6 has a pivotal role in the G1-to-S-phase cell-cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Results of pivotal phase III trials investigating palbociclib in patients with advanced-stage oestrogen receptor (ER)-positive breast cancer have demonstrated a substantial improvement in progression-free survival, with a well-tolerated toxicity profile. Mechanisms of acquired resistance to CDK4/6 inhibitors are beginning to emerge that, although unwelcome, might enable rational post-CDK4/6 inhibitor therapeutic strategies to be identified. Extending the use of CDK4/6 inhibitors beyond ER-positive breast cancer is challenging, and will likely require biomarkers that are predictive of a response, and the use of combination therapies in order to optimize CDK4/6 targeting. PMID:27030077

  15. Conformational Adaption May Explain the Slow Dissociation Kinetics of Roniciclib (BAY 1000394), a Type I CDK Inhibitor with Kinetic Selectivity for CDK2 and CDK9.

    PubMed

    Ayaz, Pelin; Andres, Dorothee; Kwiatkowski, Dennis A; Kolbe, Carl-Christian; Lienau, Philip; Siemeister, Gerhard; Lücking, Ulrich; Stegmann, Christian M

    2016-06-17

    Roniciclib (BAY 1000394) is a type I pan-CDK (cyclin-dependent kinase) inhibitor which has revealed potent efficacy in xenograft cancer models. Here, we show that roniciclib displays prolonged residence times on CDK2 and CDK9, whereas residence times on other CDKs are transient, thus giving rise to a kinetic selectivity of roniciclib. Surprisingly, variation of the substituent at the 5-position of the pyrimidine scaffold results in changes of up to 3 orders of magnitude of the drug-target residence time. CDK2 X-ray cocrystal structures have revealed a DFG-loop adaption for the 5-(trifluoromethyl) substituent, while for hydrogen and bromo substituents the DFG loop remains in its characteristic type I inhibitor position. In tumor cells, the prolonged residence times of roniciclib on CDK2 and CDK9 are reflected in a sustained inhibitory effect on retinoblastoma protein (RB) phosphorylation, indicating that the target residence time on CDK2 may contribute to sustained target engagement and antitumor efficacy. PMID:27090615

  16. Development of mice without Cip/Kip CDK inhibitors

    SciTech Connect

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

    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

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

    PubMed

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

    2016-03-10

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

  18. Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

    PubMed

    Schiemann, Kai; Mallinger, Aurélie; Wienke, Dirk; Esdar, Christina; Poeschke, Oliver; Busch, Michael; Rohdich, Felix; Eccles, Suzanne A; Schneider, Richard; Raynaud, Florence I; Czodrowski, Paul; Musil, Djordje; Schwarz, Daniel; Urbahns, Klaus; Blagg, Julian

    2016-03-01

    Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration. PMID:26852363

  19. Discovery of a novel class of 2-aminopyrimidines as CDK1 and CDK2 inhibitors.

    PubMed

    Lee, Jinho; Kim, Kyoung-Hee; Jeong, Shinwu

    2011-07-15

    A series of new 2-(2-aminopyrimidin-4-yl)phenol derivatives were synthesized as potential antitumor compounds. Substitution with pyrrolidine-3,4-diol at the 4-position of phenol provided potent inhibitory activity against CDK1 and CDK2. X-ray crystal structural studies were performed to account for the effect of the substituent on both the enzymatic and cell growth inhibitory activities. PMID:21684737

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

    PubMed

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

    2015-09-01

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

  1. Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors.

    PubMed

    Wilson, Stuart C; Atrash, Butrus; Barlow, Clare; Eccles, Susan; Fischer, Peter M; Hayes, Angela; Kelland, Lloyd; Jackson, Wayne; Jarman, Michael; Mirza, Amin; Moreno, Javier; Nutley, Bernard P; Raynaud, Florence I; Sheldrake, Peter; Walton, Mike; Westwood, Robert; Whittaker, Steven; Workman, Paul; McDonald, Edward

    2011-11-15

    The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts. PMID:21982796

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

    SciTech Connect

    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

    2013-01-25

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

  3. Kinase inhibitor screening identifies CDK4 as a potential therapeutic target for melanoma

    PubMed Central

    MAHGOUB, T.; EUSTACE, A.J.; COLLINS, D.M.; WALSH, N.; O'DONOVAN, N.; CROWN, J.

    2015-01-01

    Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma. PMID:26201960

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-01-01

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

  6. A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells.

    PubMed

    Lane, M E; Yu, B; Rice, A; Lipson, K E; Liang, C; Sun, L; Tang, C; McMahon, G; Pestell, R G; Wadler, S

    2001-08-15

    Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), a novel 3-substituted indolinone compound, binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb, an increase in caspase-3 activation (5-84%), and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents. PMID:11507069

  7. Overcoming Therapeutic Resistance in HER2-Positive Breast Cancers with CDK4/6 Inhibitors.

    PubMed

    Goel, Shom; Wang, Qi; Watt, April C; Tolaney, Sara M; Dillon, Deborah A; Li, Wei; Ramm, Susanne; Palmer, Adam C; Yuzugullu, Haluk; Varadan, Vinay; Tuck, David; Harris, Lyndsay N; Wong, Kwok-Kin; Liu, X Shirley; Sicinski, Piotr; Winer, Eric P; Krop, Ian E; Zhao, Jean J

    2016-03-14

    Using transgenic mouse models, cell line-based functional studies, and clinical specimens, we show that cyclin D1/CDK4 mediate resistance to targeted therapy for HER2-positive breast cancer. This is overcome using CDK4/6 inhibitors. Inhibition of CDK4/6 not only suppresses Rb phosphorylation, but also reduces TSC2 phosphorylation and thus partially attenuates mTORC1 activity. This relieves feedback inhibition of upstream EGFR family kinases, resensitizing tumors to EGFR/HER2 blockade. Consequently, dual inhibition of EGFR/HER2 and CDK4/6 invokes a more potent suppression of TSC2 phosphorylation and hence mTORC1/S6K/S6RP activity. The suppression of both Rb and S6RP enhances G1 arrest and a phenotype resembling cellular senescence. In vivo, CDK4/6 inhibitors sensitize patient-derived xenograft tumors to HER2-targeted therapies and delay tumor recurrence in a transgenic model of HER2-positive breast cancer. PMID:26977878

  8. Discovery of novel 5-fluoro-N2,N4-diphenylpyrimidine-2,4-diamines as potent inhibitors against CDK2 and CDK9

    PubMed Central

    Gao, Jiadi; Fang, Cheng; Xiao, Zhiyan; Huang, Li; Chen, Chin-Ho; Wang, Li-Ting; Lee, Kuo-Hsiung

    2014-01-01

    Based on a 3D-QSAR pharmacophore derived from a diverse set of known cyclin-dependent kinase 9 (CDK9) inhibitors and a composite pharmacophore extracted from the complex structure of flavopiridol (FVP)-CDK9, thirty novel 5-fluoro-N2,N4-diphenylpyrimidine-2,4-diamine derivatives were designed and synthesized. Initial tests against four tumor cell lines with the sulforhodamine B (SRB) assay identified a series of potent compounds with GI50 values at lower micromolar or submicromolar level. Most of the highly cytotoxic compounds exhibited potent inhibitory activities against both CDK2/cyclin E1 and CDK9/cyclin T1. Notably, inhibitions against the two enzymes were generally correlated well with the cytotoxicity of these compounds. Appreciable inhibition was also observed for selected compounds in the anti-HIV-1 assay. Docking studies on compounds 6d and 9g provided conducive clues to further structural optimization. PMID:25914804

  9. Discovery and SAR of novel pyrazolo[1,5-a]pyrimidines as inhibitors of CDK9.

    PubMed

    Phillipson, Louisa J; Segal, David H; Nero, Tracy L; Parker, Michael W; Wan, Soo San; de Silva, Melanie; Guthridge, Mark A; Wei, Andrew H; Burns, Christopher J

    2015-10-01

    The serine-threonine kinase CDK9 is a target of emerging interest for the development of anti-cancer drugs. There are multiple lines of evidence linking CDK9 activity to cancer, including the essential role this kinase plays in transcriptional regulation through phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Indeed, inhibition of CDK9 has been shown to result in a reduction of short-lived proteins such as the pro-survival protein Mcl-1 in malignant cells leading to the induction of apoptosis. In this work we report our initial studies towards the discovery of selective CDK9 inhibitors, starting from the known multi-kinase inhibitor PIK-75 which possesses potent CDK9 activity. Our series is based on a pyrazolo[1,5-a]pyrimidine nucleus and, importantly, the resultant lead compound 18b is devoid of the structural liabilities present in PIK-75 and possesses greater selectivity. PMID:26349627

  10. Flavonoids as CDK1 Inhibitors: Insights in Their Binding Orientations and Structure-Activity Relationship

    PubMed Central

    Navarro-Retamal, Carlos

    2016-01-01

    In the last years, the interactions of flavonoids with protein kinases (PKs) have been described by using crystallographic experiments. Interestingly, different orientations have been found for one flavonoid inside different PKs and different chemical substitutions lead to different orientations of the flavonoid scaffold inside one PK. Accordingly, orientation predictions of novel analogues could help to the design of flavonoids with high PK inhibitory activities. With this in mind, we studied the binding modes of 37 flavonoids (flavones and chalcones) inside the cyclin-dependent PK CDK1 using docking experiments. We found that the compounds under study adopted two different orientations into the active site of CDK1 (orientations I and II in the manuscript). In addition, quantitative structure–activity relationship (QSAR) models using CoMFA and CoMSIA methodologies were constructed to explain the trend of the CDK1 inhibitory activities for the studied flavonoids. Template-based and docking-based alignments were used. Models developed starting from docking-based alignment were applied for describing the whole dataset and compounds with orientation I. Adequate R2 and Q2 values were obtained by each method; interestingly, only hydrophobic and hydrogen bond donor fields describe the differential potency of the flavonoids as CDK1 inhibitors for both defined alignments and subsets. Our current application of docking and QSAR together reveals important elements to be drawn for the design of novel flavonoids with increased PK inhibitory activities. PMID:27517610

  11. Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor

    PubMed Central

    Albert, T K; Rigault, C; Eickhoff, J; Baumgart, K; Antrecht, C; Klebl, B; Mittler, G; Meisterernst, M

    2014-01-01

    BACKGROUND AND PURPOSE The cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9. EXPERIMENTAL APPROACH The selectivity of LDC000067 was established in functional kinase assays. Functions of CDK9 in gene expression were assessed with in vitro transcription experiments, single gene analyses and genome-wide expression profiling. Cultures of mouse embryonic stem cells, HeLa cells, several cancer cell lines, along with cells from patients with acute myelogenous leukaemia were also used to investigate cellular responses to LDC000067. KEY RESULTS The selectivity of LDC000067 for CDK9 over other CDKs exceeded that of the known inhibitors flavopiridol and DRB. LDC000067 inhibited in vitro transcription in an ATP-competitive and dose-dependent manner. Gene expression profiling of cells treated with LDC000067 demonstrated a selective reduction of short-lived mRNAs, including important regulators of proliferation and apoptosis. Analysis of de novo RNA synthesis suggested a wide ranging positive role of CDK9. At the molecular and cellular level, LDC000067 reproduced effects characteristic of CDK9 inhibition such as enhanced pausing of RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells. CONCLUSIONS AND IMPLICATIONS Our study provides a framework for the mechanistic understanding of cellular responses to CDK9 inhibition. LDC000067 represents a promising lead for the development of clinically useful, highly specific CDK9 inhibitors. PMID:24102143

  12. Extra precision docking, free energy calculation and molecular dynamics simulation studies of CDK2 inhibitors.

    PubMed

    Tripathi, Sunil Kumar; Muttineni, Ravikumar; Singh, Sanjeev Kumar

    2013-10-01

    Molecular docking, free energy calculation and molecular dynamics (MD) simulation studies have been performed, to explore the putative binding modes of 3,5-diaminoindazoles, imidazo(1,2-b)pyridazines and triazolo(1,5-a) pyridazines series of Cyclin-dependent kinase (CDK2) inhibitors. To evaluate the effectiveness of docking protocol in flexible docking, we have selected crystallographic bound compound to validate our docking procedure as evident from root mean square deviations (RMSDs). We found different binding sites namely catalytic, inhibitory phosphorylation, cyclin binding and CKS-binding site of the CDK2 contributing towards the binding of these compounds. Moreover, correlation between free energy of binding and biological activity yielded a statistically significant correlation coefficient. Finally, three representative protein-ligand complexes were subjected to molecular dynamics simulation to determine the stability of the predicted conformations. The low value of the RMSDs between the initial complex structure and the energy minimized final average complex structure suggests that the derived docked complexes are close to equilibrium. We suggest that the phenylacetyl type of substituents and cyclohexyl moiety make the favorable interactions with a number of residues in the active site, and show better inhibitory activity to improve the pharmacokinetic profile of compounds against CDK2. The structure-based drug design strategy described in this study will be highly useful for the development of new inhibitors with high potency and selectivity. PMID:23727278

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

    PubMed

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

    2016-06-10

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

  14. A novel multi-CDK inhibitor P1446A-05 restricts melanoma growth and produces synergistic effects in combination with MAPK pathway inhibitors.

    PubMed

    Eliades, Philip; Miller, David M; Miao, Benchun; Kumar, Raj; Taylor, Michael; Buch, Shama; Srinivasa, Sreesha P; Flaherty, Keith T; Tsao, Hensin

    2016-07-01

    Nearly 100% of melanomas have a defect in the p16(INK4A):cyclin D-CDK4/6:RB pathway, leading to abnormal cell cycle control and unregulated cellular proliferation. Here, we report that P1446A-05, a novel multi-CDK inhibitor has significant inhibitory activity against cutaneous and uveal melanoma. Mechanistic studies revealed that P1446A-05 inhibits phosphorylation targets of CDK members, and induces cell cycle arrest and apoptosis irrespective of melanoma genotype or phenotype. Additionally, we show preclinical evidence that P1446A-05 can synergize with other small molecule inhibitors previously studied in melanoma. Collectively, these data demonstrate that targeting cell cycle and transcriptional CDKs with a small molecule multi-CDK inhibitor is a viable approach for developing novel anti-melanoma therapeutics. PMID:26810603

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

    PubMed

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

    2015-01-01

    The cyclin-dependent kinase 4 (CDK4)-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1) and protein-ligand (CDK4-flavopiridol) interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL) _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment. PMID:26252490

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

    The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G0/G1 phase of the cell cycle, the block occurs primarily at the G2/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC50<1 μM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000 mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G0/G1. Its ability to

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

    PubMed Central

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

    2015-01-01

    The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G0/G1 phase of the cell cycle, the block occurs primarily at the G2/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC50 <1 μM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000 mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G0/G1. Its ability to

  19. CDK4/6 Inhibitor PD0332991 in Glioblastoma Treatment: Does It Have a Future?

    PubMed Central

    Schröder, Lisette B. W.; McDonald, Kerrie L.

    2015-01-01

    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. PMID:26649278

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

    PubMed Central

    Christensen, Camilla L.; Kwiatkowski, Nicholas; Abraham, Brian J.; Carretero, Julian; Al-shahrour, Fatima; Zhang, Tinghu; Chipumuro, Edmond; Herter-Sprie, Grit S.; Akbay, Esra A.; Altabef, Abigail; Zhang, Jianming; Shimamura, Takeshi; Capelletti, Marzia; Reibel, Jakob B.; Cavanaugh, Jillian; Gao, Peng; Liu, Yan; Michaelsen, Signe R.; Poulsen, Hans S.; Aref, Amir R.; Barbie, David A.; Bradner, James E.; George, Rani; Gray, Nathanael S.; Young, Richard A.; Wong, Kwok-Kin

    2014-01-01

    SUMMARY Small cell lung cancer (SCLC) is an aggressive disease with high mortality. The identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library we observe that SCLC is sensitive to transcription-targeting drugs, and in particular to THZ1, a recent identified covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We find that expression of super-enhancer associated transcription factor genes including MYC family proto-oncogenes and neuroendocrine lineage-specific factors are highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy. PMID:25490451

  1. Clinical Development of the CDK4/6 Inhibitors Ribociclib and Abemaciclib in Breast Cancer.

    PubMed

    Barroso-Sousa, Romualdo; Shapiro, Geoffrey I; Tolaney, Sara M

    2016-06-01

    Clinical and preclinical data support a significant role for inhibitors of the cyclin-dependent kinases (CDKs) 4 and 6 in the treatment of patients with breast cancer. Recently, based on data showing improvement in progression-free survival, the use of palbociclib (Ibrance; Pfizer, Inc.) in combination with endocrine agents was approved to treat patients with hormone receptor-positive advanced disease. Importantly, 2 other CDK4/6 inhibitors, abemaciclib (LY2835219; Lilly) and ribociclib (LEE011; Novartis), are in the late stage of clinical development. In this review, we will focus on clinical data on these 2 new drugs, highlighting their differences compared to palbociclib in terms of single-agent activity, central nervous system penetration, and common adverse events. In addition, we will present the ongoing clinical trials and discuss future directions in the field. PMID:27493615

  2. Association of CDK4 germline and BRAF somatic mutations in a patient with multiple primary melanomas and BRAF inhibitor resistance.

    PubMed

    Governa, Maurizio; Caprarella, Evelina; Dalla Pozza, Edoardo; Vigato, Enrico; Maritan, Monia; Caputo, Glenda G; Zannoni, Marina; Rosina, Paolo; Elefanti, Lisa; Stagni, Camilla; Menin, Chiara

    2015-10-01

    Many genetic alterations, including predisposing or somatic mutations, may contribute toward the development of melanoma. Although CDKN2A and CDK4 are high-penetrance genes for melanoma, MC1R is a low-penetrance gene that has been associated most consistently with the disease. Moreover, BRAF is the most frequently somatically altered oncogene and is a validated therapeutic target in melanoma. This paper reports a case of multiple primary melanoma with germline CDK4 mutation, MC1R variant, and somatic BRAF mutation in nine out of 10 melanomas, indicating that a common pathogenesis, because of a predisposing genetic background, may be shared among distinct subsequent melanomas of probable clonal origin. After 3 months of targeted therapy with BRAF inhibitor, our patient developed resistance with rapid progression of the disease leading to death. This is the first case in which early resistance to BRAF inhibitor has been reported in a patient with CDK4 germline mutation. PMID:26110554

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

    PubMed

    Spring, Laura; Bardia, Aditya; Modi, Shanu

    2016-01-01

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

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

    PubMed

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y; Wiedemeyer, W Ruprecht

    2015-01-20

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  5. Dinaciclib, a novel CDK inhibitor, demonstrates encouraging single-agent activity in patients with relapsed multiple myeloma

    PubMed Central

    LaPlant, Betsy; Chng, Wee Joo; Zonder, Jeffrey; Callander, Natalie; Fonseca, Rafael; Fruth, Briant; Roy, Vivek; Erlichman, Charles; Stewart, A. Keith

    2015-01-01

    Dysregulation of cyclin-dependent kinases is a hallmark of myeloma, and specifically, cdk5 inhibition can enhance the activity of proteasome inhibitors in vitro. Dinaciclib is a novel potent small molecule inhibitor of cyclin-dependent kinases (CDK)1, CDK2, CDK5, and CDK9. Patients with relapsed multiple myeloma and ≤5 prior lines of therapy, with measurable disease, were enrolled. Dinaciclib was administered on day 1 of a 21-day cycle at doses of 30 to 50 mg/m2. Overall, 27 evaluable patients were accrued; the median number of prior therapies was 4. The dose level of 50 mg/m2 was determined to be the maximally tolerated dose. The overall confirmed partial response rate (PR) was 3 of 27 (11%), including 1 patient at the 30 mg/m2 dose (1 very good PR [VGPR]) and 2 patients at the 40 mg/m2 dose (1 VGPR and 1 PR). In addition, 2 patients at the 50 mg/mg2 dose achieved a minimal response (clinical benefit rate, 19%). Leukopenia, thrombocytopenia, gastrointestinal symptoms, alopecia, and fatigue were the most common adverse events. The current study demonstrates single agent activity of dinaciclib in relapsed myeloma, with 2 patients achieving a deep response (VGPR) and 10 patients obtaining some degree of M protein stabilization or decrease. This trial was registered at www.clinicaltrials.gov as #NCT01096342. PMID:25395429

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

    PubMed Central

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

    2015-01-01

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  7. CDK inhibitor enhances the sensitivity to 5-fluorouracil in colorectal cancer cells.

    PubMed

    Takagi, Koichi; Sowa, Yoshihiro; Cevik, Ozgur Muhammer; Nakanishi, Ryoko; Sakai, Toshiyuki

    2008-05-01

    Thymidylate synthase (TS) is a dNTP synthetic enzyme and is also a target enzyme of 5-fluorouracil (5-FU). 5-FU is one of the anticancer agents most frequently used for the treatment of colorectal cancers. However, the clinical rate of response to its use as a single agent is not exceptionally high. Therefore, various combination chemotherapies have been devised. The elevated expression of TS in cancer cells is a serious obstacle in the clinical use of 5-FU. In the present study, TS expression was up-regulated by the knockout of the p21WAF1/CIP1 gene in human colorectal cancer HCT116 cells, suggesting that TS expression is mediated through the inhibition of cyclin-dependent kinase (CDK). Based on these findings, we tested whether the CDK inhibitor (CDKI) SU9516, acted as a suppressor of TS. SU9516 effectively reduced the expression of TS in a dose-dependent manner. Furthermore, the reduction of TS expression resulted in enhancement of the sensitivity to 5-FU in human colon cancer DLD-1 cells. Thus, SU9516 might be a promising compound for combination chemotherapy with 5-FU. PMID:18425338

  8. A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

    PubMed Central

    Slingerland, J M; Hengst, L; Pan, C H; Alexander, D; Stampfer, M R; Reed, S I

    1994-01-01

    Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses. Images PMID:8196612

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

    PubMed Central

    Liu, Shu; Premnath, Padmavathy Nandha; Bolger, Joshua K.; Perkins, Tracy; Kirkland, Lindsay O.; Kontopidis, George; McInnes, Campbell

    2013-01-01

    A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore the structure-activity relationship of a bisarylether C-terminal capping group mimicking dipeptide interactions, was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds. PMID:23323521

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

    PubMed Central

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

    2013-01-01

    Potent antiretroviral therapy (ART) has transformed HIV-1 infection into a chronic manageable disease; however drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study we utilized a combination of structure based analysis of Cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket which showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell based biological assays. Using these methods we obtained the first generation mimetic drugs and tested these compounds on HIV-1 LTR activated transcription. Using biological assays followed by similar in silico analysis to find a 2nd generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the 2nd generation mimetic against various viral isolates, and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2-/-γc-/- with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis. PMID:23247501

  11. Silencing of the polyamine catabolic key enzyme SSAT prevents CDK inhibitor-induced apoptosis in Caco-2 colon cancer cells.

    PubMed

    Çoker, A; Arısan, E D; Palavan-Ünsal, N

    2012-04-01

    Roscovitine and purvalanol are purine derivative cyclin-dependent kinase (CDK) inhibitors that induce apoptosis in various types of cancer cells. However, their impact on the apoptotic cell death mechanism requires further elucidation. Natural polyamines putrescine, spermidine and spermine play essential roles in the regulation of cell growth and proliferation. Increased levels of polyamines in cells are considered to be involved in cancer progression. Intracellular polyamine levels are under the control of several catabolic enzymes, such as spermidine/spermine-N-acetyl transferase (SSAT), acetylpolyamine oxidase (APAO) and spermine oxidase (SMO), which could be altered by several therapeutic drugs. However, the possible role of polyamines in drug-induced apoptosis has yet to be clarified. In the present study, our aim was to determine the modulation of the polyamine catabolic pathway related to CDK inhibitor-induced apoptosis in Caco-2 cells. We found that roscovitine and purvalanol (each 20 µM) induced apoptosis by activating caspase-9 and -3, and inhibiting the mitochondrial membrane potential in Caco-2 cells. CDK inhibitors decreased the intracellular putrescine and spermine levels without affecting spermidine levels. Although both roscovitine and purvalanol induced SSAT expression, they did not exert a significant effect on the APAO expression profile. SSAT transient silencing prevented roscovitine-induced apoptosis compared to parental cells. Thus, we concluded that roscovitine and purvalanol significantly induce apoptosis in Caco-2 cells by modulating the polyamine catabolism, and that SSAT could be an important target in evaluating the potential role of polyamines in apoptotic cell death. PMID:22294330

  12. Molecular modelling on small molecular CDK2 inhibitors: an integrated approach using a combination of molecular docking, 3D-QSAR and pharmacophore modelling.

    PubMed

    Yuan, H; Liu, H; Tai, W; Wang, F; Zhang, Y; Yao, S; Ran, T; Lu, S; Ke, Z; Xiong, X; Xu, J; Chen, Y; Lu, T

    2013-10-01

    Cyclin-dependent kinase 2 (CDK2) has been identified as an important target for developing novel anticancer agents. Molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) and pharmacophore modelling were combined with the ultimate goal of studying the structure-activity relationship of CDK2 inhibitors. The comparative molecular similarity indices analysis (CoMSIA) model constructed based on a set of 3-aminopyrazole derivatives as CDK2 inhibitors gave statistically significant results (q (2) = 0.700; r (2) = 0.982). A HypoGen pharmacophore model, constructed using diverse CDK2 inhibitors, also showed significant statistics ([Formula: see text]Cost = 61.483; RMSD = 0.53; Correlation coefficient = 0.98). The small residues and error values between the estimated and experimental activities of the training and test set compounds proved their strong capability of activity prediction. The structural insights obtained from these two models were consistent with each other. The pharmacophore model summarized the important pharmacophoric features required for protein-ligand binding. The 3D contour maps in combination with the comprehensive pharmacophoric features helped to better interpret the structure-activity relationship. The results will be beneficial for the discovery and design of novel CDK2 inhibitors. The simplicity of this approach provides expansion to its applicability in optimizing other classes of small molecular CDK2 inhibitors. PMID:23941641

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

    PubMed

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

    2015-12-01

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

  14. The CDK4/6 inhibitor LY2835219 has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma

    PubMed Central

    Koh, Jiae; Bae, Yeon-Hee; Sun, Jong-Mu; Lee, Se-hoon; Ahn, Jin Seok; Park, Keunchil; Ahn, Myung-Ju

    2016-01-01

    Deletion of CDKN2A (p16) or amplification of CCND1 (cyclin D1) occurs commonly in head and neck squamous cell carcinoma (HNSCC) and induces sustained cyclin-dependent kinase (CDK) 4/6 activation. Here, we report the antiproliferative activity of LY2835219, a selective CDK4/6 inhibitor through inhibition of CDK4/6-dependent Ser780 phosphorylation in retinoblastoma (RB) and induction of cell cycle arrest in HNSCC cells. In addition, we demonstrated the antitumor effects of HNSCC xenografts to LY2835219 in vivo. Given the limited effect in HNSCC as a single-agent treatment with LY2835219, a combinational strategy is required to enhance antitumor activity. At the molecular level, we found that LY2835219 inhibited activation of AKT and ERK, but not mTOR. The combination of LY2835219 with mTOR inhibitor was found to be more effective than either drug alone in vitro and in vivo. Taken together, our findings suggest that a combinational treatment with LY2835219 and mTOR inhibitor is a promising therapeutic approach for HNSCC. PMID:26909611

  15. BA-j as a novel CDK1 inhibitor selectively induces apoptosis in cancer cells by regulating ROS

    PubMed Central

    Zhang, Shixuan; Bao, Yongming; Ju, Xiulan; Li, Kangjian; Shang, Haiyan; Ha, Lisha; Qian, Yuan; Zou, Liang; Sun, Xiaodan; Li, Jing; Wang, Qianru; Fan, Qingyu

    2015-01-01

    Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in cell proliferation and a novel target in the development of anticancer drugs. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a novel selective CDK1 inhibitor with broad spectrum anti-cancer activity (IC50 12.3 μM) and 2 tumor xenografts. Because of the differential mechanisms controlling redox-states in normal and cancer cells, BA-j can capture oxygen free radicals (·O2−) and selectively increase the level of H2O2 in cancer cells, thereby specifically oxidize and activate the intrinsic apoptosis pathway bypassing the extrinsic death receptor pathway, thus inducing apoptosis in cancer cells rather than in normal cells. BA-j is different from cytotoxic anticancer drugs which can activate both the intrinsic apoptosis pathway and the extrinsic death receptor pathway, and therefore harm normal cells while killing cancer cells. The molecular and biochemical mechanisms of reactive oxygen species (ROS) regulation suggest that BA-j may be developed into a novel anticancer agent. PMID:26330167

  16. Free-energy-based methods for binding profile determination in a congeneric series of CDK2 inhibitors.

    PubMed

    Fidelak, Jérémy; Juraszek, Jarek; Branduardi, Davide; Bianciotto, Marc; Gervasio, Francesco Luigi

    2010-07-29

    Free-energy pathway methods show great promise in computing the mode of action and the free energy profile associated with the binding of small molecules with proteins, but are generally very computationally demanding. Here we apply a novel approach based on metadynamics and path collective variables. We show that this combination is able to find an optimal reaction coordinate and the free energy profile of binding with explicit solvent and full flexibility, while minimizing human intervention and computational costs. We apply it to predict the binding affinity of a congeneric series of 5 CDK2 inhibitors. The predicted binding free energy profiles are in accordance with experiment. PMID:20593892

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

    PubMed Central

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

    2016-01-01

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

  18. Assessing Cell Cycle Independent Function of the CDK Inhibitor p21(CDKN¹A) in DNA Repair.

    PubMed

    Dutto, Ilaria; Tillhon, Micol; Prosperi, Ennio

    2016-01-01

    The cyclin-dependent kinase (CDK) inhibitor p21(CDKN1A) is a small protein that is able to regulate many important cell functions, often independently of its activity of CDK inhibitor. In addition to cell cycle, this protein regulates cell transcription, apoptosis, cell motility, and DNA repair. In particular, p21 may participate in different DNA repair processes, like the nucleotide excision repair (NER), base excision repair (BER), and double-strand breaks (DSB) repair, because of its ability to interact with DNA repair proteins, such as proliferating cell nuclear antigen (PCNA), a master regulator of many DNA transactions. Although this role has been debated for a long time, the influence of p21 in DNA repair has been now established. However, it remain to be clarified how this role is coupled to proteasomal degradation that has been shown to occur after DNA damage. This chapter describes procedures to study p21 protein recruitment to localized DNA damage sites in the cell nucleus. In particular, we describe a technique based on local irrradiation with UV light through a polycarbonate filter with micropores; an in situ lysis procedure to detect chromatin-bound proteins by immunofluorescence; a cell fractionation procedure to study chromatin association of p21 by Western blot analysis, and p21 protein-protein interactions by an immunoprecipitation assay. PMID:26231713

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

    SciTech Connect

    Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi; Iwamoto, Noriko; Nakayama, Keiichi I.; Takeuchi, Takashi

    2014-01-17

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

  20. Purvalanol A, a CDK inhibitor, effectively suppresses Src-mediated transformation by inhibiting both CDKs and c-Src.

    PubMed

    Hikita, Tomoya; Oneyama, Chitose; Okada, Masato

    2010-10-01

    The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However, Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression, we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A, developed as a CDK inhibitor, potently suppressed the anchorage-independent growth of c-Src-transformed cells, indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2, and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest, whereas PP2 weakly acted on the G1-S transition. Furthermore, when compared with PP2, purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells, in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation. PMID:20825494

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

    PubMed

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

    2008-06-01

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

  2. Overcoming Endocrine Resistance in Hormone-Receptor Positive Advanced Breast Cancer-The Emerging Role of CDK4/6 Inhibitors

    PubMed Central

    O’Sullivan, Ciara C

    2015-01-01

    Dysregulation of the cyclin D and cyclin-dependent kinase (CDK) pathway in cancer cells may inhibit senescence and promote cellular proliferation. By using various different mechanisms, malignant cells may increase cyclin D-dependent activity. The cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6)-retinoblastoma (Rb) pathway controls the cell cycle restriction point, and is commonly dysregulated in breast cancer; making it a rational target for anticancer therapy. To date, three oral highly selective cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are in various stages of clinical development: PD0332991 (palbociclib), LEE011 (ribociclib) and LY2835219 (abemaciclib). Results from phase I, II and III trials in hormone-receptor (HR)-positive breast cancer have been encouraging, demonstrating convincing efficacy and a tolerable side-effect profile (mainly uncomplicated neutropenia). This article will review the preclinical and clinical development of the CDK4/6i, as well as reviewing the existing preclinical evidence regarding combination of these agents with chemotherapy and other targeted therapies. Future and ongoing clinical trials, which may expand the potential application of these agents, will also be discussed. In summary, CDK4/6i are exciting compounds which may change the therapeutic landscape of HR-positive breast cancer. PMID:26726315

  3. CDK inhibitors induce mitochondria-mediated apoptosis through the activation of polyamine catabolic pathway in LNCaP, DU145 and PC3 prostate cancer cells.

    PubMed

    Arisan, Elif Damla; Obakan, Pinar; Coker-Gurkan, Ajda; Calcabrini, Annarica; Agostinelli, Enzo; Unsal, Narcin Palavan

    2014-01-01

    Androgen signaling is critical in prostate cancer development and progression. The co-existence of hormone responsive and irresponsive cells due to functional androgen receptor (AR) in prostate gland is the major obstacle in prostate cancer therapy models. Targeting aberrant cell cycle by novel cell cycle blocking agents is a promising strategy to treat various types of malignancies. Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors able to activate apoptotic cell death by inducing cell cycle arrest at G1/S and G2/M phases in cancer cells. Polyamines are unique cationic amine derivatives involved in the regulation of cell proliferation. Although the elevated intracellular level of polyamines (putrescine, spermidine and spermine) is typical for prostate gland, abnormal regulation of polyamine metabolism might result in rapid cell proliferation and, thus in prostate cancer progression. Therefore, treatment with drug-induced depletion of intracellular polyamine levels through the activated polyamine catabolism is critical to achieve successful strategies for prostate cancer. In this study we aimed to investigate the apoptotic efficiency of CDK inhibitors in three prostate cancer cell lines (LNCaP, DU145 and PC3), showing different AR expression profile. We found that both purvalanol and roscovitine were able to induce apoptosis at moderate cytotoxic concentrations by decreasing mitochondria membrane potential. The apoptotic effect of both CDK inhibitors was due to activation of caspases by modulating Bcl-2 family members. The efficiency of drugs was quite similar on the three prostate cell lines used in this study. However, DU145 cells were found the least sensitive against CDK inhibitors while purvalanol was more potent than roscovitine. Similarly to classical chemotherapeutic agents, both drugs could up-regulate polyamine catabolic enzymes (SSAT, SMO and PAO) in cell type dependent manner. Transient silencing of SSAT and/or inhibition of PAO

  4. Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27(KIP1)-derived peptidomimetic inhibitors.

    PubMed

    Karthiga, Arumugasamy; Tripathi, Sunil Kumar; Shanmugam, Ramasamy; Suryanarayanan, Venkatesan; Singh, Sanjeev Kumar

    2015-01-01

    Functionally activated cyclin-dependent kinase 2 (CDK2)/cyclin A complex has been validated as an interesting therapeutic target to develop the efficient antineoplastic drug based on the cell cycle arrest. Cyclin A binds to CDK2 and activates the kinases as well as recruits the substrate and inhibitors using a hydrophobic cyclin-binding groove (CBG). Blocking the cyclin substrate recruitment on CBG is an alternative approach to override the specificity hurdle of the currently available ATP site targeting CDK2 inhibitors. Greater understanding of the interaction of CDK2/cyclin A complex with p27 (negative regulator) reveals that the Leu-Phe-Gly (LFG) motif region of p27 binds with the CBG site of cyclin A to arrest the malignant cell proliferation that induces apoptosis. In the present study, Replacement with Partial Ligand Alternatives through Computational Enrichment (REPLACE) drug design strategies have been applied to acquire LFG peptide-derived peptidomimetics library. The peptidomimetics function is equivalent with respect to substrate p27 protein fashion but does not act as an ATP antagonist. The combined approach of molecular docking, molecular dynamics (MD), and molecular electrostatic potential and ADME/T prediction were carried out to evaluate the peptidomimetics. Resultant interaction and electrostatic potential maps suggested that smaller substituent is desirable at the position of phenyl ring to interact with Trp217, Arg250, and Gln254 residues in the active site. The best docked poses were refined by the MD simulations which resulted in conformational changes. After equilibration, the structure of the peptidomimetic and receptor complex was stable. The results revealed that the various substrate protein-derived peptidomimetics could serve as perfect leads against CDK2 protein. PMID:25584078

  5. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    PubMed

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells. PMID:27212103

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

    PubMed

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

    2014-12-01

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

  7. Down-regulation of multiple CDK inhibitor ICK/KRP genes promotes cell proliferation, callus induction and plant regeneration in Arabidopsis

    PubMed Central

    Cheng, Yan; Liu, Han; Cao, Ling; Wang, Sheng; Li, Yongpeng; Zhang, Yuanyuan; Jiang, Wei; Zhou, Yongming; Wang, Hong

    2015-01-01

    The ICK/KRP cyclin-dependent kinase (CDK) inhibitors are important plant cell cycle regulators sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Information is still limited regarding the specific functions of different ICK/KRP genes in planta. We have shown previously that down-regulation of multiple CDK inhibitor ICK/KRP genes up-regulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis. In this study, we observed that the quintuple ick1/2/5/6/7 mutant had more cells in the cortical layer of the root apical meristem (RAM) than the wild type (Wt) while its RAM length was similar to that of the Wt, suggesting a faster cell cycle rate in the quintuple mutant. We further investigated the effects of down-regulating ICK genes on tissue culture responses. The cotyledon explants of ick1/2/5/6/7 could form callus efficiently in the absence of cytokinin and also required a lower concentration of 2,4-D for callus induction compared to the Wt plants, suggesting increased competence for callus induction in the mutant. In addition, the quintuple ick mutant showed enhanced abilities to regenerate shoots and roots, suggesting that increased competence to enter the cell cycle in the quintuple mutant might make it possible for more cells to become proliferative and be utilized to form shoots or roots. These findings indicate that CDK activity is a major factor underlying callus induction and increased cell proliferation can enhance in vitro organogenesis. PMID:26528298

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

    SciTech Connect

    Raghavan, Pavithra; Tumati, Vasu; Yu Lan; Chan, Norman; Tomimatsu, Nozomi; Burma, Sandeep; Bristow, Robert G.; Saha, Debabrata

    2012-11-15

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

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

    PubMed

    Dobeš, Petr; Fanfrlík, Jindřich; Rezáč, Jan; Otyepka, Michal; Hobza, Pavel

    2011-03-01

    A semiempirical quantum mechanical PM6-DH2 method accurately covering the dispersion interaction and H-bonding was used to score fifteen structurally diverse CDK2 inhibitors. The geometries of all the complexes were taken from the X-ray structures and were reoptimised by the PM6-DH2 method in continuum water. The total scoring function was constructed as an estimate of the binding free energy, i.e., as a sum of the interaction enthalpy, interaction entropy and the corrections for the inhibitor desolvation and deformation energies. The applied scoring function contains a clear thermodynamical terms and does not involve any adjustable empirical parameter. The best correlations with the experimental inhibition constants (ln K (i)) were found for bare interaction enthalpy (r (2) = 0.87) and interaction enthalpy corrected for ligand desolvation and deformation energies (r (2) = 0.77); when the entropic term was considered, however, the correlation becomes worse but still acceptable (r (2) = 0.52). The resulting correlation based on the PM6-DH2 scoring function is better than previously published function based on various docking/scoring, SAR studies or advanced QM/MM approach, however, the robustness is limited by number of available experimental data used in the correlation. Since a very similar correlation between the experimental and theoretical results was found also for a different system of the HIV-1 protease, the suggested scoring function based on the PM6-DH2 method seems to be applicable in drug design, even if diverse protein-ligand complexes have to be ranked. PMID:21286784

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

    PubMed

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

    2009-01-01

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

  11. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor.

    PubMed

    Christensen, Camilla L; Kwiatkowski, Nicholas; Abraham, Brian J; Carretero, Julian; Al-Shahrour, Fatima; Zhang, Tinghu; Chipumuro, Edmond; Herter-Sprie, Grit S; Akbay, Esra A; Altabef, Abigail; Zhang, Jianming; Shimamura, Takeshi; Capelletti, Marzia; Reibel, Jakob B; Cavanaugh, Jillian D; Gao, Peng; Liu, Yan; Michaelsen, Signe R; Poulsen, Hans S; Aref, Amir R; Barbie, David A; Bradner, James E; George, Rani E; Gray, Nathanael S; Young, Richard A; Wong, Kwok-Kin

    2014-12-01

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy. PMID:25490451

  12. A CDK4/6 inhibitor enhances cytotoxicity of paclitaxel in lung adenocarcinoma cells harboring mutant KRAS as well as wild-type KRAS.

    PubMed

    Zhang, Xiang-Hua; Cheng, Ying; Shin, Jung-Young; Kim, Jeong-Oh; Oh, Ji-Eun; Kang, Jin-Hyoung

    2013-07-01

    The KRAS gain-of-function mutation confers intrinsic resistance to targeted anti-cancer drugs and cytotoxic chemotherapeutic agents, ultimately leading to treatment failure. KRAS mutation frequency in lung adenocarcinoma is ~15-30%. Novel therapeutic strategies should be developed to improve clinical outcomes in these cases. Deregulation of the p16/cyclin-dependent kinase (CDK) 4/retinoblastoma (Rb) pathway is frequently observed in various cancers and it represents an attractive therapeutic target. We compared the anti-tumor efficacy of genetically knocked-down CDK4 and a pharmacological inhibitor of CDK4/6, CINK4, in KRAS mutation-positive lung adenocarcinoma cells. We also investigated changes in anti-proliferative activity and downstream molecules with these treatments in combination with paclitaxel. CDK4 short interfering RNA (siRNA) significantly increased paclitaxel sensitivity in KRAS mutation-positive H23 cells. CINK4 demonstrated concentration- and time-dependent anti-proliferative activity in 5 adenocarcinoma lines. CINK4 induced G 1 arrest by downregulating the p16/cyclin D1/Rb pathway, resulting in apoptotic induction via increased expression of cleaved caspase3, cleaved PARP and Bax. Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells. These data suggest CDK4 is a promising target for development of anti-cancer drugs and CINK4 combined with paclitaxel may be an effective therapeutic strategy for enhancing anti-tumor efficacy in KRAS mutation-positive lung adenocarcinoma. PMID:23792647

  13. PP2A inhibitors arrest G2/M transition through JNK/Sp1-dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21

    PubMed Central

    Zhi, Qiaoming; Xu, Ze-Kuan; Wang, Rong; Wang, Wen-Jie; Zong, Yang; Li, Zeng-Liang; Wu, Yadi; Zhou, Binhua P.; Chen, Kai; Tao, Min; Li, Wei

    2015-01-01

    Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors. PMID:26053095

  14. Chromium-VI arrests cell cycle and decreases granulosa cell proliferation by down-regulating cyclin-dependent kinases (CDK) and cyclins and up-regulating CDK-Inhibitors

    PubMed Central

    Stanley, Jone A.; Lee, JeHoon; Nithy, Thamizh K.; Arosh, Joe A.; Burghardt, Robert C.; Banu, Sakhila K.

    2011-01-01

    Environmental contamination with hexavalent chromium (CrVI) has been increasing in the drinking water of the USA and developing countries. CrVI causes various health problems including menstrual disorders and infertility. Recently, we reported that CrVI causes granulosa cell apoptosis through the intrinsic apoptotic pathway. Our previous studies showed that postnatal exposure to CrVI arrests follicle development. In order to explore the underlying mechanism, primary and immortalized granulosa cells from rats were treated with 10 μM potassium dichromate and analyses of the cell cycle, and cell cycle regulatory proteins were performed. CrVI decreased cell proliferation as a result of cell cycle arrest and down-regulated cyclin-dependent kinases (CDK), cyclins, and PCNA while up-regulating CDK-inhibitors and down-regulating FSH receptor and ERβ. Vitamin C mitigated the effects of CrVI. This study shows that CrVI causes cell cycle arrest in granulosa cells by altering cell cycle regulatory proteins with potential intervention by vitamin C. PMID:21621607

  15. Conformation and recognition of DNA damaged by antitumor cis-dichlorido platinum(II) complex of CDK inhibitor bohemine.

    PubMed

    Novakova, Olga; Liskova, Barbora; Vystrcilova, Jana; Suchankova, Tereza; Vrana, Oldrich; Starha, Pavel; Travnicek, Zdenek; Brabec, Viktor

    2014-05-01

    A substitution of the ammine ligands of cisplatin, cis-[Pt(NH3)2Cl2], for cyclin dependent kinase (CDK) inhibitor bohemine (boh), [2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine], results in a compound, cis-[Pt(boh)2Cl2] (C1), with the unique anticancer profile which may be associated with some features of the damaged DNA and/or its cellular processing (Travnicek Z et al. (2003) J Inorg Biochem94, 307-316; Liskova B (2012) Chem Res Toxicol25, 500-509). A combination of biochemical and molecular biology techniques was used to establish mechanistic differences between cisplatin and C1 with respect to the DNA damage they produce and their interactions with critical DNA-binding proteins, DNA-processing enzymes and glutathione. The results show that replacement of the NH3 groups in cisplatin by bohemine modulates some aspects of the mechanism of action of C1. More specifically, the results of the present work are consistent with the thesis that, in comparison with cisplatin, effects of other factors, such as: (i) slower rate of initial binding of C1 to DNA; (ii) the lower efficiency of C1 to form bifunctional adducts; (iii) the reduced bend of longitudinal DNA axis induced by the major 1,2-GG intrastrand cross-link of C1; (iv) the reduced affinity of HMG domain proteins to the major adduct of C1; (v) the enhanced efficiency of the DNA adducts of C1 to block DNA polymerization and to inhibit transcription activity of human RNA pol II and RNA transcription; (vi) slower rate of the reaction of C1 with glutathione, may partially contribute to the unique activity of C1. PMID:24675180

  16. Novel inhibitors of nuclear transport cause cell cycle arrest and decrease cyst growth in ADPKD associated with decreased CDK4 levels.

    PubMed

    Tan, Matthew; Wettersten, Hiromi I; Chu, Kristy; Huso, David L; Watnick, Terry; Friedlander, Sharon; Landesman, Yosef; Weiss, Robert H

    2014-12-01

    Autosomal-dominant polycystic kidney disease (ADPKD) is a progressive, proliferative renal disease. Kidneys from ADPKD patients are characterized by the presence of cysts that are marked by enhanced proliferation and apoptosis of renal tubular epithelial cells. Current treatment of this disease is supportive, as there are few if any clinically validated targeted therapeutics. Given the parallels between cystic disease and cancer, and in light of our findings of the efficacy of the nuclear transport inhibitors in kidney cancer, which has similarities to ADPKD, we asked whether such inhibitors show utility in ADPKD. In this study, we tested selective inhibitors of nuclear export (SINE) in two human ADPKD cell lines and in an in vivo mouse model of ADPKD. After effective downregulation of a nuclear exporter, exportin 1 (XPO1), with KPT-330, both cell lines showed dose-dependent inhibition of cell proliferation through G₀/G₁ arrest associated with downregulation of CDK4, with minimal apoptosis. To analyze mechanisms of CDK4 decrease by XPO1 inhibition, localization of various XPO1 target proteins was examined, and C/EBPβ was found to be localized in the nucleus by XPO1 inhibition, resulting in an increase of C/EBPα, which activates degradation of CDK4. Furthermore, inhibition of XPO1 with the parallel inhibitor KPT-335 attenuated cyst growth in vivo in the PKD1 mutant mouse model Pkd1(v/v). Thus, inhibition of nuclear export by KPT-330, which has shown no adverse effects in renal serum chemistries and urinalyses in animal models, and which is already in phase 1 trials for cancers, will be rapidly translatable to human ADPKD. PMID:25234309

  17. Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity

    PubMed Central

    Bajrami, Ilirjana; Frankum, Jessica R.; Konde, Asha; Miller, Rowan E.; Rehman, Farah L.; Brough, Rachel; Campbell, James; Sims, David; Rafiq, Rumana; Hooper, Sean; Chen, Lina; Kozarewa, Iwanka; Assiotis, Ioannis; Fenwick, Kerry; Natrajan, Rachael; Lord, Christopher J.; Ashworth, Alan

    2016-01-01

    Small molecule inhibitors of PARP1/2 such as olaparib have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials PARP1/2 inhibitors elicit sustained anti-tumor responses in patients with germ-line BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51 and Fanconi’s anemia susceptibility genes. Additionally, the set included genes implicated in established networks of DNA repair, DNA cohesion and chromatin remodelling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination and reduced expression of BRCA1. As one of only nine genes known to be mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its utility as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest. PMID:24240700

  18. The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia.

    PubMed

    Baker, Adele; Gregory, Gareth P; Verbrugge, Inge; Kats, Lev; Hilton, Joshua J; Vidacs, Eva; Lee, Erwin M; Lock, Richard B; Zuber, Johannes; Shortt, Jake; Johnstone, Ricky W

    2016-03-01

    Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins. PMID:26627013

  19. Down-regulation of BNIP3 by olomoucine, a CDK inhibitor, reduces LPS- and NO-induced cell death in BV2 microglial cells.

    PubMed

    Tsou, Yu-Chi; Wang, Hsiao-Hsien; Hsieh, Chii-Cheng; Sun, Kuang-Hui; Sun, Guang-Huan; Jhou, Ren-Shiang; Lin, Tz-I; Lu, Shou-Yun; Liu, Huan-Yun; Tang, Shye-Jye

    2016-08-15

    Proinflammatory responses eliciting the microglial production of cytokines and nitric oxide (NO) have been reported to play a crucial role in the acute and chronic pathogenic effects of neurodegeneration. Chemical inhibitors of cyclin-dependent kinases (CDKs) may prevent the progression of neurodegeneration by both limiting cell proliferation and reducing cell death. However, the mechanism underlying the protective effect of CDK inhibitors on microglia remains unexplored. In this study, we found that olomoucine, a CDK inhibitor, alleviated lipopolysaccharide (LPS)-induced BV2 microglial cell death by reducing the generation of NO and inhibiting the gene expression of proinflammatory cytokines. In addition, olomoucine reduced inducible NO synthase promoter activity and alleviated NF-κB- and E2F-mediated transcriptional activation. NO-induced cell death involved mitochondrial disruptions such as cytochrome c release and loss of mitochondrial membrane potential, and pretreatment with olomoucine prior to NO exposure reduced these disruptions. Microarray analysis revealed that olomoucine treatment induced prominent down-regulation of Bcl2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-apoptotic Bcl-2 family protein that is involved in mitochondrial disruption. As BNIP3 knock-down significantly increased the viability of LPS- and NO-treated BV2 cells, we conclude that olomoucine may protect cells by limiting proinflammatory responses, thereby reducing NO generation. Simultaneously, down-regulation of BNIP3 prevents NO stimulation from inducing mitochondrial disruption. PMID:27345388

  20. Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing.

    PubMed

    Brasca, Maria Gabriella; Albanese, Clara; Alzani, Rachele; Amici, Raffaella; Avanzi, Nilla; Ballinari, Dario; Bischoff, James; Borghi, Daniela; Casale, Elena; Croci, Valter; Fiorentini, Francesco; Isacchi, Antonella; Mercurio, Ciro; Nesi, Marcella; Orsini, Paolo; Pastori, Wilma; Pesenti, Enrico; Pevarello, Paolo; Roussel, Patrick; Varasi, Mario; Volpi, Daniele; Vulpetti, Anna; Ciomei, Marina

    2010-03-01

    We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors. PMID:20153204

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

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

    PubMed Central

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

    2015-01-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1–Cks1 and CDK1–cyclin B–Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodeled by cyclin binding. Relative to CDK2–cyclin A, CDK1–cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation, and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors. PMID:25864384

  3. Cellular response to antitumor cis-Dichlorido platinum(II) complexes of CDK inhibitor Bohemine and its analogues.

    PubMed

    Liskova, Barbora; Zerzankova, Lenka; Novakova, Olga; Kostrhunova, Hana; Travnicek, Zdenek; Brabec, Viktor

    2012-02-20

    The cellular and molecular pharmacology of the new class of anticancer drugs, in which the CDK inhibitor bohemine and its analogues are coordinated to Pt(II) to form cisplatin derivatives, was investigated. The results revealed the unique anticancer profile of a cisplatin-derived platinum(II) dichlorido complex involving N(7)-coordinated bohemine (C1). Although the IC(50) values were ∼6-fold higher for C1 than for cisplatin in cisplatin-sensitive tumor cells, the tumor cells in which C1 was also active are those which acquired resistance to cisplatin. In addition, among the novel conjugates of bohemine and its analogues with cisplatin, marked selectivity of C1 for tumor cells relative to the nontumorigenic, normal cells was observed. However, coordination of bohemine to platinum in C1 considerably reduced one of the dual functionalities anticipated to be effective after C1 reaches the nucleus. Further studies performed in the cells with wt p53 status show differences between cisplatin and C1 at the level of cell cycle regulation. Impedance-based real-time monitoring of the effects of C1 and cisplatin on cell growth supported the thesis that critical differences exist in the rate and mechanisms of cell kill caused by the two agents and that C1 was a more potent inducer of apoptosis and/or necrosis than cisplatin. The results also showed that the distinct differences in cell killing observed for C1 and cisplatin might be associated with processes at the DNA level. The DNA binding experiments carried out in a cell-free medium demonstrated that modification reactions resulting in the irreversible coordination of C1 to DNA were slower than that of cisplatin. Transcription mapping experiments and determination of interstrand cross-linking efficiency of C1 suggested that several aspects of DNA binding mode of C1 and cisplatin were similar. It was concluded that C1 remains a promising prototype of compounds for the generation of novel drug candidates with cytotoxicity

  4. mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells.

    PubMed

    Berrak, Ozge; Arisan, Elif Damla; Obakan-Yerlikaya, Pinar; Coker-Gürkan, Ajda; Palavan-Unsal, Narçin

    2016-10-01

    Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN(-/-) LNCaP and androgen independent (AR-), PTEN(+/-) DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (-) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent

  5. Expression and Purification of Recombinant CDKs: CDK7, CDK8, and CDK9.

    PubMed

    Pinhero, Reena; Yankulov, Krassimir

    2016-01-01

    Cyclin-dependent kinases have established roles in the regulation of cell cycle, in gene expression and in cell differentiation. Many of these kinases have been considered as drug targets and numerous efforts have been made to develop specific and potent inhibitors against them. The first step in all of these attempts and in many other biochemical analyses is the production of highly purified and reliable kinase, most frequently in a recombinant form. In this chapter we describe our experience in the cloning, expression, and purification of CDKs using CDK7/CycH, CDK8/CycC, and CDK9/CycT1 as an example. PMID:26231705

  6. Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4(+) T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb.

    PubMed

    Jamaluddin, Md Saha; Hu, Pei-Wen; Jan, Yih; Siwak, Edward B; Rice, Andrew P

    2016-02-01

    Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4(+) T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4(+) cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4(+) T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4(+) T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation. PMID:26727990

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

    PubMed

    Li, Yanxin; Pal, Rekha; Sung, Li-Ying; Feng, Haizhong; Miao, Weimin; Cheng, Shi-Yuan; Tian, Cindy; Cheng, Tao

    2012-01-01

    Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18(INK4c), is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells. PMID:23049777

  8. Synergistic effects of dexamethasone and genistein on the expression of Cdk inhibitor p21WAF1/CIP1 in human hepatocellular and colorectal carcinoma cells.

    PubMed

    Park, J H; Oh, E J; Choi, Y H; Kang, C D; Kang, H S; Kim, D K; Kang, K I; Yoo, M A

    2001-05-01

    Previous studies have shown that dexamethasone, a synthetic glucocorticoid, can induce a G1 arrest, however, genistein, a natural isoflavonoid phytoestrogen, induces a G2/M arrest in the cell cycle progression in various cancer cell lines. A block of cell cycle checkpoint by dexamethasone and genistein correlates with a selective induction of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 in a tumor suppressor p53-independent manner and abolishment of Cdk2 phosphorylation. In the present study, the effects of dexamethasone and genistein (both singly and combined) on the expression of p21 in human hepatocellular Hep G2 and colorectal Colo320 HSR carcinoma cells were evaluated. Whereas dexamethasone mildly induced the level of p21 protein, genistein strongly increased the expression of p21 protein in our experimental condition. Both compounds also activated p21 promoter reporter constructs. The combined effects of dexamethasone and genistein on the induction of p21 protein and activation of p21 promoter were synergistic in both cell lines. These findings indicate that dexamethasone and genistein act in a synergistic fashion and have potential for combination chemotherapy for the treatment of liver and colon cancer. PMID:11295047

  9. Marine steroids as potential anticancer drug candidates: In silico investigation in search of inhibitors of Bcl-2 and CDK-4/Cyclin D1.

    PubMed

    Saikia, Surovi; Kolita, Bhaskor; Dutta, Partha P; Dutta, Deep J; Neipihoi; Nath, Shyamalendu; Bordoloi, Manobjyoti; Quan, Pham Minh; Thuy, Tran Thu; Phuong, Doan Lan; Long, Pham Quoc

    2015-10-01

    Star fishes (Asteroidea) are rich in polar steroids with diverse structural characteristics. The structural modifications of star fish steroids occur at 3β, 4β, 5α, 6α (or β), 7α (or β), 8, 15α (or β) and 16β positions of the steroidal nucleus and in the side chain. Widely found polar steroids in starfishes include polyhydroxysteroids, steroidal sulfates, glycosides, steroid oligoglycosides etc. Bioactivity of these steroids is less studied; only a few reports like antibacterial, cytotoxic activity etc. are available. In continuation of our search for bioactive molecules from natural sources, we undertook in silico screening of steroids from star fishes against Bcl-2 and CDK-4/Cyclin D1 - two important targets of progression and proliferation of cancer cells. We have screened 182 natural steroids from star fishes occurring in different parts of the world and their 282 soft-derivatives by in silico methods. Their physico-chemical properties, drug-likeliness, binding potential with the selected targets, ADMET (absorption, distribution, metabolism, toxicity) were predicted. Further, the results were compared with those of existing steroidal and non steroidal drugs and inhibitors of Bcl-2 and CDK-4/Cyclin D1. The results are promising and unveil that some of these steroids can be potent leads for cancer treatments. PMID:26111591

  10. Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine.

    PubMed

    Gelbert, Lawrence M; Cai, Shufen; Lin, Xi; Sanchez-Martinez, Concepcion; Del Prado, Miriam; Lallena, Maria Jose; Torres, Raquel; Ajamie, Rose T; Wishart, Graham N; Flack, Robert Steven; Neubauer, Blake Lee; Young, Jamie; Chan, Edward M; Iversen, Philip; Cronier, Damien; Kreklau, Emiko; de Dios, Alfonso

    2014-10-01

    The G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development. PMID:24919854

  11. SUMO-specific protease 1 regulates the in vitro and in vivo growth of colon cancer cells with the upregulated expression of CDK inhibitors.

    PubMed

    Xu, Ying; Li, Jie; Zuo, Yong; Deng, Jiong; Wang, Li-Shun; Chen, Guo-Qiang

    2011-10-01

    SUMO conjugation emerges as an important mechanism in regulating protein localization, stability and activity. SUMOylation is a dynamic process and can be reversed by a family of sentrin/SUMO-specific proteases (SENPs). However, the biological roles of SENPs in cellular processes are largely unknown. Here, we show that SENP1, a member of SENP family, is overexpressed in most of colon cancer tissues. Silencing of SENP1 expression inhibits cell growth with G(1) arrest in vitro and in nude mice and colony formation in colon cancer cell line DLD-1, suggesting that SENP1 is essential for cell growth in the colon cancer cell line. Accordingly, silencing of SENP1 results in upregulation of CDK inhibitors such as p16, p19, p21 and p27. These results suggest that SENP1 might play a role in cell cycle regulation of colon cancer cells. PMID:21669491

  12. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

    PubMed

    Young, Richard J; Waldeck, Kelly; Martin, Claire; Foo, Jung H; Cameron, Donald P; Kirby, Laura; Do, Hongdo; Mitchell, Catherine; Cullinane, Carleen; Liu, Wendy; Fox, Stephen B; Dutton-Regester, Ken; Hayward, Nicholas K; Jene, Nicholas; Dobrovic, Alexander; Pearson, Richard B; Christensen, James G; Randolph, Sophia; McArthur, Grant A; Sheppard, Karen E

    2014-07-01

    We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance. PMID:24495407

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

    PubMed

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

    2004-06-01

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

  14. Phase I and Pharmacologic Study of SNS-032, a Potent and Selective Cdk2, 7, and 9 Inhibitor, in Patients With Advanced Chronic Lymphocytic Leukemia and Multiple Myeloma

    PubMed Central

    Tong, Wei-Gang; Chen, Rong; Plunkett, William; Siegel, David; Sinha, Rajni; Harvey, R. Donald; Badros, Ashraf Z.; Popplewell, Leslie; Coutre, Steven; Fox, Judith A.; Mahadocon, Kristi; Chen, Tianling; Kegley, Peggy; Hoch, Ute; Wierda, William G.

    2010-01-01

    Purpose SNS-032 is a highly selective and potent inhibitor of cyclin-dependent kinases (Cdks) 2, 7, and 9, with in vitro growth inhibitory effects and ability to induce apoptosis in malignant B cells. A phase I dose-escalation study of SNS-032 was conducted to evaluate safety, pharmacokinetics, biomarkers of mechanism-based pharmacodynamic (PD) activity, and clinical efficacy. Patients and Methods Parallel cohorts of previously treated patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) received SNS-032 as a loading dose followed by 6-hour infusion weekly for 3 weeks of each 4-week course. Results There were 19 patients with CLL and 18 with MM treated. Tumor lysis syndrome was the dose-limiting toxicity (DLT) for CLL, the maximum-tolerated dose (MTD) was 75 mg/m2, and the most frequent grade 3 to 4 toxicity was myelosuppression. One patient with CLL had more than 50% reduction in measurable disease without improvement in hematologic parameters. Another patient with low tumor burden had stable disease for four courses. For patients with MM, no DLT was observed and MTD was not identified at up to 75 mg/m2, owing to early study closure. Two patients with MM had stable disease and one had normalization of spleen size with treatment. Biomarker analyses demonstrated mechanism-based PD activity with inhibition of Cdk7 and Cdk9, decreases in Mcl-1 and XIAP expression level, and associated CLL cell apoptosis. Conclusion SNS-032 demonstrated mechanism-based target modulation and limited clinical activity in heavily pretreated patients with CLL and MM. Further single-agent, PD-based, dose and schedule modification is warranted to maximize clinical efficacy. PMID:20479412

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

    PubMed

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

    2016-03-01

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

  16. 3D-QSAR and 3D-QSSR studies of thieno[2,3-d]pyrimidin-4-yl hydrazone analogues as CDK4 inhibitors by CoMFA analysis

    PubMed Central

    Cai, Bao-qin; Jin, Hai-xiao; Yan, Xiao-jun; Zhu, Peng; Hu, Gui-xiang

    2014-01-01

    Aim: To investigate the structural basis underlying potency and selectivity of a series of novel analogues of thieno[2,3-d]pyrimidin-4-yl hydrazones as cyclin-dependent kinase 4 (CDK4) inhibitors and to use this information for drug design strategies. Methods: Three-dimensional quantitative structure-activity relationship (3D-QSAR) and three-dimensional quantitative structure-selectivity relationship (3D-QSSR) models using comparative molecular field analysis (CoMFA) were conducted on a training set of 48 compounds. Partial least squares (PLS) analysis was employed. External validation was performed with a test set of 9 compounds. Results: The obtained 3D-QSAR model (q2=0.724, r2=0.965, r2pred=0.945) and 3D-QSSR model (q2=0.742, r2=0.923, r2pred=0.863) were robust and predictive. Contour maps with good compatibility to active binding sites provided insight into the potentially important structural features required to enhance activity and selectivity. The contour maps indicated that bulky groups at R1 position could potentially enhance CDK4 inhibitory activity, whereas bulky groups at R3 position have the opposite effect. Appropriate incorporation of bulky electropositive groups at R4 position is favorable and could improve both potency and selectivity to CDK4. Conclusion: These two models provide useful information to guide drug design strategies aimed at obtaining potent and selective CDK4 inhibitors. PMID:24122012

  17. BREED: Generating novel inhibitors through hybridization of known ligands. Application to CDK2, p38, and HIV protease.

    PubMed

    Pierce, Albert C; Rao, Govinda; Bemis, Guy W

    2004-05-20

    In this work we describe BREED, a method for the generation of novel inhibitors from structures of known ligands bound to a common target. The method is essentially an automation of the common medicinal chemistry practice of joining fragments of two known ligands to generate a new inhibitor. The ligand-bound target structures are overlaid, all overlapping bonds in all pairs of ligands are found, and the fragments on each side of each matching bond are swapped to generate the new molecules. Since the method is automated, it can be applied recursively to generate all possible combinations of known ligands. In an application of this method to HIV protease inhibitors and protein kinase inhibitors, hundreds of new molecular structures were generated. These included known inhibitor scaffolds not included in the initial set, entirely novel scaffolds, and novel substituents on known scaffolds. The method is fast, and since all of the ligand functional groups are known to bind the target in the precise position and orientation present in the novel ligand, the success rate of this method should be superior to more traditional de novo design techniques. In an era of increasingly high-throughput structural biology, such methods for high-throughput utilization of structural information will become increasingly valuable. PMID:15139755

  18. Preclinical Characterization of G1T28: A Novel CDK4/6 Inhibitor for Reduction of Chemotherapy-Induced Myelosuppression.

    PubMed

    Bisi, John E; Sorrentino, Jessica A; Roberts, Patrick J; Tavares, Francis X; Strum, Jay C

    2016-05-01

    Chemotherapy-induced myelosuppression continues to represent the major dose-limiting toxicity of cytotoxic chemotherapy, which can be manifested as neutropenia, lymphopenia, anemia, and thrombocytopenia. As such, myelosuppression is the source of many of the adverse side effects of cancer treatment including infection, sepsis, bleeding, and fatigue, thus resulting in the need for hospitalizations, hematopoietic growth factor support, and transfusions (red blood cells and/or platelets). Moreover, clinical concerns raised by myelosuppression commonly lead to chemotherapy dose reductions, therefore limiting therapeutic dose intensity, and reducing the antitumor effectiveness of the treatment. Currently, the only course of treatment for myelosuppression is growth factor support which is suboptimal. These treatments are lineage specific, do not protect the bone marrow from the chemotherapy-inducing cytotoxic effects, and the safety and toxicity of each agent is extremely specific. Here, we describe the preclinical development of G1T28, a novel potent and selective CDK4/6 inhibitor that transiently and reversibly regulates the proliferation of murine and canine bone marrow hematopoietic stem and progenitor cells and provides multilineage protection from the hematologic toxicity of chemotherapy. Furthermore, G1T28 does not decrease the efficacy of cytotoxic chemotherapy on RB1-deficient tumors. G1T28 is currently in clinical development for the reduction of chemotherapy-induced myelosuppression in first- and second-line treatment of small-cell lung cancer. Mol Cancer Ther; 15(5); 783-93. ©2016 AACR. PMID:26826116

  19. Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

    PubMed

    Mushtaq, Gohar; Greig, Nigel H; Anwar, Firoz; Al-Abbasi, Fahad A; Zamzami, Mazin A; Al-Talhi, Hasan A; Kamal, Mohammad A

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  20. Neuroprotective Mechanisms Mediated by CDK5 Inhibition

    PubMed Central

    Mushtaq, Gohar; Greig, Nigel H.; Anwar, Firoz; Al-Abbasi, Fahad A.; Zamzami, Mazin A.; Al-Talhi, Hasan A.; Kamal, Mohammad A.

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not well-understood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine-induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  1. Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4.

    PubMed

    Ding, Q M; Ko, T C; Evers, B M

    1998-11-01

    The cellular mechanisms regulating intestinal proliferation and differentiation remain largely undefined. Previously, we showed an early induction of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1) in Caco-2 cells, a human colon cancer line that spontaneously differentiates into a small bowel phenotype. The purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation in Caco-2 cells and to examine the mechanisms responsible for CDK inactivation. Caco-2 cells undergo a relative G1/S block and cease to proliferate at day 3 postconfluency; an increase in the activity of terminally differentiated brush-border enzymes (sucrase and alkaline phosphatase) was noted at day 6 postconfluency. Cell cycle block was associated with suppression of both CDK2 and CDK4 activities, which are important for G1/S progression. Treatment of the CDK immune complexes with the detergent deoxycholate (DOC) resulted in restoration of CDK2, but not CDK4, activity at day 3 postconfluency, suggesting the presence of inhibitory protein(s) binding to the cyclin/CDK2 complex at this time point. An increased binding of p21(Waf1/Cip1) to CDK2 complexes at day 3 postconfluency was noted, suggesting a potential role for p21(Waf1/Cip1) in CDK2 inactivation; however, immunodepletion of p21(Waf1/Cip1) from Caco-2 protein extracts demonstrated that p21(Waf1/Cip1) is only partially responsible for CDK2 suppression at day 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears to contribute to the CDK2 inactivation noted at days 6 and 12 postconfluency. Taken together, our results suggest that multiple mechanisms contribute to CDK suppression during Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leads to G1 arrest and inhibition of proliferation that precede Caco-2 cell differentiation. PMID:9814966

  2. Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1

    SciTech Connect

    Yu, Eun-Young; Lee, Ju-Hee; Kang, Won-Hwa; Park, Yun-Hee; Kim, Lila; Park, Hee-Moon

    2013-03-01

    Highlights: ► Deletion of lkh1{sup +} made cells pass the G1/S phase faster than the wild type. ► Lkh1 can interact with a cyclin-dependent kinase inhibitor (CKI) Rum1. ► Lkh1 can phosphorylate Rum1 to activate its CKI activity. ► Thr110 was confirmed as the Lkh1-dependent phosphorylation site of Rum1. ► Positive acting mechanism for the Rum1 activation is reported for the first time. - Abstract: In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1{sup T110A} confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

  3. CDK9 inhibitors selectively target estrogen receptor-positive breast cancer cells through combined inhibition of MYB and MCL-1 expression

    PubMed Central

    Mitra, Partha; Yang, Ren-Ming; Sutton, James; Ramsay, Robert G.; Gonda, Thomas J.

    2016-01-01

    Our previous studies showed that MYB is required for proliferation of, and confers protection against apoptosis on, estrogen receptor-positive (ER+ve) breast cancer cells, which are almost invariably also MYB+ve. We have also shown that MYB expression in ER+ve breast cancer cells is regulated at the level of transcriptional elongation and as such, is suppressed by CDK9i. Here we examined the effects of CDK9i on breast cancer cells and the involvement of MYB in these effects. ER+ve breast cancer cell lines including MCF-7 were much more sensitive (> 10 times) to killing by CDK9i than ER−ve/MYB−ve cells. Moreover, surviving cells showed a block at the G2/M phase of the cell cycle. Importantly, ectopic MYB expression conferred resistance to apoptosis induction, cell killing and G2/M accumulation. Expression of relevant MYB target genes including BCL2 and CCNB1 was suppressed by CDK9 inhibition, and this too was reversed by ectopic MYB expression. Nevertheless, inhibition of BCL2 alone either by MYB knockdown or by ABT-199 treatment was insufficient for significant induction of apoptosis. Further studies implied that suppression of MCL-1, a well-documented target of CDK9 inhibition, was additionally required for apoptosis induction, while maximal levels of apoptosis induced by CDK9i are likely to also involve inhibition of BCL2L1 expression. Taken together these data suggest that MYB regulation of BCL2 underlies the heightened sensitivity of ER+ve compared to ER−ve breast cancer cells to CDK9 inhibition, and that these compounds represent a potential therapeutic for ER+ve breast cancers and possibly other MYB-dependent cancers. PMID:26812885

  4. Insights into the structural basis of 3,5-diaminoindazoles as CDK2 inhibitors: prediction of binding modes and potency by QM-MM interaction, MESP and MD simulation.

    PubMed

    Tripathi, Sunil Kumar; Singh, Sanjeev Kumar

    2014-08-01

    The novel 3,5-diaminoindazole derivatives are well-known as potent and anti-proliferative cyclin-dependent kinase 2 inhibitors. We report a combined quantum mechanics/molecular mechanics study to determine the protein-ligand interaction energy, and some quantum chemical descriptors to successfully rank these inhibitors. The results in this work show that the QM-MM interaction energy is strongly correlated to the biological activity and can be used as a predictor, which was further validated by Spearman's rank correlation coefficient. An exhaustive analysis of the protein-ligand structures obtained from molecular dynamics simulations shows specific interactions within the active site. Furthermore, the docking study was supported by electronic property analysis using density functional theory at the B3LYP/3-21*G level. The results obtained from molecular docking and surface analysis shed some insight on steric and electronic complementarities of these molecules to CDK2. Aqueous solvation energy values give an indication of the solubility and can be used as a guide for the pharmacokinetic optimization of these molecules. Furthermore, ADME/T properties calculated are in the desirable range, so these compounds are predicted to be drug like with low toxicity potential. Overall, the approach was successful in the cases considered, and it could be useful for the design of inhibitors in the lead optimization phase of drug discovery against CDK2. PMID:24909777

  5. Targeting CDK4/6 in patients with cancer.

    PubMed

    Hamilton, Erika; Infante, Jeffrey R

    2016-04-01

    The cyclin D-cyclin dependent kinase (CDK) 4/6-inhibitor of CDK4 (INK4)-retinoblastoma (Rb) pathway controls cell cycle progression by regulating the G1-S checkpoint. Dysregulation of the cyclin D-CDK4/6-INK4-Rb pathway results in increased proliferation, and is frequently observed in many types of cancer. Pathway activation can occur through a variety of mechanisms, including gene amplification or rearrangement, loss of negative regulators, epigenetic alterations, and point mutations in key pathway components. Due to the importance of CDK4/6 activity in cancer cells, CDK4/6 inhibitors have emerged as promising candidates for cancer treatment. Moreover, combination of a CDK4/6 inhibitor with other targeted therapies may help overcome acquired or de novo treatment resistance. Ongoing studies include combinations of CDK4/6 inhibitors with endocrine therapy and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors for hormone receptor-positive (HR+) breast cancers, and with selective RAF and MEK inhibitors for tumors with alterations in the mitogen activated protein kinase (MAPK) pathway such as melanoma. In particular, the combination of CDK4/6 inhibitors with endocrine therapy, such as palbociclib's recent first-line approval in combination with letrozole, is expected to transform the treatment of HR+ breast cancer. Currently, three selective CDK4/6 inhibitors have been approved or are in late-stage development: palbociclib (PD-0332991), ribociclib (LEE011), and abemaciclib (LY2835219). Here we describe the current preclinical and clinical data for these novel agents and discuss combination strategies with other agents for the treatment of cancer. PMID:27017286

  6. Dual CDK4/CDK6 Inhibition Induces Cell Cycle Arrest and Senescence in Neuroblastoma

    PubMed Central

    Rader, JulieAnn; Russell, Mike R.; Hart, Lori S.; Nakazawa, Michael S.; Belcastro, Lili T.; Martinez, Daniel; Li, Yimei; Carpenter, Erica L.; Attiyeh, Edward F.; Diskin, Sharon J.; Kim, Sunkyu; Parasuraman, Sudha; Caponigro, Giordano; Schnepp, Robert W.; Wood, Andrew C.; Pawel, Bruce; Cole, Kristina A.; Maris, John M.

    2013-01-01

    Purpose Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. Experimental Procedures We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011, a highly specific CDK4/6 inhibitor. Results Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nM in sensitive lines). LEE011 caused cell cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p = 0.01), the identification of additional clinically accessible biomarkers is of high importance. Conclusions Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. PMID:24045179

  7. CDK8 kinase--An emerging target in targeted cancer therapy.

    PubMed

    Rzymski, Tomasz; Mikula, Michał; Wiklik, Katarzyna; Brzózka, Krzysztof

    2015-10-01

    Cyclin-dependent kinase (CDK) inhibitors have been developed as potential anticancer therapeutics and several nonselective compounds are currently in advanced clinical trials. This review is focused on the key biological roles of CDK8 kinase, which provide a proof-of-principle for continued efforts toward effective cancer treatment, targeting activity of this CDK family member. Among currently identified kinase inhibitors, several displayed significant selectivity for CDK8 and notably the effectiveness in targeting cancer specific gene expression programs. Structural features of CDK8 and available ligands were discussed from a perspective of the rational drug design process. Current state of the art confirms that further development of CDK8 inhibitors will translate into targeted therapies in oncology. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases. PMID:26006748

  8. Design, synthesis and biological evaluation of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors.

    PubMed

    Ganga Reddy, V; Srinivasa Reddy, T; Lakshma Nayak, V; Prasad, Budaganaboyina; Reddy, Adiyala Praveen; Ravikumar, A; Taj, Shaik; Kamal, Ahmed

    2016-10-21

    A series of new (N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives (8-35) were designed, synthesized and evaluated as CDK1/Cdc2 inhibitors. Biological evaluation assays indicated that compounds 16 and 27 showed the most potent growth inhibitory activity against human cancer cell lines (MIAPaCa-2, MCF-7 and HeLa) with GI50 values ranging from 0.13 to 0.7 μM, compared with the positive control nocodazole (0.81-0.95 μM). Flow cytometric analysis revealed that these compounds induce cell cycle arrest in the G2/M phase and Western blot analysis suggested that compound treatment resulted in reduction of CDK1 expression levels in MCF-7 cell line. Moreover, the apoptosis inducing effect of the compounds was studied using Hoechst staining, Rhodamine 123 staining (MMP), carboxy-DCFDA staining (ROS), Annexin V-FITC assay. Based on these studies, two compounds 16 and 27 have been identified as promising new molecules that have the potential to be developed as leads. PMID:27344493

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

    PubMed

    Wang, HaiYang; Kim, Nam-Hyung

    2016-08-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  13. Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach.

    PubMed

    Tripathi, Sunil Kumar; Singh, Sanjeev Kumar; Singh, Poonam; Chellaperumal, Palanisamy; Reddy, Karnati Konda; Selvaraj, Chandrabose

    2012-10-01

    Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508-8522) have found a ligand BS194 that has a high affinity with CDK2 (IC(50) = 3 nM) but shows low affinity with CDK1 (IC(50) = 30 nM). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free-energy calculation to analyze the interactions. A rational three-dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non-polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1. PMID:22996593

  14. Design, synthesis and biological evaluation of N-alkyl or aryl substituted isoindigo derivatives as potential dual cyclin-dependent kinase 2 (CDK2)/glycogen synthase kinase 3β (GSK-3β) phosphorylation inhibitors.

    PubMed

    Zhao, Ping; Li, Yanzhong; Gao, Guangwei; Wang, Shuai; Yan, Yun; Zhan, Xiaoping; Liu, Zenglu; Mao, Zhenmin; Chen, Shaoxiong; Wang, Liqun

    2014-10-30

    A series of N-alkyl or aryl substituted isoindigo derivatives have been synthesized and their anti-proliferative activity was evaluated by Sulforhodamine B (SRB) assay. Some of the target compounds exhibited significant antitumor activity, including compounds 6h and 6k (against K562 cells), 6i (against HeLa cells) and 6j (against A549 cells). N-(p-methoxy-phenyl)-isoindigo (6k) exhibited a high and selective anti-proliferative activity against K562 cells (IC50 7.8 μM) and induced the apoptosis of K562 cells in a dose-dependent manner. Compound 6k arrested the cell cycle at S phase in K562 cells by decreasing the expression of cyclin A and CDK2, which played critical roles in DNA replication and passage through G2 phase. Moreover, compound 6k down-regulated the expression of p-GSK-3β (Ser9), β-catenin and c-myc proteins, up-regulated the expression of GSK-3β, consequently, suppressed Wnt/β-catenin signaling pathway and induced the apoptosis of K562 cells. The binding mode of compound 6k with GSK-3β was simulated using molecular docking tools. All of these studies gave a better understanding to the molecular mechanisms of this class of agents and clues to develop dual CDK2/GSK-3β (Ser9) phosphorylation inhibitors applied in cancer chemotherapy. PMID:25151579

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

    PubMed Central

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

    2002-01-01

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

  16. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation.

    PubMed

    Baumli, Sonja; Lolli, Graziano; Lowe, Edward D; Troiani, Sonia; Rusconi, Luisa; Bullock, Alex N; Debreczeni, Judit E; Knapp, Stefan; Johnson, Louise N

    2008-07-01

    The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis. PMID:18566585

  17. Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

    PubMed Central

    Perez, Marco; Muñoz-Galván, Sandra; Jiménez-García, Manuel P.; Marín, Juan J.; Carnero, Amancio

    2015-01-01

    Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors. PMID:26528855

  18. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease

    PubMed Central

    Esdar, Christina; Waalboer, Dennis; Adeniji-Popoola, Olajumoke; Ortiz-Ruiz, Maria-Jesus; Mallinger, Aurélie; Samant, Rahul S.; Czodrowski, Paul; Musil, Djordje; Schwarz, Daniel; Schneider, Klaus; Stubbs, Mark; Ewan, Ken; Fraser, Elizabeth; TePoele, Robert; Court, Will; Box, Gary; Valenti, Melanie; de Haven Brandon, Alexis; Gowan, Sharon; Rohdich, Felix; Raynaud, Florence; Schneider, Richard; Poeschke, Oliver; Blaukat, Andree; Workman, Paul; Schiemann, Kai; Eccles, Suzanne A.; Wienke, Dirk; Blagg, Julian

    2015-01-01

    There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small molecule WNT-pathway inhibitor discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a Type 1 binding mode involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues alter WNT-pathway regulated gene expression and other on-target effects of modulating CDK8/19 including genes regulated by STAT1. Consistent with this we find that phosphorylation of STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors. PMID:26502155

  19. The Proliferation-Quiescence Decision Is Controlled by a Bifurcation in CDK2 Activity at Mitotic Exit

    PubMed Central

    Spencer, Sabrina L.; Cappell, Steven D.; Tsai, Feng-Chiao; Overton, K. Wesley; Wang, Clifford L.; Meyer, Tobias

    2014-01-01

    SUMMARY Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity. PMID:24075009

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

    PubMed Central

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

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5−/− embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5−/− brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  1. Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer.

    PubMed

    Herrera-Abreu, Maria Teresa; Palafox, Marta; Asghar, Uzma; Rivas, Martín A; Cutts, Rosalind J; Garcia-Murillas, Isaac; Pearson, Alex; Guzman, Marta; Rodriguez, Olga; Grueso, Judit; Bellet, Meritxell; Cortés, Javier; Elliott, Richard; Pancholi, Sunil; Baselga, José; Dowsett, Mitch; Martin, Lesley-Ann; Turner, Nicholas C; Serra, Violeta

    2016-04-15

    Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR. PMID:27020857

  2. Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism

    PubMed Central

    Lu, Huasong; Xue, Yuhua; Yu, Guoying K; Arias, Carolina; Lin, Julie; Fong, Susan; Faure, Michel; Weisburd, Ben; Ji, Xiaodan; Mercier, Alexandre; Sutton, James; Luo, Kunxin; Gao, Zhenhai; Zhou, Qiang

    2015-01-01

    CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. DOI: http://dx.doi.org/10.7554/eLife.06535.001 PMID:26083714

  3. Inhibition of Eimeria tenella CDK-related Kinase 2: From Target Identification to Lead Compounds

    PubMed Central

    Engels, Kristin; Beyer, Carsten; Fernández, Maria L. Suárez; Bender, Frank; Gaßel, Michael; Unden, Gottfried; Marhöfer, Richard J.; Mottram, Jeremy C.; Selzer, Paul M.

    2011-01-01

    Apicomplexan parasites encompass several human-pathogenic as well as animal-pathogenic protozoans like Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella is the causative agent of coccidiosis a disease of chickens, which causes tremendous economic losses to the world poultry industry. Considerable increase of drug resistance makes it necessary to develop and pursue new therapeutic strategies. Cyclin-dependent kinases (CDKs) are key molecules in the regulation of the cell cycle and are therefore prominent target proteins in parasitic diseases. Bioinformatic analysis revealed four potential CDK-like proteins of which one – E. tenella CDK-related kinase 2 (EtCRK2) – is already cloned, expressed and characterized.[1] Using the CDK specific inhibitor Flavopiridol in EtCRK2 enzyme assays and schizont maturation assays we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as template to built protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP-binding site between EtCRK2 and HsCDK2 as well as chicken CDK3 have been addressed for the optimization of selective ATP-competitive inhibitors. Virtual screening and “wet-bench” high throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized leading to a set of four promising lead compounds inhibiting EtCRK2. PMID:20575139

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

    PubMed Central

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

    2016-01-01

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

  5. miR-30e controls DNA damage-induced stress responses by modulating expression of the CDK inhibitor p21WAF1/CIP1 and caspase-3

    PubMed Central

    Sohn, Dennis; Peters, Dominik; Piekorz, Roland P.; Budach, Wilfried; Jänicke, Reiner U.

    2016-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3′-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value. PMID:26895377

  6. Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin

    SciTech Connect

    Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula

    2005-01-10

    Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with anti-mitotic and/or anti-neurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinity and specificity.

  7. Roscovitine in cancer and other diseases.

    PubMed

    Cicenas, Jonas; Kalyan, Karthik; Sorokinas, Aleksandras; Stankunas, Edvinas; Levy, Josh; Meskinyte, Ingrida; Stankevicius, Vaidotas; Kaupinis, Algirdas; Valius, Mindaugas

    2015-06-01

    Roscovitine [CY-202, (R)-Roscovitine, Seliciclib] is a small molecule that inhibits cyclin-dependent kinases (CDKs) through direct competition at the ATP-binding site. It is a broad-range purine inhibitor, which inhibits CDK1, CDK2, CDK5 and CDK7, but is a poor inhibitor for CDK4 and CDK6. Roscovitine is widely used as a biological tool in cell cycle, cancer, apoptosis and neurobiology studies. Moreover, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, inflammation, viral infections, polycystic kidney disease and glomerulonephritis. This review focuses on the use of roscovitine in the disease model as well as clinical model research. PMID:26207228

  8. CDK2 Inhibition Causes Anaphase Catastrophe in Lung Cancer through the Centrosomal Protein CP110

    PubMed Central

    Hu, Shanhu; Danilov, Alexey V.; Godek, Kristina; Orr, Bernardo; Tafe, Laura J.; Rodriguez-Canales, Jaime; Behrens, Carmen; Mino, Barbara; Moran, Cesar A.; Memoli, Vincent A.; Mustachio, Lisa Maria; Galimberti, Fabrizio; Ravi, Saranya; DeCastro, Andrew; Lu, Yun; Sekula, David; Andrew, Angeline S; Wistuba, Ignacio I.; Freemantle, Sarah; Compton, Duane A.; Dmitrovsky, Ethan

    2015-01-01

    Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacological targeting of aneuploidy is an attractive therapeutic strategy as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. Small interfering RNA (siRNA)-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor-mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2-inhibition-driven anaphase catastrophe. Independent examination of murine and human paired normal-malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared to KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. PMID:25808870

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-07-01

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

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

    PubMed

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

    2015-05-20

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

    PubMed Central

    Montagnoli, Alessia; Fiore, Francesca; Eytan, Esther; Carrano, Andrea C.; Draetta, Giulio F.; Hershko, Avram; Pagano, Michele

    1999-01-01

    The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin–proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin–proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK−)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. PMID:10323868

  17. Discovery of 8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x) as a Potent Inhibitor of Cyclin-Dependent Kinase 4 (CDK4) and AMPK-Related Kinase 5 (ARK5)

    PubMed Central

    2015-01-01

    The success of imatinib, a BCR-ABL inhibitor for the treatment of chronic myelogenous leukemia, has created a great impetus for the development of additional kinase inhibitors as therapeutic agents. However, the complexity of cancer has led to recent interest in polypharmacological approaches for developing multikinase inhibitors with low toxicity profiles. With this goal in mind, we analyzed more than 150 novel cyano pyridopyrimidine compounds and identified structure–activity relationship trends that can be exploited in the design of potent kinase inhibitors. One compound, 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x), was found to be the most active, inducing apoptosis of tumor cells at a concentration of approximately 30–100 nM. In vitro kinase profiling revealed that 7x is a multikinase inhibitor with potent inhibitory activity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we report the synthesis, structure–activity relationship, kinase inhibitory profile, in vitro cytotoxicity, and in vivo tumor regression studies by this lead compound. PMID:24417566

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

    PubMed Central

    Li, Yan; Zhang, Jingxiao; Gao, Weimin; Zhang, Lilei; Pan, Yanqiu; Zhang, Shuwei; Wang, Yonghua

    2015-01-01

    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. PMID:25918937

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

    PubMed

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

    2009-01-23

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

  20. A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

    PubMed Central

    Diehl, J A; Sherr, C J

    1997-01-01

    Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting

  1. Programmed cell death protein 4 suppresses CDK1/cdc2 via induction of p21(Waf1/Cip1).

    PubMed

    Göke, R; Barth, P; Schmidt, A; Samans, B; Lankat-Buttgereit, B

    2004-12-01

    We show that the recently discovered tumor suppressor pdcd4 represses the transcription of the mitosis-promoting factor cyclin-dependent kinase (CDK)1/cdc2 via upregulation of p21(Waf1/Cip1). p21(Waf1/Cip1) inhibits CDK4/6 and CDK2. Decrease of CDK4/6 and CDK2 enhances the binding of pRb to E2F/DP, which in turn together bind to and repress the cdc2 promoter. Upregulation of CDK1/cdc2 accompanied by a malignant change was previously reported in colon cancer. We show that expression of pdcd4 as an indirect suppressor of CDK1/cdc2 is lost in progressed carcinomas of lung, breast, colon, and prostate. Furthermore, it seems that localization and expression of pdcd4 directly correlate with tumor progression. Finally, the CDK1/cdc2 inhibitor roscovitine reduces the proliferation of several tumor cell lines, suggesting that inhibition of CDK1/cdc2 may be a useful strategy against malignant transformation. Therefore, pdcd4 might serve as a novel target for antineoplastic therapies. PMID:15317660

  2. Cyclin-dependent kinase inhibitor drugs as potential novel anti-inflammatory and pro-resolution agents

    PubMed Central

    Leitch, AE; Haslett, C; Rossi, AG

    2009-01-01

    The cyclin-dependent kinase inhibitor (CDKi) drugs such as R-roscovitine have emerged as potential anti-inflammatory, pharmacological agents that can influence the resolution of inflammation. Usually, once an inciting inflammatory stimulus has been eliminated, resolution proceeds by prompt, safe removal of dominant inflammatory cells. This is accomplished by programmed cell death (apoptosis) of prominent effector, inflammatory cells typified by the neutrophil. Apoptosis of neutrophils ensures that toxic neutrophil granule contents are securely packaged in apoptotic bodies and expedites phagocytosis by professional phagocytes such as macrophages. A panel of CDKi drugs have been shown to promote neutrophil apoptosis in a concentration- and time-dependent manner and the archetypal CDKi drug, R-roscovitine, overrides the anti-apoptotic effects of powerful survival factors [including lipopolysaccharide (LPS) and granulocyte macrophage-colony stimulating factor (GM-CSF)]. Inflammatory cell longevity and survival signalling is integral to the inflammatory process and any putative anti-inflammatory agent must unravel a complex web of redundancy in order to be effective. CDKi drugs have also been demonstrated to have significant effects on other cell types including lymphocytes and fibroblasts indicating that they may have pleiotropic anti-inflammatory, pro-resolution activity. In keeping with this, CDKi drugs like R-roscovitine have been reported to be efficacious in resolving established animal models of neutrophil-dominant and lymphocyte-driven inflammation. However, the mechanism of action behind these powerful effects has not yet been fully elucidated. CDKs play an integral role in the regulation of the cell cycle but are also recognized as participants in processes such as apoptosis and transcriptional regulation. Neutrophils have functional CDKs, are transcriptionally active and demonstrate augmented apoptosis in response to CDKi drugs, while lymphocyte proliferation

  3. Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas.

    PubMed

    Bartkowiak, Bartlomiej; Yan, Christopher; Greenleaf, Arno L

    2015-09-01

    The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12's roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies. PMID:26189575

  4. Cdk9 T-loop Phosphorylation is Regulated by the Calcium Signaling Pathway

    PubMed Central

    Ramakrishnan, Rajesh; Rice, Andrew P.

    2011-01-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Calcium/Calmodulin- dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4+ T lymphocytes, we found that the Ca2+ signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca2+ signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca2+ signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca2+ pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function. PMID:21448926

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

    PubMed

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

    2013-09-27

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

  6. S-nitrosylation of Cdk5

    PubMed Central

    Qu, Jing; Nakamura, Tomohiro; Holland, Emily A.; McKercher, Scott R.; Lipton, Stuart A.

    2012-01-01

    Aberrant activation of Cdk5 has been implicated in the process of neurodegenerative diseases such as Alzheimer's disease (AD). We recently reported that S-nitrosylation of Cdk5 (forming SNO-Cdk5) at specific cysteine residues results in excessive activation of Cdk5, contributing to mitochondrial dysfunction, synaptic damage, and neuronal cell death in models of AD. Furthermore, SNO-Cdk5 acts as a nascent S-nitrosylase, transnitrosylating the mitochondrial fission protein Drp1 and enhancing excessive mitochondrial fission in dendritic spines. However, a molecular mechanism that leads to the formation of SNO-Cdk5 in neuronal cells remained obscure. Here, we demonstrate that neuronal nitric oxide synthase (NOS1) interacts with Cdk5 and that the close proximity of the two proteins facilitates the formation of SNO-Cdk5. Interestingly, as a negative feedback mechanism, Cdk5 phosphorylates and suppresses NOS1 activity. Thus, together with our previous report, these findings delineate an S-nitrosylation pathway wherein Cdk5/NOS1 interaction enhances SNO-Cdk5 formation, mediating mitochondrial dysfunction and synaptic loss during the etiology of AD. PMID:22874667

  7. Nuclear accumulation of cyclin E/Cdk2 triggers a concentration-dependent switch for the destruction of p27Xic1

    PubMed Central

    Swanson, Craig; Ross, John; Jackson, Peter K.

    2000-01-01

    The action of cyclin-dependent kinases (CDKs) is regulated by phosphorylation, cyclin levels, the abundance of CDK inhibitors, and, as recently has been shown for cyclin B/cdc2, their localization. It is unclear how localization regulates the action of cyclin E/Cdk2 and its inhibitors. Here, we show that the closest known Xenopus laevis homolog of mammalian Cdk2 inhibitors p27Kip1 and p21CIP1, Xic1, is concentrated, ubiquitinated, and destroyed in the nucleus. Furthermore, Xic1 destruction requires nuclear import, but not nuclear export, and requires the formation of a transport-competent nuclear envelope, but not interactions between the lamina and chromatin. We show that (i) cyclin E/Cdk2 and Xic1 are transported into the nucleus as a complex and that Xic1 destruction requires the activity of cyclin E, (ii) that phosphorylation of Xic1 by cyclin E/Cdk2 bypasses the requirement for nuclear formation, and (iii) that the phosphorylation of Xic1 by cyclin E/Cdk2 is concentration dependent and likely realized through second-order interactions between stable cyclin E/Cdk2/Xic1 ternary complexes. Based on these results we propose a model wherein nuclear accumulation of the cyclin E/Cdk2/Xic1 complex triggers a concentration-dependent switch that promotes the phosphorylation of Xic1 and, consequently, its ubiquitination and destruction, thus allowing subsequent activation of cyclin E/Cdk2. PMID:10884410

  8. MYC pathway activation in triple-negative breast cancer is synthetic lethal with CDK inhibition

    PubMed Central

    Horiuchi, Dai; Kusdra, Leonard; Huskey, Noelle E.; Chandriani, Sanjay; Lenburg, Marc E.; Gonzalez-Angulo, Ana Maria; Creasman, Katelyn J.; Bazarov, Alexey V.; Smyth, James W.; Davis, Sarah E.; Yaswen, Paul; Mills, Gordon B.; Esserman, Laura J.

    2012-01-01

    Estrogen, progesterone, and HER2 receptor-negative triple-negative breast cancers encompass the most clinically challenging subtype for which targeted therapeutics are lacking. We find that triple-negative tumors exhibit elevated MYC expression, as well as altered expression of MYC regulatory genes, resulting in increased activity of the MYC pathway. In primary breast tumors, MYC signaling did not predict response to neoadjuvant chemotherapy but was associated with poor prognosis. We exploit the increased MYC expression found in triple-negative breast cancers by using a synthetic-lethal approach dependent on cyclin-dependent kinase (CDK) inhibition. CDK inhibition effectively induced tumor regression in triple-negative tumor xenografts. The proapoptotic BCL-2 family member BIM is up-regulated after CDK inhibition and contributes to this synthetic-lethal mechanism. These results indicate that aggressive breast tumors with elevated MYC are uniquely sensitive to CDK inhibitors. PMID:22430491

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Pisani, Pasquale; Rastelli, Giulio

    2016-01-01

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

  11. ING5 Is Phosphorylated by CDK2 and Controls Cell Proliferation Independently of p53

    PubMed Central

    Linzen, Ulrike; Lilischkis, Richard; Pandithage, Ruwin; Schilling, Britta; Ullius, Andrea; Lüscher-Firzlaff, Juliane; Kremmer, Elisabeth; Lüscher, Bernhard; Vervoorts, Jörg

    2015-01-01

    Inhibitor of growth (ING) proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2) in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53. PMID:25860957

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

    PubMed

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

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle. PMID:26549230

  13. CDK8 expression in 470 colorectal cancers in relation to beta-catenin activation, other molecular alterations and patient survival.

    PubMed

    Firestein, Ron; Shima, Kaori; Nosho, Katsuhiko; Irahara, Natsumi; Baba, Yoshifumi; Bojarski, Emeric; Giovannucci, Edward L; Hahn, William C; Fuchs, Charles S; Ogino, Shuji

    2010-06-15

    Alterations in the Wnt/beta-catenin pathway define a key event in the pathogenesis of colon cancer. We have recently shown that CDK8, the gene encoding a cyclin-dependent kinase (CDK) component of the Mediator complex, acts as a colon cancer oncogene that is necessary for beta-catenin activity. Here, we tested the hypothesis that colorectal cancers with CDK8 expression have distinct clinical, prognostic and molecular attributes. Among 470 colorectal cancers identified in 2 prospective cohort studies, CDK8 expression was detected in 329 (70%) tumors by immunohistochemistry. Cox proportional hazards model and backward stepwise elimination were used to compute hazard ratio (HR) of deaths according to CDK8 status, initially adjusted for various patient and molecular features, including beta-catenin, p53, p21, p27 (CDK inhibitors), cyclin D1, fatty acid synthase (FASN), cyclooxygenase-2 (COX-2), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), LINE-1 methylation, and mutations in KRAS, BRAF and PIK3CA. CDK8 expression in colorectal cancer was independently associated with beta-catenin activation (p = 0.0002), female gender (p < 0.0001) and FASN overexpression (p = 0.0003). Among colon cancer patients, CDK8 expression significantly increased colon cancer-specific mortality in both univariate analysis [HR 1.70; 95% confidence interval (CI), 1.03-2.83; p = 0.039] and multivariate analysis (adjusted HR 2.05; 95% CI, 1.18-3.56; p = 0.011) that was adjusted for potential confounders including beta-catenin, COX-2, FASN, LINE-1 hypomethylation, CIMP and MSI. CDK8 expression was unrelated with clinical outcome among rectal cancer patients. These data support a potential link between CDK8 and beta-catenin, and suggest that CDK8 may identify a subset of colon cancer patients with a poor prognosis. PMID:19790197

  14. 1α,25 dihydroxi-vitamin D{sub 3} modulates CDK4 and CDK6 expression and localization

    SciTech Connect

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

    2015-03-27

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

  15. Direct interactions with both p27 and Cdk2 regulate Spy1-mediated proliferation in vivo and in vitro.

    PubMed

    Al Sorkhy, Mohammad; Fifield, Bre-Anne; Myers, Dorothy; Porter, Lisa A

    2016-01-01

    Families of cyclin-like proteins have emerged that bind and activate cyclin dependent kinases (Cdk)s, directing the phosphorylation of noncanonical Cdk substrates. One of these proteins, Spy1, has demonstrated the unique ability to directly bind and activate both Cdk1 and Cdk2, as well as binding and promoting the degradation of at least one Cdk inhibitor, p27(Kip1). Spy1 accelerates somatic cell growth and proliferation and is implicated in a number of human cancers including the breast, brain and liver. Herein we isolate key residues mediating the direct interaction with p27. We use mutants of Spy1 to determine the physiological role of direct interactions with distinct binding partners Cdk2 and p27. We demonstrate that disrupting the direct interaction with either Spy1 binding partner decreased endogenous activity of Cdk2, as well as Spy1-mediated proliferation. However, only the direct interaction with p27 was essential for Spy1-mediated effects on p27 stability. In vivo neither mutation completely prevented tumorigenesis, although each mutation slowed the rate of Spy1-mediated tumorigenesis and decreased overall tumor volumes. This work supports the conclusion that direct interaction with both p27 and Cdk2 contribute to Spy1-mediated effects on cell growth. It is important to elucidate the dynamics of these interactions and to consider these data when assessing functional outcomes. PMID:26771716

  16. Expression of CDK8 and CDK8-interacting Genes as Potential Biomarkers in Breast Cancer.

    PubMed

    Broude, Eugenia V; Győrffy, Balázs; Chumanevich, Alexander A; Chen, Mengqian; McDermott, Martina S J; Shtutman, Michael; Catroppo, James F; Roninson, Igor B

    2015-01-01

    CDK8 and its paralog CDK19, in complex with CCNC, MED12 and MED13, are transcriptional regulators that mediate several carcinogenic pathways and the chemotherapy-induced tumor-supporting paracrine network. Following up on our previous observation that CDK8, CDK19 and CCNC RNA expression is associated with shorter relapse-free survival (RFS) in breast cancer, we now found by immunohistochemical analysis that CDK8/19 protein is overexpressed in invasive ductal carcinomas relative to non-malignant mammary tissues. Meta-analysis of transcriptomic data revealed that higher CDK8 expression is associated with shorter RFS in all molecular subtypes of breast cancer. These correlations were much stronger in patients who underwent systemic adjuvant therapy, suggesting that CDK8 impacts the failure of systemic therapy. The same associations were found for CDK19, CCNC and MED13. In contrast, MED12 showed the opposite association with a longer RFS. The expression levels of CDK8 in breast cancer samples were directly correlated with the expression of MYC, as well as CDK19, CCNC and MED13 but inversely correlated with MED12. CDK8, CDK19 and CCNC expression was strongly increased and MED12 expression was decreased in tumors with mutant p53. Gene amplification is the most frequent type of genetic alterations of CDK8, CDK19, CCNC and MED13 in breast cancers (9.7% of which have amplified MED13), whereas point mutations are more common in MED12. These results suggest that the expression of CDK8 and its interactive genes has a profound impact on the response to adjuvant therapy in breast cancer in accordance with the role of CDK8 in chemotherapy-induced tumor-supporting paracrine activities. PMID:26452386

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

    SciTech Connect

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

    2014-08-15

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

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

    PubMed Central

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

    2016-01-01

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

  19. NUAK2 Amplification Coupled with PTEN Deficiency Promotes Melanoma Development via CDK Activation.

    PubMed

    Namiki, Takeshi; Yaguchi, Tomonori; Nakamura, Kenta; Valencia, Julio C; Coelho, Sergio G; Yin, Lanlan; Kawaguchi, Masakazu; Vieira, Wilfred D; Kaneko, Yasuhiko; Tanemura, Atsushi; Katayama, Ichiro; Yokozeki, Hiroo; Kawakami, Yutaka; Hearing, Vincent J

    2015-07-01

    The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes, but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplification in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulated p21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivation specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Finally, pharmacologic inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results show how CDK2 blockade may offer a promising therapy for genetically defined melanomas, where NUAK2 is amplified and PTEN is deleted. PMID:25832654

  20. Evidence for a CDK4-dependent checkpoint in a conditional model of cellular senescence

    PubMed Central

    Brookes, Sharon; Gagrica, Sladjana; Sanij, Elaine; Rowe, Janice; Gregory, Fiona J; Hara, Eiji; Peters, Gordon

    2015-01-01

    Cellular senescence, the stable cell cycle arrest elicited by various forms of stress, is an important facet of tumor suppression. Although much is known about the key players in the implementation of senescence, including the pRb and p53 axes and the cyclin dependent kinase inhibitors p16INK4a and p21CIP1, many details remain unresolved. In studying conditional senescence in human fibroblasts that express a temperature sensitive SV40 large T-antigen (T-Ag), we uncovered an unexpected role for CDK4. At the permissive temperature, where pRb and p53 are functionally compromised by T-Ag, cyclin D-CDK4 complexes are disrupted by the high p16INK4a levels and reduced expression of p21CIP1. In cells arrested at the non-permissive temperature, p21CIP1 promotes reassembly of cyclin D-CDK4 yet pRb is in a hypo-phosphorylated state, consistent with cell cycle arrest. In exploring whether the reassembled cyclin D-CDK4-p21 complexes are functional, we found that shRNA-mediated knockdown or chemical inhibition of CDK4 prevented the increase in cell size associated with the senescent phenotype by allowing the cells to arrest in G1 rather than G2/M. The data point to a role for CDK4 kinase activity in a G2 checkpoint that contributes to senescence. PMID:25695870

  1. Structural Basis for the Modulation of CDK-Dependent/Independent Activity of Cyclin D1

    PubMed Central

    Ferrer, Jean-Luc; Dupuy, Jérôme; Borel, Franck; Jacquamet, Lilian; Noel, Joseph P.; Dulic, Vjekoslav

    2010-01-01

    D-type cyclins are key regulators of the cell division cycle. In association with Cyclin Dependent Kinases (CDK) 2/4/6, they control the G1/S-phase transition in part by phosphorylation and inactivation of tumor suppressor of retinoblastoma family. Defective regulation of the G1/S transition is a well-known cause of cancer, making the cyclin D1-CDK4/6 complex a promising therapeutic target. Our objective is to develop inhibitors that would block the formation or the activation of the cyclin D1-CDK4/6 complex, using in silico docking experiments on a structural homology model of the cyclin D1-CDK4/6 complex. To this end we focused on the cyclin subunit in three different ways: (1) targeting the part of the cyclin D1 facing the N-terminal domain of CDK4/6, in order to prevent the dimer formation; (2) targeting the part of the cyclin D1 facing the C-terminal domain of CDK4/6, in order to prevent the activation of CDK4/6 by blocking the T-loop in an inactive conformation, and also to destabilize the dimer; (3) targeting the groove of cyclin D1 where p21 binds, in order to mimic its inhibition mode by preventing binding of cyclin D1-CDK4/6 complex to its targets. Our strategy, and the tools we developed, will provide a computational basis to design lead compounds for novel cancer therapeutics, targeting a broad range of proteins involved in the regulation of the cell cycle. PMID:17172845

  2. Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells.

    PubMed

    Pauls, Eduardo; Ruiz, Alba; Badia, Roger; Permanyer, Marc; Gubern, Albert; Riveira-Muñoz, Eva; Torres-Torronteras, Javier; Alvarez, Mar; Mothe, Beatriz; Brander, Christian; Crespo, Manel; Menéndez-Arias, Luis; Clotet, Bonaventura; Keppler, Oliver T; Martí, Ramon; Posas, Francesc; Ballana, Ester; Esté, José A

    2014-08-15

    Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain-containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67(+)) primary CD4(+) T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability. PMID:25015816

  3. Delayed Treatment with Systemic (S)-Roscovitine Provides Neuroprotection and Inhibits In Vivo CDK5 Activity Increase in Animal Stroke Models

    PubMed Central

    Menn, Bénédicte; Bach, Stéphane; Blevins, Teri L.; Campbell, Mark; Meijer, Laurent; Timsit, Serge

    2010-01-01

    Background Although quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment. Methodology/Principal Findings In the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms. Conclusions/Significance Altogether, our data bring new evidences for the further development of pharmacological CDK inhibitors in stroke therapy. PMID:20711428

  4. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Jena, Nilamani; Sheng, Jinghao; Hu, Jamie K.; Li, Wei; Zhou, Wenhui; Lee, Gene; Tsichlis, Nicolaos; Pathak, Aparna; Brown, Nelson; Deshpande, Amit; Luo, Chi; Hu, Guo-fu; Hinds, Philip W.; Van Etten, Richard A.; Hu, Miaofen G.

    2015-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

    Nabti, Ibtissem; Reis, Alexandra; Levasseur, Mark; Stemmann, Olaf; Jones, Keith T

    2008-09-15

    Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1. PMID:18639540

  12. MEN1 tumorigenesis in the pituitary and pancreatic islet requires Cdk4 but not Cdk2.

    PubMed

    Gillam, M P; Nimbalkar, D; Sun, L; Christov, K; Ray, D; Kaldis, P; Liu, X; Kiyokawa, H

    2015-02-12

    Recent studies suggest that physiological and tumorigenic proliferation of mammalian cells is controlled by multiple cyclin-dependent kinases (CDKs) largely in tissue-specific manners. We and others previously demonstrated that adult mice deficient for the Cyclin D partner CDK4 (Cdk4(-/-) mice) exhibit hypoplasia in the pituitary and pancreatic islet due to primary postnatal defects in proliferation. Intriguingly, those neuroendocrine tissues affected in Cdk4(-/-) mice are the primary targets of tumorigenesis in the syndrome of multiple endocrine neoplasia type-1 (MEN1). Mice with heterozygous disruption of the tumor suppressor Men1 gene (Men1(+/-)) develop tumors in the pituitary, pancreatic islets and other neuroendocrine tissues, which is analogous to humans with MEN1 mutations. To explore the genetic interactions between loss of Men1 and activation of CDKs, we examined the impact of Cdk4 or Cdk2 disruption on tumorigenesis in Men1(+/-) mice. A majority of Men1(+/-) mice with wild-type CDKs developed pituitary and islet tumors by 15 months of age. Strikingly, Men1(+/-); Cdk4(-/-) mice did not develop any tumors, and their islets and pituitaries remained hypoplastic with decreased proliferation. In contrast, Men1(+/-); Cdk2(-/-) mice showed pituitary and islet tumorigenesis comparable to those in Men1(+/-) mice. Pituitaries of Men1(+/-); Cdk4(-/-) mice showed no signs of loss of heterozygosity (LOH) in the Men1 locus, whereas tumors in Men1(+/-) mice and Men1(+/-); Cdk2(-/-) mice exhibited LOH. Consistently, CDK4 knockdown in INS-1 insulinoma cells inhibited glucose-stimulated cell cycle progression with a significant decrease in phosphorylation of retinoblastoma protein (RB) at specific sites including Ser780. CDK2 knockdown had minimum effects on RB phosphorylation and cell cycle progression. These data suggest that CDK4 is a critical downstream target of MEN1-dependent tumor suppression and is required for tumorigenic proliferation in the pituitary and

  13. Inhibition of CDK1 activity by sumoylation.

    PubMed

    Xiao, Yuxuan; Lucas, Benjamin; Molcho, Elana; Schiff, Tania; Vigodner, Margarita

    2016-09-16

    Sumoylation (a covalent modification by Small Ubiquitin-like Modifiers or SUMO proteins) has been implicated in the regulation of various cellular events including cell cycle progression. We have recently identified CDK1, a master regulator of mitosis and meiosis, as a SUMO target both in vivo and in vitro, supporting growing evidence concerning a close cross talk between sumoylation and phosphorylation during cell cycle progression. However, any data regarding the effect of sumoylation upon CDK1 activity have been missing. In this study, we performed a series of in vitro experiments to inhibit sumoylation by three different means (ginkgolic acid, physiological levels of oxidative stress, and using an siRNA approach) and assessed the changes in CDK1 activity using specific antibodies and a kinase assay. We have also tested for an interaction between SUMO and active and/or inactive CDK1 isoforms in addition to having assessed the status of CDK1-interacting sumoylated proteins upon inhibition of sumoylation. Our data suggest that inhibition of sumoylation increases the activity of CDK1 probably through changes in sumoylated status and/or the ability of specific proteins to bind CDK1 and inhibit its activity. PMID:27520372

  14. A novel approach to the discovery of small molecule ligands of CDK2

    PubMed Central

    Martin, Mathew P.; Alam, Riazul; Betzi, Stephane; Ingles, Donna J.; Zhu, Jin-Yi

    2012-01-01

    In an attempt to identify novel small molecule ligands of CDK2 with potential as allosteric inhibitors, we devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds which displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1,453 compounds led to the discovery of 12 compounds with displacement activities (EC50 values) ranging from 6 to 44 μM, all of which were classified as ATP site-directed ligands. Four new Type I inhibitor scaffolds were confirmed by X-ray crystallography. While this small compound library contained only ATP-site directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action. PMID:22893598

  15. Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer

    PubMed Central

    Wang, Bo-Yong; Liu, Quan-Yan; Cao, Jun; Chen, Ji-Wei; Liu, Zhi-Su

    2016-01-01

    Cyclin-dependent kinase (CDK) family members have been considered as attractive therapeutic targets for cancer. In this study, we aim to investigate the anticancer effects of a selective CDK7 inhibitor, BS-181, in gastric cancer (GC) cell line. Human GC cells (BGC823) were cultured with or without BS-181 at different concentrations for 24–72 hours. BS-181 significantly reduced the activity of CDK7 with downregulation of cyclin D1 and XIAP in GC cells. Treatment with BS-181 induced cell cycle arrest and apoptosis. The expression of Bax and caspase-3 was significantly increased, while Bcl-2 expression was decreased in cells treated with BS-181. In addition, the inhibition of CDK7 with BS-181 resulted in reduced rates of proliferation, migration, and invasion of gastric cells. Those results demonstrated the anticancer activities of selective CDK7 inhibitor BS-181 in BGC823 cells, suggesting that CDK7 may serve as a novel therapeutic target or the treatment of GC. PMID:27042010

  16. Cdk5r1 Overexpression Induces Primary β-Cell Proliferation

    PubMed Central

    Draney, Carrie; Hobson, Amanda E.; Grover, Samuel G.; Jack, Benjamin O.; Tessem, Jeffery S.

    2016-01-01

    Decreased β-cell mass is a hallmark of type 1 and type 2 diabetes. Islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. Understanding the pathways controlling islet proliferation may be used to increase functional β-cell mass through transplantation or by enhanced growth of endogenous β-cells. We have shown that the transcription factor Nkx6.1 induces β-cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to define Nkx6.1-independent mechanisms by which Nr4a1 and Nr4a3 induce β-cell proliferation, we demonstrated that cyclin-dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1. Overexpression of Cdk5r1 is sufficient to induce primary rat β-cell proliferation while maintaining glucose stimulated insulin secretion. Overexpression of Cdk5r1 in β-cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. The Cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that Cdk5r1 mediated β-cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the Cdk5 kinase complex is sufficient to induce β-cell proliferation while maintaining glucose stimulated insulin secretion. PMID:26788519

  17. INHIBITION OF CDK9 PREVENTS MECHANICAL INJURY-INDUCED INFLAMMATION, APOPTOSIS AND MATRIX DEGRADATION IN CARTILAGE EXPLANTS

    PubMed Central

    Hu, Z.; Yik, J.H.N.; Cissell, D.D.; Michelier, P.V.; Athanasiou, K.A.; Haudenschild, D.R.

    2016-01-01

    Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acute injury responses to trauma induce production of pro-inflammatory cytokines and catabolic enzymes, which promote chondrocyte apoptosis and degrade cartilage to potentiate PTOA development. Recent studies show that the rate-limiting step for transcriptional activation of injury response genes is controlled by cyclin-dependent kinase 9 (CDK9), and thus it is an attractive target for limiting the injury response. Here, we determined the effects of CDK9 inhibition in suppressing the injury response in mechanically-injured cartilage explants. Bovine cartilage explants were injured by a single compressive load of 30 % strain at 100 %/s, and then treated with the CDK9 inhibitor Flavopiridol. To assess acute injury responses, we measured the mRNA expression of pro-inflammatory cytokines, catabolic enzymes, and apoptotic genes by RT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. For long-term outcome, cartilage matrix degradation was assessed by soluble glycosaminoglycan release, and by determining the mechanical properties with instantaneous and relaxation moduli. Our data showed CDK9 inhibitor markedly reduced injury-induced inflammatory cytokine and catabolic gene expression. CDK9 inhibitor also attenuated chondrocyte apoptosis and reduced cartilage matrix degradation. Lastly, the mechanical properties of the injured explants were preserved by CDK9 inhibitor. Our results provide a temporal profile connecting the chain of events from mechanical impact, acute injury responses, to the subsequent induction of chondrocyte apoptosis and cartilage matrix deterioration. Thus, CDK9 is a potential disease-modifying agent for injury response after knee trauma to prevent or delay PTOA development. PMID:26859911

  18. The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription.

    PubMed

    Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej; Oikonomou, Catherine; Cross, Frederick R

    2016-04-01

    Throughout cell-cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell-cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low. PMID:27058667

  19. Expression, Purification, and Identification of Associated Proteins of the Full-length hCDK12/CyclinK Complex*

    PubMed Central

    Bartkowiak, Bartlomiej; Greenleaf, Arno L.

    2015-01-01

    The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3′-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a “2A” peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors. PMID:25429106

  20. An Erk/Cdk5 axis controls the diabetogenic actions of PPARγ

    PubMed Central

    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.

    2014-01-01

    Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ at serine 273 by Cdk5 stimulates diabetogenic gene expression in adipose tissues1. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic PPARγ ligand drugs, such as the thiazolidinediones and PPARγ partial/non-agonists2. To better understand the importance of this obesity-linked PPARγ phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. Surprisingly, these mice have both a paradoxical increase in PPARγ phosphorylation at S273 and worsened insulin resistance. Unbiased proteomic studies show that ERK kinases are activated in these KO animals. We show here that ERK directly phosphorylates S273 of PPARγ in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MEK, the ERK kinase. Importantly, pharmacological MEK and ERK inhibition 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. PMID:25409143

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

    PubMed

    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. PMID:25409143

  2. Chloroquine alleviates etoposide-induced centrosome amplification by inhibiting CDK2 in adrenocortical tumor cells

    PubMed Central

    Chen, T-Y; Syu, J-S; Lin, T-C; Cheng, H-l; Lu, F-l; Wang, C-Y

    2015-01-01

    The antitumor drug etoposide (ETO) is widely used in treating several cancers, including adrenocortical tumor (ACT). However, when used at sublethal doses, tumor cells still survive and are more susceptible to the recurring tumor due to centrosome amplification. Here, we checked the effect of sublethal dose of ETO in ACT cells. Sublethal dose of ETO treatment did not induce cell death but arrested the ACT cells in G2/M phase. This resulted in centrosome amplification and aberrant mitotic spindle formation leading to genomic instability and cellular senescence. Under such conditions, Chk2, cyclin A/CDK2 and ERK1/2 were aberrantly activated. Pharmacological inactivation of Chk2, CDK2 or ERK1/2 or depletion of CDK2 or Chk2 inhibited the centrosome amplification in ETO-treated ACT cells. In addition, autophagy was activated by ETO and was required for ACT cell survival. Chloroquine, the autophagy inhibitor, reduced ACT cell growth and inhibited ETO-induced centrosome amplification. Chloroquine alleviated CDK2 and ERK, but not Chk2, activation and thus inhibited centrosome amplification in either ETO- or hydroxyurea-treated ACT cells. In addition, chloroquine also inhibited centrosome amplification in osteosarcoma U2OS cell lines when treated with ETO or hydroxyurea. In summary, we have demonstrated that chloroquine inhibited ACT cell growth and alleviated DNA damage-induced centrosome amplification by inhibiting CDK2 and ERK activity, thus preventing genomic instability and recurrence of ACT. PMID:26690546

  3. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma. PMID:27203461

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Graf, Franziska; Koehler, Lena; Kniess, Torsten; Wuest, Frank; Mosch, Birgit; Pietzsch, Jens

    2009-01-01

    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 124I-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 [124I]CKIA and [124I]CKIB observed in human tumor cells are promising prerequisites for in vivo biodistribution and imaging studies. PMID:19551155

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2007-10-15

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

  9. Inhibition of cdk9 during Herpes Simplex Virus 1 Infection Impedes Viral Transcription

    PubMed Central

    Ou, Mark; Sandri-Goldin, Rozanne M.

    2013-01-01

    During herpes simplex virus 1 (HSV-1) infection there is a loss of the serine-2 phosphorylated form of RNA polymerase II (RNAP II) found in elongation complexes. This occurs in part because RNAP II undergoes ubiquitination and proteasomal degradation during times of highly active viral transcription, which may result from stalled elongating complexes. In addition, a viral protein, ICP22, was reported to trigger a loss of serine-2 RNAP II. These findings have led to some speculation that the serine-2 phosphorylated form of RNAP II may not be required for HSV-1 transcription, although this form is required for cellular transcription elongation and RNA processing. Cellular kinase cdk9 phosphorylates serine-2 in the C-terminal domain (CTD) of RNAP II. To determine if serine-2 phosphorylated RNAP II is required for HSV-1 transcription, we inhibited cdk9 during HSV-1 infection and measured viral gene expression. Inhibition was achieved by adding cdk9 inhibitors 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB) or flavopiridol (FVP) or by expression of a dominant–negative cdk9 or HEXIM1, which in conjunction with 7SK snRNA inhibits cdk9 in complex with cyclin 1. Here we report that inhibition of cdk9 resulted in decreased viral yields and levels of late proteins, poor formation of viral transcription-replication compartments, reduced levels of poly(A)+ mRNA and decreased RNA synthesis as measured by uptake of 5-bromouridine into nascent RNA. Importantly, a global reduction in viral mRNAs was seen as determined by microarray analysis. We conclude that serine-2 phosphorylation of the CTD of RNAP II is required for HSV-1 transcription. PMID:24205359

  10. Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events

    PubMed Central

    McCloy, Rachael A; Rogers, Samuel; Caldon, C Elizabeth; Lorca, Thierry; Castro, Anna; Burgess, Andrew

    2014-01-01

    Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G2 phase onwards. Addition of low doses of RO3306 in G2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A. PMID:24626186

  11. Proliferation and Cdk4 expression in microsatellite unstable colon cancers with TGFBR2 mutations.

    PubMed

    Grady, William M; Willis, Joseph E; Trobridge, Patty; Romero-Gallo, Judith; Munoz, Nina; Olechnowicz, Joseph; Ferguson, Kelly; Gautam, Shiva; Markowitz, Sanford D

    2006-02-01

    Approximately 15% of human colon cancers have microsatellite instability (MSI) and carry frameshift mutations in a polyadenine tract (BAT-RII) in the type II transforming growth factor beta (TGF-beta) receptor (TGFBR2), a required component of the TGF-beta receptor. The BAT-RII mutations in MSI colon cancers make the tumors resistant to the effects of TGF-beta. In cultured epithelial cells, TGF-beta can inhibit cell proliferation and induce apoptosis, and in vitro it can regulate the expression of a variety of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors. These effects are context- and tissue type-dependent, raising questions about which of these in vitro effects of TGF-beta signaling inactivation contribute to the formation of primary colon cancer. Thus, this study sought to determine the pathogenetically relevant effects of TGFBR2 inactivation in primary MSI colon cancers with mutant BAT-RII. Colon cancers with mutant BAT-RII were found to have increased proliferation compared to cancers with wild-type BAT-RII. Assessment of cdk4, cyclin D1 and p27(kip1) expression revealed that only cdk4 expression was increased in the cancers with mutant BAT-RII. In order to determine if TGFBR2 inactivation was the cause of these changes, TGFBR2 was reconstituted in an MSI colon cancer cell line, resulting in decreased proliferation and decreased cdk4 expression and kinase activity. These results suggest that TGFBR2 mutations in primary colon cancers may be responsible for the increased proliferation and cdk4 expression in these tumors and provide evidence that deregulation of cdk4 is a pathogenic in vivo consequence of TGFBR2 inactivation in primary colon cancer. PMID:16108056

  12. Inhibition of Cdk5 in the nucleus accumbens enhances the locomotor-activating and incentive-motivational effects of cocaine.

    PubMed

    Taylor, Jane R; Lynch, Wendy J; Sanchez, Hayde; Olausson, Peter; Nestler, Eric J; Bibb, James A

    2007-03-01

    Neuronal adaptations in striatal dopamine signaling have been implicated in enhanced responses to addictive drugs. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine signaling and is a downstream target gene of the transcription factor DeltaFosB, which accumulates in striatal neurons after chronic cocaine exposure. Here we investigated the role of Cdk5 activity in the nucleus accumbens (NAc) on cocaine-induced locomotor sensitization, responding for reward-associated stimuli (conditioned reinforcement), and cocaine self-administration under a progressive ratio schedule. Repeated infusions of the Cdk5 inhibitor roscovitine into the NAc before cocaine injections augmented both the development and expression of cocaine sensitization without having any intrinsic stimulant actions of its own. Additionally, repeated intra-NAc infusions of roscovitine to saline-injected rats enhanced locomotor responses to a subsequent cocaine challenge. Similar effects were found after infusions of another Cdk5 inhibitor, olomoucine, but not its inactive congener, iso-olomoucine. Repeated inhibition of Cdk5 within the NAc also robustly enhanced the incentive-motivational effects of cocaine, similar to the effect of prior repeated cocaine exposure. The enhanced responding with conditioned reinforcement induced by cocaine persisted at least 2 weeks after the final roscovitine infusion. NAc infusions of olomoucine also produced acute and enduring increases in "breakpoints" achieved on a progressive ratio schedule for cocaine reinforcement. These results demonstrate profound and persistent effects of NAc Cdk5 inhibition on locomotor sensitization and incentive-motivational processes and provide direct evidence for a role for striatal Cdk5-induced alterations in the brain's long-term adaptations to cocaine. PMID:17360491

  13. Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

    PubMed Central

    Hou, Hailong; Sun, Lu; Siddoway, Benjamin A.; Petralia, Ronald S.; Yang, Hongtian; Gu, Hua; Nairn, Angus C.

    2013-01-01

    The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-d-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity. PMID:24189275

  14. Targeting the AKT/GSK3{beta}/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy

    SciTech Connect

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

    2011-06-01

    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 GSK3{beta}-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3{beta}/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/GSK3{beta}/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor

  15. Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5.

    PubMed

    Bibb, J A; Chen, J; Taylor, J R; Svenningsson, P; Nishi, A; Snyder, G L; Yan, Z; Sagawa, Z K; Ouimet, C C; Nairn, A C; Nestler, E J; Greengard, P

    2001-03-15

    Cocaine enhances dopamine-mediated neurotransmission by blocking dopamine re-uptake at axon terminals. Most dopamine-containing nerve terminals innervate medium spiny neurons in the striatum of the brain. Cocaine addiction is thought to stem, in part, from neural adaptations that act to maintain equilibrium by countering the effects of repeated drug administration. Chronic exposure to cocaine upregulates several transcription factors that alter gene expression and which could mediate such compensatory neural and behavioural changes. One such transcription factor is DeltaFosB, a protein that persists in striatum long after the end of cocaine exposure. Here we identify cyclin-dependent kinase 5 (Cdk5) as a downstream target gene of DeltaFosB by use of DNA array analysis of striatal material from inducible transgenic mice. Overexpression of DeltaFosB, or chronic cocaine administration, raised levels of Cdk5 messenger RNA, protein, and activity in the striatum. Moreover, injection of Cdk5 inhibitors into the striatum potentiated behavioural effects of repeated cocaine administration. Our results suggest that changes in Cdk5 levels mediated by DeltaFosB, and resulting alterations in signalling involving D1 dopamine receptors, contribute to adaptive changes in the brain related to cocaine addiction. PMID:11268215

  16. Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

    PubMed Central

    Lee, Young-Sam; Huang, Kexin; Quiocho, Florante A; O’Shea, Erin K

    2008-01-01

    When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin–cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action. PMID:18059263

  17. Regulation of CDK4 activity by a novel CDK4-binding protein, p34SEI-1

    PubMed Central

    Sugimoto, Masataka; Nakamura, Takeshi; Ohtani, Naoko; Hampson, Lynne; Hampson, Ian N.; Shimamoto, Akira; Furuichi, Yasuhiro; Okumura, Ko; Niwa, Shinichiro; Taya, Yoichi; Hara, Eiji

    1999-01-01

    The p16INK4a tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6). Here we report the isolation of a novel gene, SEI-1, whose product (p34SEI-1) appears to antagonize the function of p16INK4a. Addition of p34SEI-1 to cyclin D1–CDK4 renders the complex resistant to inhibition by p16INK4a. Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34SEI-1 enables fibroblasts to proliferate even in low serum concentrations. p34SEI-1 seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D–CDK complexes in the face of inhibitory levels of INK4 proteins. PMID:10580009

  18. Cyclin B–Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase–independent mechanism

    PubMed Central

    Okumura, Eiichi; Morita, Atsushi; Wakai, Mizuho; Mochida, Satoru; Hara, Masatoshi

    2014-01-01

    Entry into M phase is governed by cyclin B–Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B–Cdk1–dependent inhibition of the protein phosphatase 2A (PP2A)–B55, which antagonizes cyclin B–Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B–Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B–Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B–Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B–Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles. PMID:24616226

  19. KNIME-CDK: Workflow-driven cheminformatics

    PubMed Central

    2013-01-01

    Background Cheminformaticians have to routinely process and analyse libraries of small molecules. Among other things, that includes the standardization of molecules, calculation of various descriptors, visualisation of molecular structures, and downstream analysis. For this purpose, scientific workflow platforms such as the Konstanz Information Miner can be used if provided with the right plug-in. A workflow-based cheminformatics tool provides the advantage of ease-of-use and interoperability between complementary cheminformatics packages within the same framework, hence facilitating the analysis process. Results KNIME-CDK comprises functions for molecule conversion to/from common formats, generation of signatures, fingerprints, and molecular properties. It is based on the Chemistry Development Toolkit and uses the Chemical Markup Language for persistence. A comparison with the cheminformatics plug-in RDKit shows that KNIME-CDK supports a similar range of chemical classes and adds new functionality to the framework. We describe the design and integration of the plug-in, and demonstrate the usage of the nodes on ChEBI, a library of small molecules of biological interest. Conclusions KNIME-CDK is an open-source plug-in for the Konstanz Information Miner, a free workflow platform. KNIME-CDK is build on top of the open-source Chemistry Development Toolkit and allows for efficient cross-vendor structural cheminformatics. Its ease-of-use and modularity enables researchers to automate routine tasks and data analysis, bringing complimentary cheminformatics functionality to the workflow environment. PMID:24103053

  20. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6.

    PubMed

    Uras, Iris Z; Walter, Gina J; Scheicher, Ruth; Bellutti, Florian; Prchal-Murphy, Michaela; Tigan, Anca S; Valent, Peter; Heidel, Florian H; Kubicek, Stefan; Scholl, Claudia; Fröhling, Stefan; Sexl, Veronika

    2016-06-01

    Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD(+) acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration-approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors. PMID:27099147

  1. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

    PubMed Central

    Uras, Iris Z.; Walter, Gina J.; Scheicher, Ruth; Bellutti, Florian; Prchal-Murphy, Michaela; Tigan, Anca S.; Valent, Peter; Heidel, Florian H.; Kubicek, Stefan; Scholl, Claudia; Fröhling, Stefan

    2016-01-01

    Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD+ acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration–approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors. PMID:27099147

  2. Regulation of CDK9 activity by phosphorylation and dephosphorylation.

    PubMed

    Nekhai, Sergei; Petukhov, Michael; Breuer, Denitra

    2014-01-01

    HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1. PMID:24524087

  3. CDK5RAP2 is required for spindle checkpoint function.

    PubMed

    Zhang, Xiaoying; Liu, Dongyun; Lv, Shuang; Wang, Haibo; Zhong, Xueyan; Liu, Bo; Wang, Bo; Liao, Ji; Li, Jing; Pfeifer, Gerd P; Xu, Xingzhi

    2009-04-15

    The combination of paclitaxel and doxorubicin is among the most successful chemotherapy regimens in cancer treatment. CDK5RAP2, when mutated, causes primary microcephaly. We show here that inhibition of CDK5RAP2 expression causes chromosome mis-segregation, fails to maintain the spindle checkpoint, and is associated with reduced expression of the spindle checkpoint proteins BUBR1 and MAD2 and an increase in chromatin-associated CDC20. CDK5RAP2 resides on the BUBR1 and MAD2 promoters and regulates their transcription. Furthermore, CDK5RAP2-knockdown cells have increased resistance to paclitaxel and doxorubicin, and this resistance is partially rescued upon restoration of CDK5RAP2 expression. Cancer cells cultured in the presence of paclitaxel or doxorubicin exhibit dramatically decreased CDK5RAP2 levels. These results suggest that CDK5RAP2 is required for spindle checkpoint function and is a common target in paclitaxel and doxorubicin resistance. PMID:19282672

  4. Functional, chemical genomic, and super-enhancer screening identify sensitivity to cyclin D1/CDK4 pathway inhibition in Ewing sarcoma

    PubMed Central

    Crompton, Brian; Cowley, Glenn; Vazquez, Francisca; Weir, Barbara A.; Tsherniak, Aviad; Parasuraman, Sudha; Kim, Sunkyu; Alexe, Gabriela; Stegmaier, Kimberly

    2015-01-01

    Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1. While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease. PMID:26337082

  5. Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread—Identification of a Promising Novel therapeutic target1

    PubMed Central

    Bisht, Savita; Nolting, Jens; Schütte, Ute; Haarmann, Jens; Jain, Prashi; Shah, Dhruv; Brossart, Peter; Flaherty, Patrick; Feldmann, Georg

    2015-01-01

    Despite considerable progress in recent years, the overall prognosis of metastatic malignant melanoma remains poor, and curative therapeutic options are lacking. Therefore, better understanding of molecular mechanisms underlying melanoma progression and metastasis, as well as identification of novel therapeutic targets that allow inhibition of metastatic spread, are urgently required. The current study provides evidence for aberrant cyclin-dependent kinase 5 (CDK5) activation in primary and metastatic melanoma lesions by overexpression of its activator protein CDK5R1/p35. Moreover, using melanoma in vitro model systems, shRNA-mediated inducible knockdown of CDK5 was found to cause marked inhibition of cell motility, invasiveness, and anchorage-independent growth, while at the same time net cell growth was not affected. In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases. Inhibition of lung metastasis was further validated in a syngenic murine melanoma model. CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype. Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells. Thus, experimental data presented here provide strong evidence for a crucial role of aberrantly activated CDK5 in melanoma progression and metastasis and establish CDK5 as promising target for therapeutic intervention. PMID:26310376

  6. Transforming growth factor beta 1 increases the stability of p21/WAF1/CIP1 protein and inhibits CDK2 kinase activity in human colon carcinoma FET cells.

    PubMed

    Gong, JianGen; Ammanamanchi, Sudhakar; Ko, Tien C; Brattain, Michael G

    2003-06-15

    We examined transforming growth factor-beta 1 (TGF-beta 1) effects on cell cycle progression of human colon carcinoma FET cells. TGF-beta 1 inhibited DNA synthesis and cyclin-dependent kinase (CDK) activity after release from growth arrest in association with induction of the p21 CDK inhibitor, whereas cyclins, CDKs, and p27 protein levels remained relatively unchanged. The decrease in CDK2 kinase activity was the result of increased p21 association with cyclin A-CDK2 and cyclin E-CDK2. TGF-beta 1 treatment in late G(1) showed reduced induction of p21 protein levels in association with increased DNA synthesis. Consequently, p21 induction in early G(1) is critical for TGF-beta 1 inhibition of CDK2 kinase activity. Although TGF-beta 1 treatments in late G(1) failed to induce p21 protein, p21 mRNA induction was observed in late G(1) and in S phase. Further analysis showed that TGF-beta 1 treatment in early G(1) increases p21 protein stability throughout the G(1) and S phases of the cell cycle. Our results demonstrate that TGF-beta 1 stimulation of p21 is regulated at the posttranscriptional and transcriptional levels. This is a novel mechanism of TGF-beta 1 inhibition requiring early G(1) induction and stabilization of p21 protein, which binds to and inhibits cyclin E-CDK2 and cyclin A-CDK2 kinase activity rather than direct modulation of cyclin or CDK protein levels as seen in other systems. PMID:12810668

  7. NSun2 delays replicative senescence by repressing p27 (KIP1) translation and elevating CDK1 translation

    PubMed Central

    Xing, Junyue; Liu, Zhenyun; Jiang, Bin; Dou, Yali; Gorospe, Myriam; Wang, Wengong

    2015-01-01

    A rise in the levels of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is important for the growth arrest of senescent cells, but the mechanisms responsible for this increase are poorly understood. Here, we show that the tRNA methyltransferase NSun2 represses the expression of p27 in replicative senescence. NSun2 methylated the 5′-untranslated region (UTR) of p27 mRNA at cytosine C64 in vitro and in cells, thereby repressing the translation of p27. During replicative senescence, increased p27 protein levels were accompanied by decreased NSun2 protein levels. Knockdown of NSun2 in human diploid fibroblasts (HDFs) elevated p27 levels and reduced the expression of CDK1 (encoded by CDK1 mRNA, a previously reported target of NSun2), which in turn further repressed cell proliferation and accelerated replicative senescence, while overexpression of NSun2 exerted the opposite effect. Ectopic overexpression of the p27 5′UTR fragment rescued the effect of NSun2 overexpression in lowering p27, increasing CDK1, promoting cell proliferation, and delaying replicative senescence. Our findings indicate that NSun2-mediated mRNA methylation regulates p27 and CDK1 levels during replicative senescence. PMID:26687548

  8. Cyclin E/Cdk2-dependent phosphorylation of Mcl-1 determines its stability and cellular sensitivity to BH3 mimetics

    PubMed Central

    Choudhary, Gaurav S.; Tat, Trinh T.; Misra, Saurav; Hill, Brian T.; Smith, Mitchell R.; Almasan, Alexandru; Mazumder, Suparna

    2015-01-01

    Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E−/− mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors. PMID:26219338

  9. Enhanced sensitivity to irinotecan by Cdk1 inhibition in the p53-deficient HT29 human colon cancer cell line.

    PubMed

    Abal, Miguel; Bras-Goncalves, Rui; Judde, Jean-Gabriel; Fsihi, Hafida; De Cremoux, Patricia; Louvard, Daniel; Magdelenat, Henri; Robine, Sylvie; Poupon, Marie-France

    2004-03-01

    Mutations in the tumor-suppressor gene p53 have been associated with advanced colorectal cancer (CRC). Irinotecan (CPT-11), a DNA topoisomerase 1 inhibitor, has been recently incorporated to the adjuvant therapy. Since the DNA-damage checkpoint depends on p53 activation, the status of p53 might critically influence the response to CPT-11. We analysed the sensitivity to CPT-11 in the human colon cancer cell line HT29 (mut p53) and its wild-type (wt)-p53 stably transfected subclone HT29-A4. Cell-cycle analysis in synchronised cells demonstrated the activation of transfected wt-p53 and a p21(WAF1/CIP1)-dependent cell-cycle blockage in the S phase. Activated wt-p53 increased apoptosis and enhanced sensitivity to CPT-11. In p53-deficient cells, cDNA-macroarray analysis and western blotting showed an accumulation of the cyclin-dependent kinase (cdk)1/cyclin B complex. Subsequent p53-independent activation of the cdk-inhibitor (cdk-I) p21(WAF1/CIP1) prevented cell-cycle progression. Cdk1 induction was exploited in vivo to improve the sensitivity to CPT-11 by additional treatment with the cdk-I CYC-202. We demonstrate a gain of sensitivity to CPT-11 in a p53-mutated colon cancer model either by restoring wild-type p53 function or by sequential treatment with cdk-Is. Considering that mutations in p53 are among the most common genetic alterations in CRC, a therapeutic approach specifically targeting p53-deficient tumors could greatly improve the treatment outcomes. PMID:15001986

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

    PubMed

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

    2016-03-01

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

  11. Mechanism of p27 Unfolding for CDK2 Reactivation

    PubMed Central

    Rath, Soumya Lipsa; Senapati, Sanjib

    2016-01-01

    Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid β-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27. PMID:27211815

  12. Functional specialization of chordate CDK1 paralogs during oogenic meiosis

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  14. Mechanism of p27 Unfolding for CDK2 Reactivation.

    PubMed

    Rath, Soumya Lipsa; Senapati, Sanjib

    2016-01-01

    Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid β-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27. PMID:27211815

  15. Phylogenetic analysis of CDK and cyclin proteins in premetazoan lineages

    PubMed Central

    2014-01-01

    Background The molecular history of animal evolution from single-celled ancestors remains a major question in biology, and little is known regarding the evolution of cell cycle regulation during animal emergence. In this study, we conducted a comprehensive evolutionary analysis of CDK and cyclin proteins in metazoans and their unicellular relatives. Results Our analysis divided the CDK family into eight subfamilies. Seven subfamilies (CDK1/2/3, CDK5, CDK7, CDK 20, CDK8/19, CDK9, and CDK10/11) are conserved in metazoans and fungi, with the remaining subfamily, CDK4/6, found only in eumetazoans. With respect to cyclins, cyclin C, H, L, Y subfamilies, and cyclin K and T as a whole subfamily, are generally conserved in animal, fungi, and amoeba Dictyostelium discoideum. In contrast, cyclin subfamilies B, A, E, and D, which are cell cycle-related, have distinct evolutionary histories. The cyclin B subfamily is generally conserved in D. discoideum, fungi, and animals, whereas cyclin A and E subfamilies are both present in animals and their unicellular relatives such as choanoflagellate Monosiga brevicollis and filasterean Capsaspora owczarzaki, but are absent in fungi and D. discoideum. Although absent in fungi and D. discoideum, cyclin D subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan Thecamonas trahens. Within opisthokonta, the cyclin D subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan Amphimedon queenslandica. Conclusions Our data indicate that the CDK4/6 subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin D subfamily also tightly linked with eumetazoan appearance. Establishment of the CDK4/6-cyclin D complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence. PMID:24433236

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

    PubMed

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

    2016-07-01

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

  17. 6,7,4'-trihydroxyisoflavone inhibits HCT-116 human colon cancer cell proliferation by targeting CDK1 and CDK2.

    PubMed

    Lee, Dong Eun; Lee, Ki Won; Jung, Sung Keun; Lee, Eun Jung; Hwang, Jung A; Lim, Tae-Gyu; Kim, Bo Yeon; Bode, Ann M; Lee, Hyong Joo; Dong, Zigang

    2011-04-01

    Colon cancer is a common epithelial malignancies worldwide. Epidemiologic evidence has shown that nutrition and dietary components are important environmental factors involved in the development of this disease. We investigated the biological activity of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF, a metabolite of daidzein) in in vitro and in vivo models of human colon cancer. 6,7,4'-THIF suppressed anchorage-dependent and -independent growth of HCT-116 and DLD1 human colon cancer cells more effectively than daidzein. In addition, 6,7,4'-THIF induced cell cycle arrest at the S and G2/M phases in HCT-116 human colon cancer cells. Western blot analysis revealed that 6,7,4'-THIF effectively suppressed the expression of cyclin-dependent kinase (CDK) 2, but had no effect on other S- or G2/M-phase regulatory proteins such as cyclin A, cyclin B1 or CDK1. Daidzein did not affect the expression of any of these proteins. In kinase and pull-down assays, 6,7,4'-THIF, but not daidzein, inhibited CDK1 and CDK2 activities in HCT-116 cells by directly interacting with CDK1 and CDK2. In a xenograft mouse model, 6,7,4'-THIF significantly decreased tumor growth, volume and weight of HCT-116 xenografts. 6,7,4'-THIF bound directly to CDK1 and CDK2 in vivo, resulting in the suppression of CDK1 and CDK2 activity in tumors corresponding with our in vitro results. Collectively, these results suggest that CDK1 and CDK2 are potential molecular targets of 6,7,4'-THIF to suppress HCT-116 cell proliferation in vitro and in vivo. These findings provide insight into the biological actions of 6,7,4'-THIF and might establish a molecular basis for the development of new cancer therapeutic agents. PMID:21258042

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

    SciTech Connect

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

    2012-08-17

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

  19. Downregulation of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by silica is mediated through the ERK and JNK pathway.

    PubMed

    Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Gao, Ai; Du, Hongju; Ye, Meng; You, Baorong; Huang, Chuanshu; Shi, Xianglin

    2008-10-01

    Silica is a factor in the induction of acute injury and chronic pulmonary fibrosis. In 1996, silica was also listed as a human carcinogen by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved in its pathologic effects are not well understood. We found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica for 2h decreased cyclin D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Extracellular signal-regulated protein kinase (ERKs), c-Jun NH2-terminal amino kinase (JNKs), and p38 kinase, as well as their downstream transcription factor, AP-1, had different effects on the regulation of expression levels of cyclin D1 and CDK4 alterations induced by silica. Silica activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We established the requirements for ERK and JNK, members of the mitogen-activated protein kinase (MAPK) family, in mediating G1 phase arrest of HELF induced by silica. Silica treatment activated ERK in a dose-dependent manner. AG126 (a chemical inhibitor of the ERK signaling pathway) and the dominant negative mutant of ERK2 (a molecular inhibitor of ERK2) prevented decreases in cyclin D1 and CDK4 expression levels. A chemical inhibitor of JNK, SP600125, prevented the decreased expression of both cyclin D1 and CDK4, whereas SB203580, a chemical inhibitor of p38, did not. Interestingly, curcumin prevented the decrease in DK4 expression, but not in cyclin D1. These results demonstrate that ERKs and JNKs are responsible for the decrease of cyclin D1 and CDK4 expression levels in HELF induced by silica. Activator protein-1 (AP-1) was responsible for the decrease of CDK4 expression level, but not that of cyclin D1. The findings help to explain the mechanisms of diseases induced by silica. PMID:18703151

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

    PubMed

    Albert, Thomas K; Antrecht, Claudia; Kremmer, Elisabeth; Meisterernst, Michael

    2016-01-01

    CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription. PMID:26745862

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

    PubMed Central

    Albert, Thomas K.; Antrecht, Claudia; Kremmer, Elisabeth; Meisterernst, Michael

    2016-01-01

    CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed—with significant delay—by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription. PMID:26745862

  2. CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development.

    PubMed

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

    2008-08-01

    It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21(Cip1) and p27(Kip1). Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4(D158N) mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4(D158N), but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21(Cip1) in K5Cdk2, but not in K5Cdk4(D158N), epidermis, suggesting that CDK2 overexpression elicits a p21(Cip1) response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis. PMID:18599613

  3. Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics.

    PubMed

    de la Fuente, Cynthia; Maddukuri, Anil; Kehn, Kylene; Baylor, Shanese Y; Deng, Longwen; Pumfery, Anne; Kashanchi, Fatah

    2003-04-01

    Human immunodeficiency virus type 1 (HIV-1) can infect quiescent cells; however, viral production is restricted to actively proliferating cells. Recent evidence has indicated that HIV-1 viral proteins, Vpr and Tat, perturb the cell cycle to optimize HIV-1 replication. Vpr arrests the cell cycle at G2 by inactivating the cyclin B/cdk1 complex. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation (i.e. the cdk inhibitor p21/waf1) and associating with cyclin/cdk complexes (i.e. cyclin E/cdk2, cyclin H/cdk7, and cyclin T/cdk9). These studies indicate the importance of host cellular factors, such as cyclin/cdk complexes, in regulating HIV-1 replication and therefore represent novel targets for antiviral therapeutics. Recently, the efficacy of pharmalogical cdk inhibitors (PCIs) in abrogating viral replication has been under development. To date there are 25-30 PCIs that have been synthesized against known cdks, several of which have been shown to inhibit HIV-1 and other AIDS-associated viruses in vitro and in vivo. Targeting these critical cyclin/cdk complexes needed for viral propagation may solve the problems inherent in current HAART therapy, including the emergence of drug-resistant viruses. Thus, PCIs have the potential to become novel therapeutic antiviral drugs that can inhibit HIV-1 transcription and opens the possibility of new avenues of treatment. PMID:15043199

  4. Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases

    PubMed Central

    Schonbrunn, Ernst; Betzi, Stephane; Alam, Riazul; Martin, Mathew P.; Becker, Andreas; Han, Huijong; Francis, Rawle; Chakrasali, Ramappa; Jakkaraj, Sudhakar; Kazi, Aslamuzzaman; Sebti, Said M.; Cubitt, Christopher L.; Gebhard, Anthony W.; Hazlehurst, Lori A.; Tash, Joseph S.; Georg, Gunda I.

    2013-01-01

    Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009 – 0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 co-crystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4 and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics. PMID:23600925

  5. Cyclin D1-Cdk4 controls glucose metabolism independently of cell cycle progression.

    PubMed

    Lee, Yoonjin; Dominy, John E; Choi, Yoon Jong; Jurczak, Michael; Tolliday, Nicola; Camporez, Joao Paulo; Chim, Helen; Lim, Ji-Hong; Ruan, Hai-Bin; Yang, Xiaoyong; Vazquez, Francisca; Sicinski, Piotr; Shulman, Gerald I; Puigserver, Pere

    2014-06-26

    Insulin constitutes a principal evolutionarily conserved hormonal axis for maintaining glucose homeostasis; dysregulation of this axis causes diabetes. PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α) links insulin signalling to the expression of glucose and lipid metabolic genes. The histone acetyltransferase GCN5 (general control non-repressed protein 5) acetylates PGC-1α and suppresses its transcriptional activity, whereas sirtuin 1 deacetylates and activates PGC-1α. Although insulin is a mitogenic signal in proliferative cells, whether components of the cell cycle machinery contribute to its metabolic action is poorly understood. Here we report that in mice insulin activates cyclin D1-cyclin-dependent kinase 4 (Cdk4), which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high-throughput chemical screen, we identify a Cdk4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK-3β (glycogen synthase kinase 3-beta) signalling induces cyclin D1 protein stability by sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 messenger RNA transcripts. Activated cyclin D1-Cdk4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycaemia. In diabetic models, cyclin D1-Cdk4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycaemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division. PMID:24870244

  6. Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    PubMed Central

    Lee, Yoonjin; Dominy, John E.; Choi, Yoon Jong; Jurczak, Michael; Tolliday, Nicola; Camporez, Joao Paulo; Chim, Helen; Lim, Ji-Hong; Ruan, Hai-Bin; Yang, Xiaoyong; Vazquez, Francisca; Sicinski, Piotr; Shulman, Gerald I.; Puigserver, Pere

    2014-01-01

    Insulin constitutes a major evolutionarily conserved hormonal axis for maintaining glucose homeostasis1-3; dysregulation of this axis causes diabetes2,4. PGC-1α links insulin signaling to the expression of glucose and lipid metabolic genes5-7. GCN5 acetylates PGC-1α and suppresses its transcriptional activity, whereas SIRT1 deacetylates and activates PGC-1α8,9. Although insulin is a mitogenic signal in proliferative cells10,11, whether components of the cell cycle machinery contribute to insulin’s metabolic action is poorly understood. Herein, we report that insulin activates cyclin D1-CDK4, which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high throughput chemical screen, we identified a CDK4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK3β signaling induces cyclin D1 protein stability via sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 mRNA transcripts. Activated cyclin D1-CDK4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycemia. In diabetic models, cyclin D1-CDK4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division. PMID:24870244

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

    PubMed

    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. PMID:25277198

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

    PubMed Central

    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

    2014-01-01

    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. PMID:25277198

  9. Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2

    PubMed Central

    Du, William W.; Yang, Weining; Liu, Elizabeth; Yang, Zhenguo; Dhaliwal, Preet; Yang, Burton B.

    2016-01-01

    Most RNAs generated by the human genome have no protein-coding ability and are termed non-coding RNAs. Among these include circular RNAs, which include exonic circular RNAs (circRNA), mainly found in the cytoplasm, and intronic RNAs (ciRNA), predominantly detected in the nucleus. The biological functions of circular RNAs remain largely unknown, although ciRNAs have been reported to promote gene transcription, while circRNAs may function as microRNA sponges. We demonstrate that the circular RNA circ-Foxo3 was highly expressed in non-cancer cells and were associated with cell cycle progression. Silencing endogenous circ-Foxo3 promoted cell proliferation. Ectopic expression of circ-Foxo3 repressed cell cycle progression by binding to the cell cycle proteins cyclin-dependent kinase 2 (also known as cell division protein kinase 2 or CDK2) and cyclin-dependent kinase inhibitor 1 (or p21), resulting in the formation of a ternary complex. Normally, CDK2 interacts with cyclin A and cyclin E to facilitate cell cycle entry, while p21works to inhibit these interactions and arrest cell cycle progression. The formation of this circ-Foxo3-p21-CDK2 ternary complex arrested the function of CDK2 and blocked cell cycle progression. PMID:26861625

  10. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis.

    PubMed

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  11. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis

    PubMed Central

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D.; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  12. Pharmacological inhibitors of cyclin-dependent kinases.

    PubMed

    Knockaert, Marie; Greengard, Paul; Meijer, Laurent

    2002-09-01

    Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription and differentiation in addition to functions in the nervous system. Deregulation of CDKs in various diseases has stimulated an intensive search for selective pharmacological inhibitors of these kinases. More than 50 inhibitors have been identified, among which >20 have been co-crystallized with CDK2. These inhibitors all target the ATP-binding pocket of the catalytic site of the kinase. The actual selectivity of most known CDK inhibitors, and thus the underlying mechanism of their cellular effects, is poorly known. Pharmacological inhibitors of CDKs are currently being evaluated for therapeutic use against cancer, alopecia, neurodegenerative disorders (e.g. Alzheimer's disease, amyotrophic lateral sclerosis and stroke), cardiovascular disorders (e.g. atherosclerosis and restenosis), glomerulonephritis, viral infections (e.g. HCMV, HIV and HSV) and parasitic protozoa (Plasmodium sp. and Leishmania sp.). PMID:12237154

  13. Abnormal expression of CDK11p58 in prostate cancer

    PubMed Central

    2014-01-01

    Background CDK11p58 is one of the large families of p34cdc2-related kinases whose functions are linked with cell cycle progression, tumorigenesis and apoptotic signaling. Our previous investigation demonstrated that CDK11p58 repressed androgen receptor (AR) transcriptional activity and was involved in the negative regulation of AR function. Methods CDK11p58 expression was examined in the prostate cancer tissues and adjacent tissues by IHC and qRT-PCR. Cell apoptosis was detected by flow cytometry. The metastasis of cancer cells was evaluated by the Transwell Assay. Finally we further investigated the underlying molecular mechanisms by examining expression levels of relevant proteins using western blot analysis. Results We found that both RNA and protein expression of CDK11p58 were low in prostate cancer tissues compared with its adjacent noncancerous tissues. CDK11p58 promoted the prostate cancer cell apoptosis and inhibited its metastasis in a kinase dependent way. And finally CDK11p58 could inhibit the metastasis of AR positive prostate cancer cells through inhibition of integrin β3 and MMP2. Conclusions These data indicate that CDK11p58 is an anti-metastasis gene product in prostate cancer. PMID:24397471

  14. CDK4 is an essential insulin effector in adipocytes

    PubMed Central

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

    2015-01-01

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

  15. Physiological and pathological phosphorylation of tau by Cdk5.

    PubMed

    Kimura, Taeko; Ishiguro, Koichi; Hisanaga, Shin-Ichi

    2014-01-01

    Hyperphosphorylation of microtubule-associated protein tau is one of the major pathological events in Alzheimer's disease (AD) and other related neurodegenerative diseases, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Mutations in the tau gene MAPT are a cause of FTDP-17, and the mutated tau proteins are hyperphosphorylated in patient brains. Thus, it is important to determine the molecular mechanism of hyperphosphorylation of tau to understand the pathology of these diseases collectively called tauopathy. Tau is phosphorylated at many sites via several protein kinases, and a characteristic is phosphorylation at Ser/Thr residues in Ser/Thr-Pro sequences, which are targeted by proline-directed protein kinases such as ERK, GSK3β, and Cdk5. Among these kinases, Cdk5 is particularly interesting because it could be abnormally activated in AD. Cdk5 is a member of the cyclin-dependent kinases (Cdks), but in contrast to the major Cdks, which promote cell cycle progression in proliferating cells, Cdk5 is activated in post-mitotic neurons via the neuron-specific activator p35. Cdk5-p35 plays a critical role in brain development and physiological synaptic activity. In contrast, in disease brains, Cdk5 is thought to be hyperactivated by p25, which is the N-terminal truncated form of p35 and is generated by cleavage with calpain. Several reports have indicated that tau is hyperphosphorylated by Cdk5-p25. However, normal and abnormal phosphorylation of tau by Cdk5 is still not completely understood. In this article, we summarize the physiological and pathological phosphorylation of tau via Cdk5. PMID:25076872

  16. Physiological and pathological phosphorylation of tau by Cdk5

    PubMed Central

    Kimura, Taeko; Ishiguro, Koichi; Hisanaga, Shin-ichi

    2014-01-01

    Hyperphosphorylation of microtubule-associated protein tau is one of the major pathological events in Alzheimer’s disease (AD) and other related neurodegenerative diseases, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Mutations in the tau gene MAPT are a cause of FTDP-17, and the mutated tau proteins are hyperphosphorylated in patient brains. Thus, it is important to determine the molecular mechanism of hyperphosphorylation of tau to understand the pathology of these diseases collectively called tauopathy. Tau is phosphorylated at many sites via several protein kinases, and a characteristic is phosphorylation at Ser/Thr residues in Ser/Thr-Pro sequences, which are targeted by proline-directed protein kinases such as ERK, GSK3β, and Cdk5. Among these kinases, Cdk5 is particularly interesting because it could be abnormally activated in AD. Cdk5 is a member of the cyclin-dependent kinases (Cdks), but in contrast to the major Cdks, which promote cell cycle progression in proliferating cells, Cdk5 is activated in post-mitotic neurons via the neuron-specific activator p35. Cdk5-p35 plays a critical role in brain development and physiological synaptic activity. In contrast, in disease brains, Cdk5 is thought to be hyperactivated by p25, which is the N-terminal truncated form of p35 and is generated by cleavage with calpain. Several reports have indicated that tau is hyperphosphorylated by Cdk5-p25. However, normal and abnormal phosphorylation of tau by Cdk5 is still not completely understood. In this article, we summarize the physiological and pathological phosphorylation of tau via Cdk5. PMID:25076872

  17. p57(Kip2) knock-in mouse reveals CDK-independent contribution in the development of Beckwith-Wiedemann syndrome.

    PubMed

    Duquesnes, Nicolas; Callot, Caroline; Jeannot, Pauline; Daburon, Virginie; Nakayama, Keiichi I; Manenti, Stephane; Davy, Alice; Besson, Arnaud

    2016-07-01

    CDKN1C encodes the cyclin-CDK inhibitor p57(Kip2) (p57), a negative regulator of the cell cycle and putative tumour suppressor. Genetic and epigenetic alterations causing loss of p57 function are the most frequent cause of Beckwith-Wiedemann syndrome (BWS), a genetic disorder characterized by multiple developmental anomalies and increased susceptibility to tumour development during childhood. So far, BWS development has been attributed entirely to the deregulation of proliferation caused by loss of p57-mediated CDK inhibition. However, a fraction of BWS patients have point mutations in CDKN1C located outside of the CDK inhibitory region, suggesting the involvement of other parts of the protein in the disease. To test this possibility, we generated knock-in mice deficient for p57-mediated cyclin-CDK inhibition (p57(CK) (-) ), the only clearly defined function of p57. Comparative analysis of p57(CK) (-) and p57(KO) mice provided clear evidence for CDK-independent roles of p57 and revealed that BWS is not caused entirely by CDK deregulation, as several features of BWS are caused by the loss of CDK-independent roles of p57. Thus, while the genetic origin of BWS is well understood, our results underscore that the underlying molecular mechanisms remain largely unclear. To probe these mechanisms further, we determined the p57 interactome. Several partners identified are involved in genetic disorders with features resembling those caused by CDKN1C mutation, suggesting that they could be involved in BWS pathogenesis and revealing a possible connection between seemingly distinct syndromes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27015986

  18. RNA interference directed to CDK2 inhibits HIV-1 transcription.

    PubMed

    Ammosova, Tatyana; Berro, Reem; Kashanchi, Fatah; Nekhai, Sergei

    2005-10-25

    We previously reported that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro. In the present study, CDK2-specific RNA interference in cultured HEK293T cells inhibited CDK2 expression and Tat-induced HIV-1 transcription from non-integrated HIV-1 promoter but not basal HIV-1 transcription or transcription from CMV or beta-actin promoters. Also, CDK2-specific RNA interference inhibited Tat-induced transcription from the integrated HIV-1 promoter in HeLa-CD4-LTR-beta-gal cells and potently blocked TNFalpha-induced HIV-1 viral replication in OM10.1 cells. CDK2-specific RNA interference did not have an effect on cell cycle progression, but it augmented TNFalpha-induced apoptosis of OM10.1 cells. Our results indicate that CDK2 participates in Tat-mediated HIV-1 transcription and may serve as a potential therapeutic target. PMID:16085226

  19. Mutations in CDK5RAP2 cause Seckel syndrome

    PubMed Central

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

    2015-01-01

    Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152. PMID:26436113

  20. Mutations in CDK5RAP2 cause Seckel syndrome.

    PubMed

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

    2015-09-01

    Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152. PMID:26436113

  1. An RNAi-based screen reveals PLK1, CDK1 and NDC80 as potential therapeutic targets in malignant pleural mesothelioma

    PubMed Central

    Linton, A; Cheng, Y Y; Griggs, K; Kirschner, M B; Gattani, S; Srikaran, S; Chuan-Hao Kao, S; McCaughan, B C; Klebe, S; van Zandwijk, N; Reid, G

    2014-01-01

    Background: Malignant pleural mesothelioma (MPM) is an aggressive tumour originating in the thoracic mesothelium. Prognosis remains poor with 9- to 12-month median survival, and new targets for treatments are desperately needed. Methods: Utilising an RNA interference (RNAi)-based screen of 40 genes overexpressed in tumours, including genes involved in the control of cell cycle, DNA replication and repair, we investigated potential therapeutic targets for MPM. Following in vitro characterisation of the effects of target silencing on MPM cells, candidates were assessed in tumour samples from 154 patients. Results: Gene knockdown in MPM cell lines identified growth inhibition following knockdown of NDC80, CDK1 and PLK1. Target knockdown induced cell-cycle arrest and increased apoptosis. Using small-molecule inhibitors specific for these three proteins also led to growth inhibition of MPM cell lines, and Roscovitine (inhibitor of CDK1) sensitised cells to cisplatin. Protein expression was also measured in tumour samples, with markedly variable levels of CDK1 and PLK1 noted. PLK1 expression in over 10% of cells correlated significantly with a poor prognosis. Conclusion: These results suggest that RNAi-based screening has utility in identifying new targets for MPM, and that inhibition of NDC80, CDK1 and PLK1 may hold promise for treatment of this disease. PMID:24327015

  2. Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer

    PubMed Central

    Balakrishnan, Archana; Vyas, Arpita; Deshpande, Kaivalya; Vyas, Dinesh

    2016-01-01

    Colorectal cancer accounts for a significant proportion of cancer deaths worldwide. The need to develop more chemotherapeutic agents to combat this disease is critical. Cyclin dependent kinases (CDKs), along with its binding partner cyclins, serve to control the growth of cells through the cell cycle. A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers like colorectal cancer. CDK article provides overview of the emerging drug class of CDK inhibitors and provides a list of ones that are currently in clinical trials. PMID:26900281

  3. Recurrent CDK1 overexpression in laryngeal squamous cell carcinoma.

    PubMed

    Bednarek, K; Kiwerska, K; Szaumkessel, M; Bodnar, M; Kostrzewska-Poczekaj, M; Marszalek, A; Janiszewska, J; Bartochowska, A; Jackowska, J; Wierzbicka, M; Grenman, R; Szyfter, K; Giefing, M; Jarmuz-Szymczak, M

    2016-08-01

    In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis. PMID:26912061

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

    SciTech Connect

    Bjoerklund, Mia A.; Vaahtomeri, Kari; Peltonen, Karita; Viollet, Benoit; Maekelae, Tomi P.; Band, Arja M.; Laiho, Marikki

    2010-03-10

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

  5. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  6. Trisubstituted Pyrazolopyrimidines as Novel Angiogenesis Inhibitors

    PubMed Central

    Weitensteiner, Sabine B.; Liebl, Johanna; Krystof, Vladimir; Havlíček, Libor; Gucký, Tomáš; Strnad, Miroslav; Fürst, Robert; Vollmar, Angelika M.; Zahler, Stefan

    2013-01-01

    Current inhibitors of angiogenesis comprise either therapeutic antibodies (e.g. bevacicumab binding to VEGF-A) or small molecular inhibitors of receptor tyrosin kinases like e.g. sunitinib, which inhibits PDGFR and VEGFR. We have recently identified cyclin-dependent kinase 5 (Cdk5) as novel alternative and pharmacologically accessible target in the context of angiogenesis. In the present work we demonstrate that trisubstituted pyrazolo[4,3-d]pyrimidines constitute a novel class of compounds which potently inhibit angiogenesis. All seven tested compounds inhibited endothelial cell proliferation with IC50 values between 1 and 18 µM. Interestingly, this seems not to be due to cytotoxicity, since none of them showed acute cytotoxic effects on endothelial cells at a concentration of 10 µM,. The three most potent compounds (LGR1404, LGR1406 and LGR1407) also inhibited cell migration (by 27, 51 and 31%, resp.), chemotaxis (by 50, 70 and 60% in accumulative distance, resp.), and tube formation (by 25, 60 and 30% of total tube length, resp.) at the non-toxic concentration of 10 µM. Furthermore, angiogenesis was reduced in vivo in the CAM assay by these three compounds. A kinase selectivity profiling revealed that the compounds prevalently inhibit Cdk2, Cdk5 and Cdk9. The phenotype of the migrating cells (reduced formation of lamellipodia, loss of Rac-1 translocation to the membrane) resembles the previously described effects of silencing of Cdk5 in endothelial cells. We conclude that especially LGR1406 and LGR1407 are highly attractive anti-angiogenic compounds, whose effects seem to largely depend on their Cdk5 inhibiting properties. PMID:23336010

  7. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

    PubMed Central

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-01-01

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors. PMID:26508632

  8. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila.

    PubMed

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-11-10

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors. PMID:26508632

  9. Novel Alternative Splice Variants of Mouse Cdk5rap2

    PubMed Central

    Kraemer, Nadine; Issa-Jahns, Lina; Neubert, Gerda; Ravindran, Ethiraj; Mani, Shyamala; Ninnemann, Olaf; Kaindl, Angela M.

    2015-01-01

    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. PMID:26322982

  10. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    PubMed

    Kraemer, Nadine; Issa-Jahns, Lina; Neubert, Gerda; Ravindran, Ethiraj; Mani, Shyamala; Ninnemann, Olaf; Kaindl, Angela M

    2015-01-01

    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. PMID:26322982