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

PubMed

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

2018-01-01

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

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

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

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

2013-01-25

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

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

PubMed

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

2015-01-06

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

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

Spring, Laura; Bardia, Aditya; Modi, Shanu

2017-01-01

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

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

PubMed

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

2016-06-01

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

Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

PubMed Central

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

2016-01-01

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

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

PubMed

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

2010-12-17

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

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

PubMed

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

2002-10-01

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

Discovery of potent and selective CDK8 inhibitors through FBDD approach.

PubMed

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

2017-09-15

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

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

PubMed

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

2016-11-01

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

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

PubMed

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

2010-12-23

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

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

PubMed

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

2007-09-01

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

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

PubMed

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

2011-04-01

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

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

PubMed

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

2015-09-01

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

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

PubMed

Lolli, Graziano; Johnson, Louise N

2005-04-01

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

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

PubMed Central

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

2018-01-01

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

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

PubMed

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

2016-02-01

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

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

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

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

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

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

Pisani, Pasquale; Rastelli, Giulio

2016-01-01

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

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

PubMed

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

2016-01-01

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

Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.

PubMed

Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

2016-04-12

Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2014-07-30

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

Evaluation of the neuronal apoptotic pathways involved in cytoskeletal disruption-induced apoptosis.

PubMed

Jordà, Elvira G; Verdaguer, Ester; Jimenez, Andrés; Arriba, S Garcia de; Allgaier, Clemens; Pallàs, Mercè; Camins, Antoni

2005-08-01

The cytoskeleton is critical to neuronal functioning and survival. Cytoskeletal alterations are involved in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We studied the possible pathways involved in colchicine-induced apoptosis in cerebellar granule neurons (CGNs). Although colchicine evoked an increase in caspase-3, caspase-6 and caspase-9 activation, selective caspase inhibitors did not attenuate apoptosis. Inhibitors of other cysteine proteases such as PD150606 (a calpain-specific inhibitor), Z-Phe-Ala fluoromethyl ketone (a cathepsins-inhibitors) and N(alpha)-p-tosyl-l-lysine chloromethyl ketone (serine-proteases inhibitor) also had no effect on cell death/apoptosis induced by colchicine. However, BAPTA-AM 10 microM (intracellular calcium chelator) prevented apoptosis mediated by cytoskeletal alteration. These data indicate that calcium modulates colchicine-induced apoptosis in CGNs. PARP-1 inhibitors did not prevent apoptosis mediated by colchicine. Finally, colchicine-induced apoptosis in CGNs was attenuated by kenpaullone, a cdk5 inhibitor. Kenpaullone and indirubin also prevented cdk5/p25 activation mediated by colchicine. These findings indicate that cytoskeletal alteration can compromise cdk5 activation, regulating p25 formation and suggest that cdk5 inhibitors attenuate apoptosis mediated by cytoskeletal alteration. The present data indicate the potential therapeutic value of drugs that prevent the formation of p25 for the treatment of neurodegenerative disorders.

The Formation of Tight Tumor Clusters Affects the Efficacy of Cell Cycle Inhibitors: A Hybrid Model Study

PubMed Central

Kim, MunJu; Reed, Damon; Rejniak, Katarzyna A.

2014-01-01

Cyclin-dependent kinases (CDKs) are vital in regulating cell cycle progression, and, thus, in highly proliferating tumor cells CDK inhibitors are gaining interest as potential anticancer agents. Clonogenic assay experiments are frequently used to determine drug efficacy against the survival and proliferation of cancer cells. While the anticancer mechanisms of drugs are usually described at the intracellular single-cell level, the experimental measurements are sampled from the entire cancer cell population. This approach may lead to discrepancies between the experimental observations and theoretical explanations of anticipated drug mechanisms. To determine how individual cell responses to drugs that inhibit CDKs affect the growth of cancer cell populations, we developed a spatially explicit hybrid agent-based model. In this model, each cell is equipped with internal cell cycle regulation mechanisms, but it is also able to interact physically with its neighbors. We model cell cycle progression, focusing on the G1 and G2/M cell cycle checkpoints, as well as on related essential components, such as CDK1, CDK2, cell size, and DNA damage. We present detailed studies of how the emergent properties (e.g., cluster formation) of an entire cell population depend on altered physical and physiological parameters. We analyze the effects of CDK1 and CKD2 inhibitors on population growth, time-dependent changes in cell cycle distributions, and the dynamic evolution of spatial cell patterns. We show that cell cycle inhibitors that cause cell arrest at different cell cycle phases are not necessarily synergistically super-additive. Finally, we demonstrate that the physical aspects of cell population growth, such as the formation of tight cell clusters versus dispersed colonies, alter the efficacy of cell cycle inhibitors, both in 2D and 3D simulations. This finding may have implications for interpreting the treatment efficacy results of in vitro experiments, in which treatment is applied before the cells can grow to produce clusters, especially because in vivo tumors, in contrast, form large masses before they are detected and treated. PMID:24607745

Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-cell Acute Lymphoblastic Leukemia.

PubMed

Pikman, Yana; Alexe, Gabriela; Roti, Giovanni; Conway, Amy Saur; Furman, Andrew; Lee, Emily S; Place, Andrew E; Kim, Sunkyu; Saran, Chitra; Modiste, Rebecca; Weinstock, David M; Harris, Marian; Kung, Andrew L; Silverman, Lewis B; Stegmaier, Kimberly

2017-02-15

Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease. Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity. Results: We first determined that both NOTCH1 -mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice. Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012-24. ©2016 AACR See related commentary by Carroll et al., p. 873 . ©2016 American Association for Cancer Research.

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

PubMed Central

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

2005-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2011-01-01

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

Evaluation of the first cytostatically active 1-aza-9-oxafluorenes as novel selective CDK1 inhibitors with P-glycoprotein modulating properties.

PubMed

Brachwitz, Kristin; Voigt, Burkhardt; Meijer, Laurent; Lozach, Olivier; Schächtele, Christoph; Molnár, Josef; Hilgeroth, Andreas

2003-02-27

The first series of synthetic 1-aza-9-oxafluorenes with cytostatic activities in the micromolar range was evaluated as cyclin-dependent kinase (CDK1) inhibitors. Activity was found to be selective in comparison to the inhibition of other kinases within the CDK family. Compounds were shown to inhibit the membrane-efflux pump P-glycoprotein responsible for multidrug resistance in cancer cells. First structure-activity relationships are discussed.

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

PubMed

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

2018-03-25

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

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

PubMed

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

2013-06-01

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

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

PubMed

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

2014-01-01

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

Pharmacological targeting of CDK9 in cardiac hypertrophy.

PubMed

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

2010-07-01

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-08-02

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2015-01-30

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

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

PubMed Central

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

2009-01-01

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

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

PubMed

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

2017-04-01

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

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

PubMed Central

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

2017-01-01

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

Treating ER+ Breast Cancer with CDK4/6 Inhibitors.

PubMed

2017-08-01

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

Inhibitors of cyclin-dependent kinases as cancer therapeutics.

PubMed

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

2017-05-01

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

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

PubMed

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

2014-04-01

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

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

PubMed Central

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

2009-01-01

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

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

PubMed

Nahálková, Jarmila; Tomkinson, Birgitta

2014-12-15

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

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

PubMed

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

2001-01-05

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

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

PubMed

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

2017-10-01

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

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

PubMed Central

2010-01-01

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

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

PubMed

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

2010-08-12

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

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

PubMed

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

2018-01-01

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

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

PubMed

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

2015-04-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

Neuveut, C; Low, K G; Maldarelli, F; Schmitt, I; Majone, F; Grassmann, R; Jeang, K T

1998-06-01

Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a).

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines.

PubMed

Zúñiga, Rafael; Valenzuela, Claudio; Concha, Guierdy; Brown, Nelson; Zúñiga, Leandro

2018-03-29

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

PubMed Central

Zúñiga, Rafael; Valenzuela, Claudio; Concha, Guierdy; Brown, Nelson; Zúñiga, Leandro

2018-01-01

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment. PMID:29596383

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

PubMed

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

2018-04-02

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

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

PubMed Central

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

2018-01-01

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

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2016-03-01

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

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

DTIC Science & Technology

2010-09-01

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

Structural Basis of the Interaction of Cyclin-Dependent Kinase 2 with Roscovitine and Its Analogues Having Bioisosteric Central Heterocycles.

PubMed

Nekardová, Michaela; Vymětalová, Ladislava; Khirsariya, Prashant; Kováčová, Silvia; Hylsová, Michaela; Jorda, Radek; Kryštof, Vladimír; Fanfrlík, Jindřich; Hobza, Pavel; Paruch, Kamil

2017-04-05

The structural basis for the interaction of roscovitine and analogues containing 13 different bioisosteric central heterocycles with the enzyme cyclin-dependent kinase 2 (CDK2) is elucidated. Although all the central scaffolds are very similar to the purine core of roscovitine, the experimentally determined IC 50 values of the inhibitors span three orders of magnitude. By using an extensive computational chemistry approach, the affinities of the inhibitors to CDK2 are determined as calculated binding scores of complexes of the inhibitors with the protein. The interactions of the inhibitors with CDK2 are computationally described by using a hybrid quantum mechanics/semi-empirical quantum mechanics method (QM/SQM), which combines the DFT-D method for the QM part and the PM6-D3H4X method for the SQM part. The solvent effect is described by the COSMO implicit solvation model at the SQM level for the whole system. The contributions of the scaffolds and the individual substituents, quantified and evaluated in relation to conformations of optimized protein-inhibitor complexes, are found not to be simply additive. The inhibitory activity of the selected candidates, including two newly prepared compounds, is tested against CDK2. The results of the calculations are in close agreement with the experimental data. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular Modeling Studies of 4,5-Dihydro-1H-pyrazolo[4,3-h] quinazoline Derivatives as Potent CDK2/Cyclin A Inhibitors Using 3D-QSAR and Docking

PubMed Central

Ai, Yong; Wang, Shao-Teng; Sun, Ping-Hua; Song, Fa-Jun

2010-01-01

CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r2cv values of 0.747 and 0.518 and r2 values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity. PMID:21152296

Molecular modeling studies of 4,5-dihydro-1H-pyrazolo[4,3-h] quinazoline derivatives as potent CDK2/Cyclin a inhibitors using 3D-QSAR and docking.

PubMed

Ai, Yong; Wang, Shao-Teng; Sun, Ping-Hua; Song, Fa-Jun

2010-09-28

CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r(2) (cv) values of 0.747 and 0.518 and r(2) values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity.

Whether to target single or multiple CDKs for therapy? That is the question.

PubMed

Węsierska-Gądek, Józefa; Maurer, Margarita; Zulehner, Nora; Komina, Oxana

2011-02-01

Complexes consisting of cyclin-dependent kinases (CDKs) and their regulatory subunits (the cyclins) control the progression of normal mammalian cells through the cell cycle. However, during malignant transformation this regulatory apparatus malfunctions, allowing cells to undergo unchecked proliferation. In many cases, the high mitotic potential of malignant cells is due to the constitutive activation of CDK-cyclin complexes, facilitated by the inactivation of cellular CDK inhibitors, such as p16(INK4A) or p27(Kip1), and the loss of functional tumor suppressors, such as the p53 and pRb proteins. It has recently been suggested that pharmacological intervention based on remedying the deficiency or loss of activity of these negative regulators of the cell cycle could be a very effective therapeutic option in the treatment of cancer. Multiple CDK inhibitors have been synthesized over the last two decades, spanning at least five classes of compounds. While these inhibitors can be classified on the basis of their chemical structure, it may be more interesting to categorize them according to their pharmacological nature, as broad-spectrum unspecific, pan-specific, or very selective antagonists. This review offers a critical assessment of the advantages and disadvantages of both pan-specific and highly selective CDK inhibitors in therapy. © 2010 Wiley-Liss, Inc.

The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

PubMed

Fong, Clifford W

2016-08-01

Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2009-01-01

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

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

PubMed Central

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

2012-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-04-01

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

Ubiquitylation and proteasomal degradation of the p21(Cip1), p27(Kip1) and p57(Kip2) CDK inhibitors.

PubMed

Lu, Zhimin; Hunter, Tony

2010-06-15

The expression levels of the p21(Cip1) family CDK inhibitors (CKIs), p21(Cip1), p27(Kip1) and p57(Kip2), play a pivotal role in the precise regulation of cyclin-dependent kinase (CDK) activity, which is instrumental to proper cell cycle progression. The stabilities of p21(Cip1), p27(Kip1) and p57(Kip2) are all tightly and differentially regulated by ubiquitylation and proteasome-mediated degradation during various stages of the cell cycle, either in steady state or in response to extracellular stimuli, which often elicit site-specific phosphorylation of CKIs triggering their degradation.

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

PubMed Central

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

2017-01-01

Abstract Selective inhibition of exclusively transcription‐regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY‐958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer. PMID:28961375

Early Intervention with cdk9 Inhibitors to Prevent Post-Traumatic Osteoarthritis

DTIC Science & Technology

2014-10-01

proinflammatory stimuli (interleukin-1! [IL-1!], lipopolysaccharides, and tumor necrosis factor ") in the presence or absence of either the CDK-9 inhibitor...interleukin 1! (IL-1!) and tumor necrosis factor " (TNF"), elicit a cascade of events that activate inflam- matory mediator genes and apoptosis in...IL-1!), lipopolysaccharide (LPS), and tumor necrosis factor " (TNF"), activate their respective cell surface receptors. These signals are then

Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

DOEpatents

Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

2003-06-03

The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2014-12-01

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

Role of p53 in cdk Inhibitor VMY-1-103-induced Apoptosis in Prostate Cancer

DTIC Science & Technology

2013-11-01

DAOY medulloblastoma cells, which have a p53 mutation (6). In order to examine if this holds true in prostate cancer cell lines, I stably transfected...disrupts chromosome organization and delays metaphase progression in medulloblastoma cells. Cancer Biol Ther. 2011 Nov 1;12(9):818-26  Other...1-103 is a novel CDK inhibitor that disrupts chromosome organization and delays metaphase progression in medulloblastoma cells. Cancer Biol Ther

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

PubMed

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

2017-10-01

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

A novel PKC-ι inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma.

PubMed

Pillai, Prajit; Desai, Shraddha; Patel, Rekha; Sajan, Mini; Farese, Robert; Ostrov, David; Acevedo-Duncan, Mildred

2011-05-01

Protein Kinase C-iota (PKC-ι), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-ι confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-ι inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-ι but not that of PKC-zeta (PKC-ζ), the closely related atypical PKC family member. The IC(50) for the kinase activity assay was approximately 0.1μM which is 1000 times less than that of aurothiomalate, a known PKC-ι inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-ι is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-ι was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-ι/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1μM) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-ι inhibitor and chemotherapeutic agent for neuroblastoma. Published by Elsevier Ltd.

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

PubMed

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

1995-10-01

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

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

PubMed Central

1995-01-01

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

Cyclin-dependent kinase inhibitor Dinaciclib (SCH727965) inhibits pancreatic cancer growth and progression in murine xenograft models

PubMed Central

Bisht, Savita; Karikari, Collins; Garrido-Laguna, Ignacio; Rasheed, Zeshaan; Ottenhof, Niki A; Dadon, Tikva; Alvarez, Hector; Fendrich, Volker; Rajeshkumar, NV; Matsui, William; Brossart, Peter; Hidalgo, Manuel; Bannerji, Rajat

2011-01-01

Pancreatic cancer is one of the most lethal of human malignancies, and potent therapeutic options are lacking. Inhibition of cell cycle progression through pharmacological blockade of cyclin-dependent kinases (CDK) has been suggested as a potential treatment option for human cancers with deregulated cell cycle control. Dinaciclib (SCH727965) is a novel small molecule multi-CDK inhibitor with low nanomolar potency against CDK1, CDK2, CDK5 and CDK9 that has shown favorable toxicity and efficacy in preliminary mouse experiments, and has been well tolerated in Phase I clinical trials. In the current study, the therapeutic efficacy of SCH727965 on human pancreatic cancer cells was tested using in vitro and in vivo model systems. Treatment with SCH727965 significantly reduced in vitro cell growth, motility and colony formation in soft agar of MIAPaCa-2 and Pa20C cells. These phenotypic changes were accompanied by marked reduction of phosphorylation of Retinoblastoma (Rb) and reduced activation of RalA. Single agent therapy with SCH727965 (40 mg/kg i.p. twice weekly) for 4 weeks significantly reduced subcutaneous tumor growth in 10/10 (100%) of tested low-passage human pancreatic cancer xenografts. Treatment of low passage pancreatic cancer xenografts with a combination of SCH727965 and gemcitabine was significantly more effective than either agent alone. Gene Set Enrichment Analysis identified overrepresentation of the Notch and Transforming Growth Factor-β (TGFβ) signaling pathways in the xenografts least responsive to SCH727965 treatment. Treatment with the cyclin-dependent kinase inhibitor SCH727965 alone or in combination is a highly promising novel experimental therapeutic strategy against pancreatic cancer. PMID:21768779

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-06-01

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

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

PubMed Central

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

2016-01-01

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

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2017-06-01

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

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

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2005-02-01

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

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

PubMed

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

2014-09-15

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

Specific Calpain Inhibition by Calpastatin Prevents Tauopathy and Neurodegeneration and Restores Normal Lifespan in Tau P301L Mice

PubMed Central

McBrayer, Mary Kate; Campbell, Jabbar; Kumar, Asok; Hashim, Audrey; Sershen, Henry; Stavrides, Philip H.; Ohno, Masuo; Hutton, Michael; Nixon, Ralph A.

2014-01-01

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states. PMID:25009256

Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors.

PubMed

Starostina, Natalia G; Kipreos, Edward T

2012-01-01

The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2012-01-01

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

Evaluation of the interaction of cyclin-dependent kinase 5 with activator p25 and with p25-derived inhibitor CIP.

PubMed

Cardone, Antonio; Albers, R Wayne; Sriram, Ram D; Pant, Harish C

2010-05-01

A high-affinity inhibitor protein called CIP, produced by small truncations of p35, was experimentally identified. P35 is a physiological activator of the cyclin-dependent kinase cdk5. P25 is derived from proteolytic truncation of p35 within "stressed" neurons, and it is associated with the hyperphosphorylation of specific neuronal proteins, typically occurring in neurodegenerative diseases such as Alzheimer's. Here, we report a study of the binding mechanisms of the cdk5-p25 and cdk5-CIP complexes. This provides a better understanding of the source of the inhibitory activity of the protein CIP. We use a geometry-based technique to test the hypothesis that p25's truncation increases the flexibility of CIP and thus prevents cdk5 from reaching its active conformation. Our study is based on a geometry-based alignment algorithm, which aligns two given protein conformations with respect to their interfaces. Our results support the flexibility hypothesis and will be used as a basis for targeted molecular dynamics simulations.

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

PubMed

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

2002-05-01

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

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

PubMed Central

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

2002-01-01

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

Osteosarcoma cells induce endothelial cell proliferation during neo-angiogenesis.

PubMed

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

2013-04-01

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

PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

PubMed

Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

2001-10-11

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen.

PubMed

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

2015-01-01

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen

PubMed Central

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

2015-01-01

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure. PMID:26192324

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

PubMed

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

2017-01-27

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

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

PubMed

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

2018-04-20

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

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

PubMed

Risal, Sanjiv; Adhikari, Deepak; Liu, Kui

2016-01-01

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

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

PubMed

Coqueret, Olivier

2003-02-01

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

RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation

PubMed Central

Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E.; Liau, Brian B.; Mack, Stephen C.; Yang, Kailin; Factor, Daniel C.; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E.; Bao, Shideng; Rich, Jeremy N.

2016-01-01

Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor–initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH. PMID:27322055

Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2).

PubMed

Jiang, P; Stone, S; Wagner, R; Wang, S; Dayananth, P; Kozak, C A; Wold, B; Kamb, A

1995-12-01

Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor-beta (TGF-beta). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.

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

PubMed Central

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

2014-01-01

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

Insights into the structural basis of N2 and O6 substituted guanine derivatives as cyclin-dependent kinase 2 (CDK2) inhibitors: prediction of the binding modes and potency of the inhibitors by docking and ONIOM calculations.

PubMed

Alzate-Morales, Jans H; Caballero, Julio; Vergara Jague, Ariela; González Nilo, Fernando D

2009-04-01

N2 and O6 substituted guanine derivatives are well-known as potent and selective CDK2 inhibitors. The ability of molecular docking using the program AutoDock3 and the hybrid method ONIOM, to obtain some quantum chemical descriptors with the aim to successfully rank these inhibitors, was assessed. The quantum chemical descriptors were used to explain the affinity, of the series studied, by a model of the CDK2 binding site. The initial structures were obtained from docking studies and the ONIOM method was applied with only a single point energy calculation on the protein-ligand structure. We obtained a good correlation model between the ONIOM derived quantum chemical descriptor "H-bond interaction energy" and the experimental biological activity, with a correlation coefficient value of R = 0.80 for 75 compounds. To the best of our knowledge, this is the first time that both methodologies are used in conjunction in order to obtain a correlation model. The model suggests that electrostatic interactions are the principal driving force in this protein-ligand interaction. Overall, the approach was successful for the cases considered, and it suggests that could be useful for the design of inhibitors in the lead optimization phase of drug discovery.

Taking Aim at Glycolysis with CDK8 Inhibitors.

PubMed

Fisher, Robert P

2018-05-01

Dependence on glycolysis under aerobic conditions, a frequent metabolic derangement in cancer cells, suggests a therapeutic opportunity. Now, through chemical genetics, CDK8, a kinase associated with the Mediator transcriptional coactivator complex, has emerged as an upstream inducer of glycolysis and a possible target for anticancer drug discovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed

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

2018-01-04

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

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

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

Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi

2014-01-17

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

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

PubMed

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

2013-10-01

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

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

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

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

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

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

PubMed

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

2015-12-25

Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2001-01-01

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

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

PubMed

Byun, Mi Ran; Choi, Jin Woo

2018-01-02

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

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

PubMed

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

2010-04-01

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

Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21

PubMed Central

Adeyemi, Richard O.

2012-01-01

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G2/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK. PMID:22623787

Hematological adverse effects in breast cancer patients treated with cyclin-dependent kinase 4 and 6 inhibitors: a systematic review and meta-analysis.

PubMed

Kassem, Loay; Shohdy, Kyrillus S; Lasheen, Shaimaa; Abdel-Rahman, Omar; Bachelot, Thomas

2018-01-01

The introduction of specific cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly improved progression-free survival in hormone receptor-positive metastatic breast cancer. CDK 4/6 inhibitors induce cell cycle arrest via liberating the tumor suppressor retinoblastoma protein from CDK4/6 inhibitory effect. Preliminary studies suggested an increase in the hematological toxicities which might affect the quality of life in such palliative setting. We searched PubMed, ASCO, ESMO and San Antonio meeting databases for randomized phase II/III trials in metastatic breast cancer receiving CDK4/6 inhibitors with safety data provided on the incidence of hematological adverse effects. Our search identified 1012 citations that were screened for relevance. Thirty-six studies were found to be potentially eligible. After excluding the ineligible studies, six studies were deemed to be eligible for meta-analysis. The risk ratio (RR) was 11.31 [95% confidence interval (CI) 8.06-15.87; p < 0.0001] for all-grade leucopenia, 14.86 (95% CI 11.37-19.41; p < 0.0001) for all-grade neutropenia, 9.04 (95% CI 3.78-21.63; p < 0.0001) for all-grade thrombocytopenia and 3.57 (95% CI 2.65-4.81; p < 0.0001) for all-grade anemia. The RR for grade 3/4 leucopenia was 33.86 (95% CI 14.59-78.57; p < 0.0001), for grade 3/4 neutropenia was 44.00 (95% CI 24.72-78.33; p < 0.0001), for grade 3/4 thrombocytopenia was 5.70 (95% CI 2.03-16.01; p = 0.001) and for grade 3/4 anemia was 2.80 (95% CI 1.45-5.41; p = 0.002). There was no significant increase in the RR of febrile neutropenia with RR of 3.29 (95% CI 0.93-11.57; p = 0.06). Our analysis provides evidence that the use of CDK 4/6 inhibitors is associated with an increased risk of all-grade and high-grade hematological adverse events, which seems to be a class-effect, but not of febrile neutropenia compared with hormonal therapy alone.

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

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

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

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

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

PubMed

Miyamoto, Yuki; Yamauchi, Junji; Tanoue, Akito

2008-08-13

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

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

PubMed

Dessalew, Nigus; Bharatam, Prasad V

2007-07-01

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

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

PubMed

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

2015-11-01

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

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

PubMed

Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

2008-01-01

The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

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

PubMed Central

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

2014-01-01

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

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

PubMed Central

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

2016-01-01

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

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

PubMed

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

2017-08-31

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

Early circulating tumor DNA dynamics and clonal selection with palbociclib and fulvestrant for breast cancer.

PubMed

O'Leary, Ben; Hrebien, Sarah; Morden, James P; Beaney, Matthew; Fribbens, Charlotte; Huang, Xin; Liu, Yuan; Bartlett, Cynthia Huang; Koehler, Maria; Cristofanilli, Massimo; Garcia-Murillas, Isaac; Bliss, Judith M; Turner, Nicholas C

2018-03-01

CDK4/6 inhibition substantially improves progression-free survival (PFS) for women with advanced estrogen receptor-positive breast cancer, although there are no predictive biomarkers. Early changes in circulating tumor DNA (ctDNA) level may provide early response prediction, but the impact of tumor heterogeneity is unknown. Here we use plasma samples from patients in the randomized phase III PALOMA-3 study of CDK4/6 inhibitor palbociclib and fulvestrant for women with advanced breast cancer and show that relative change in PIK3CA ctDNA level after 15 days treatment strongly predicts PFS on palbociclib and fulvestrant (hazard ratio 3.94, log-rank p = 0.0013). ESR1 mutations selected by prior hormone therapy are shown to be frequently sub clonal, with ESR1 ctDNA dynamics offering limited prediction of clinical outcome. These results suggest that early ctDNA dynamics may provide a robust biomarker for CDK4/6 inhibitors, with early ctDNA dynamics demonstrating divergent response of tumor sub clones to treatment.

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

PubMed

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

2007-07-01

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

Abemaciclib gets new approval for breast cancer

Cancer.gov

DA has approved the CDK4/6 inhibitor abemaciclib (Verzenio) as a first-line treatment in some women with advanced or metastatic breast cancer. Under the approval, it must be used in combination with an aromatase inhibitor, as this Cancer Currents blog post explains.

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

PubMed

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

2014-09-01

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

Cdk5 regulates PSD-95 ubiquitination in neurons

PubMed Central

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

2011-01-01

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

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

PubMed Central

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

2006-01-01

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

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

PubMed Central

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

2016-01-01

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

A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN

DTIC Science & Technology

2015-09-01

Through publications in scientific journals (see Journal publications). What do you plan to do during the next reporting period to accomplish the goals...We plan to elucidate the mechanism of action of the synergy between ALK and CDK inhibitors, validate CDK7 inhibition as a tractable therapeutic...delays and actions or plans to resolve them Nothing to Report Changes that had a significant impact on expenditures Nothing to Report Significant

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

PubMed

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-12-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3(r)CDDP(1000) in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.

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

PubMed Central

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-01-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3rCDDP1000 in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases. PMID:24466371

The new indirubin derivative inhibitors of glycogen synthase kinase-3, 6-BIDECO and 6-BIMYEO, prevent tau phosphorylation and apoptosis induced by the inhibition of protein phosphatase-2A by okadaic acid in cultured neurons.

PubMed

Martin, Ludovic; Magnaudeix, Amandine; Wilson, Cornelia M; Yardin, Catherine; Terro, Faraj

2011-11-01

Alterations in glycogen synthase kinase-3β (GSK3β) and protein phosphatase-2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6-BIDECO (6-bromoindirubin-3'-[O-(N,N-diethylcarbamyl)-oxime] and 6-BIMYEO (6-bromoindirubin-3'-[O-(2-morpholin-1-ylethyl)-oxime] hydrochloride) on OKA-induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6-BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6-BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6-BIDECO and 6-BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD. Copyright © 2011 Wiley-Liss, Inc.

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

PubMed

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

2017-10-01

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

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

PubMed Central

Gérard, Claude; Goldbeter, Albert

2009-01-01

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

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

PubMed

Morgan, D O

1997-01-01

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

A current and comprehensive review of cyclin-dependent kinase inhibitors for the treatment of metastatic breast cancer.

PubMed

Bilgin, Burak; Sendur, Mehmet A N; Şener Dede, Didem; Akıncı, Muhammed Bülent; Yalçın, Bülent

2017-09-01

Resistance to endocrine treatment generally occurs over time, especially in the metastatic stage. In this paper, we aimed to review the mechanisms of cyclin-dependent kinase (CDK) 4/6 inhibition and clinical usage of new agents in the light of recent literature updates. A literature search was carried out using PubMed, Medline and ASCO and ESMO annual-meeting abstracts by using the following search keywords; "palbociclib", "abemaciclib", "ribociclib", "cyclin-dependent kinase inhibitors" and "CDK 4/6" in metastatic breast cancer (MBC). The last search was on 10 June 2017. CDKs and cyclins are two molecules that have a key role in cell cycle progression. Today, there are three highly selective CDK4/6 inhibitors in clinical development - palbociclib, ribociclib and abemaciclib. Palbociclib and ribociclib were recently approved by the US FDA in combination with letrozole for the treatment of MBC in a first-line setting, as well as palbociclib in combination with fulvestrant for hormone-receptor (HR)-positive MBC that had progressed while on previous endocrine therapy according to the PALOMA-1, MONALEESA-2 and PALOMA-3 trials, respectively. In the recently published randomized phase III MONARCH 2 trial, abemaciclib plus letrozole had longer progression-free survival and higher objective response rates with less serious adverse events in advanced HR-positive breast cancer previously treated with hormonal treatment. CDK4/6 inhibition is a new and promising target for patients with hormone-receptor-positive MBC. Both palbociclib and ribociclib showed significant additive benefit for patients receiving first-line treatment for HR-positive, epidermal growth factor receptor-2-negative advanced breast cancer. Palbociclib and abemaciclib also had significant activity in combination with fulvestrant for patients with MBC that progressed on previous endocrine therapy.

Progress with palbociclib in breast cancer: latest evidence and clinical considerations

PubMed Central

Rocca, Andrea; Schirone, Alessio; Maltoni, Roberta; Bravaccini, Sara; Cecconetto, Lorenzo; Farolfi, Alberto; Bronte, Giuseppe; Andreis, Daniele

2016-01-01

Deregulation of the cell cycle is a hallmark of cancer, and research on cell cycle control has allowed identification of potential targets for anticancer treatment. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which are involved, with their coregulatory partners cyclin D, in the G1-S transition. Inhibition of this step halts cell cycle progression in cells in which the involved pathway, including the retinoblastoma protein (Rb) and the E2F family of transcription factors, is functioning, although having been deregulated. Among breast cancers, those with functioning cyclin D-CDK4/6-Rb-E2F are mainly hormone-receptor (HR) positive, with some HER2-positive and rare triple-negative cases. Deregulation results from genetic or otherwise occurring hyperactivation of molecules subtending cell cycle progression, or inactivation of cell cycle inhibitors. Based on results of randomized clinical trials, palbociclib was granted accelerated approval by the US Food and Drug Administration (FDA) for use in combination with letrozole as initial endocrine-based therapy for metastatic disease in postmenopausal women with HR-positive, HER2-negative breast cancer, and was approved for use in combination with fulvestrant in women with HR-positive, HER2-negative advanced breast cancer with disease progression following endocrine therapy. This review provides an update of the available knowledge on the cell cycle and its regulation, on the alterations in cyclin D-CDK4/6-Rb-E2F axis in breast cancer and their roles in endocrine resistance, on the preclinical activity of CDK4/6 inhibitors in breast cancer, both as monotherapy and as partners of combinatorial synergic treatments, and on the clinical development of palbociclib in breast cancer. PMID:28203301

Cell Cycle Regulators during Human Atrial Development

PubMed Central

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

1998-01-01

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

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

PubMed

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

2017-04-26

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

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

PubMed

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

2015-01-01

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

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

PubMed

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

2010-06-01

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

Correlation between Cyclin Dependent Kinases and Artemisinin-Induced Dormancy in Plasmodium falciparum In Vitro

PubMed Central

Gray, Karen-Ann; Gresty, Karryn J.; Chen, Nanhua; Zhang, Veronica; Gutteridge, Clare E.; Peatey, Christopher L.; Chavchich, Marina; Waters, Norman C.; Cheng, Qin

2016-01-01

Background Artemisinin-induced dormancy provides a plausible explanation for recrudescence following artemisinin monotherapy. This phenomenon shares similarities with cell cycle arrest where cyclin dependent kinases (CDKs) and cyclins play an important role. Methods Transcription profiles of Plasmodium falciparum CDKs and cyclins before and after dihydroartemisinin (DHA) treatment in three parasite lines, and the effect of CDK inhibitors on parasite recovery from DHA-induced dormancy were investigated. Results After DHA treatment, parasites enter a dormancy phase followed by a recovery phase. During the dormancy phase parasites up-regulate pfcrk1, pfcrk4, pfcyc2 and pfcyc4, and down-regulate pfmrk, pfpk5, pfpk6, pfcrk3, pfcyc1 and pfcyc3. When entering the recovery phase parasites immediately up-regulate all CDK and cyclin genes. Three CDK inhibitors, olomoucine, WR636638 and roscovitine, produced distinct effects on different phases of DHA-induced dormancy, blocking parasites recovery. Conclusions The up-regulation of PfCRK1 and PfCRK4, and down regulation of other CDKs and cyclins correlate with parasite survival in the dormant state. Changes in CDK expression are likely to negatively regulate parasite progression from G1 to S phase. These findings provide new insights into the mechanism of artemisinin-induced dormancy and cell cycle regulation of P. falciparum, opening new opportunities for preventing recrudescence following artemisinin treatment. PMID:27326764

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

PubMed

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

2008-02-28

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

The mTOR kinase inhibitor everolimus synergistically enhances the anti-tumor effect of the Bruton's tyrosine kinase (BTK) inhibitor PLS-123 on Mantle cell lymphoma.

PubMed

Li, Jiao; Wang, Xiaogan; Xie, Yan; Ying, Zhitao; Liu, Weiping; Ping, Lingyan; Zhang, Chen; Pan, Zhengying; Ding, Ning; Song, Yuqin; Zhu, Jun

2018-01-01

Mantle cell lymphoma (MCL) is an aggressive and incurable malignant disease. Despite of general chemotherapy, relapse and mortality are common, highlighting the need for the development of novel targeted drugs or combination of therapeutic regimens. Recently, several drugs that target the B-cell receptor (BCR) signaling pathway, especially the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicate that pharmacological inhibition of BCR pathway holds promise in MCL treatment. Here, we have developed a novel irreversible BTK inhibitor, PLS-123, that has more potent and selective anti-tumor activity than ibrutinib in vitro and in vivo. Using in vitro screening, we discovered that the combination of PLS-123 and the mammalian target of rapamycin (mTOR) inhibitor everolimus exert synergistic activity in attenuating proliferation and motility of MCL cell lines. Simultaneous inhibition of BTK and mTOR resulted in marked induction of apoptosis and cell cycle arrest in the G1 phase, which were accompanied by upregulation of pro-apoptotic proteins (cleaved Caspase-3, cleaved PARP and Bax), repression of anti-apoptotic proteins (Mcl-1, Bcl-xl and XIAP), and downregulation of regulators of the G1/S phase transition (CDK2, CDK4, CDK6 and Cyclin D1). Gene expression profile analysis revealed simultaneous treatment with these agents led to inhibition of the JAK2/STAT3, AKT/mTOR signaling pathways and SGK1 expression. Finally, the anti-tumor and pro-apoptotic activities of combination strategy have also been demonstrated using xenograft mice models. Taken together, simultaneous suppression of BTK and mTOR may be indicated as a potential therapeutic modality for the treatment of MCL. © 2017 UICC.

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

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

Zhang, Yue; Li, Hongbo; Hao, Jun

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have notmore » yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr-1 decreased p35 expression in HG-cultured podocytes. • Inhibition of Cdk5 activity alleviated podocyte apoptosis induced by HG or TGF-β1.« less

Development of mice without Cip/Kip CDK inhibitors.

PubMed

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

2012-10-19

Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G(0) to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage). Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-01-01

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

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

PubMed

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

2014-10-01

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

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

PubMed

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

2016-08-01

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

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

PubMed

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

2012-06-01

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

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

PubMed Central

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

2016-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP's E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIPS20A, but not CHIPWT, attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli. PMID:26206088

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

PubMed Central

Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N. R.; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M.; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L.; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon

2013-01-01

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P+ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P+ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency. PMID:23888052

Selective antitumor activity of roscovitine in head and neck cancer

PubMed Central

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

2016-01-01

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

[Current Status of Targeted Treatment in Breast Cancer].

PubMed

Seiffert, Katharina; Schmalfeldt, Barbara; Müller, Volkmar

2017-11-01

Within the last years, significant improvements have been achieved in breast cancer treatment, particularly with the development of targeted therapies. Major progress has been made in identifying the drivers malignant growth in oestrogen-receptor-positive breast cancer and the mechanisms of resistance to endocrine therapy. This progress has translated into several targeted therapies that enhance the efficacy of endocrine therapy; inhibitors of the cyclin-dependent kinases CDK4 and CDK6 like palbociclib and inhibitors of mTOR substantially improve progression-free survival. For patients with HER2-positive disease the addition of Pertuzumab to Trastuzumab in combination with chemotherapy has been a significant improvement in anti-HER2 therapy in early as well as metastatic breast cancer. Evidence-based further line therapy options in the metastatic setting include T-DM1 and in later lines Lapatinib. For triple negative disease the angiogenesis inhibitor Bevacizumab is approved, which increases progression free survival. Immune checkpoint inhibitors, PARP-inhibitors or anti-androgens represent promising strategies, all of which are currently being evaluated in clinical trials. The development of predictive biomarkers to guide targeted therapies is still the subject of research. © Georg Thieme Verlag KG Stuttgart · New York.

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers

PubMed Central

Blakely, Collin M.; Watkins, Thomas B.K.; Wu, Wei; Gini, Beatrice; Chabon, Jacob J.; McCoach, Caroline E.; McGranahan, Nicholas; Wilson, Gareth A.; Birkbak, Nicolai J.; Olivas, Victor R.; Rotow, Julia; Maynard, Ashley; Wang, Victoria; Gubens, Matthew A.; Banks, Kimberly C.; Lanman, Richard B.; Caulin, Aleah F.; John, John St.; Cordero, Anibal R.; Giannikopoulos, Petros; Simmons, Andrew D.; Mack, Philip C.; Gandara, David R.; Husain, Hatim; Doebele, Robert C.; Riess, Jonathan W.; Diehn, Maximilian; Swanton, Charles; Bivona, Trever G.

2017-01-01

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant protein product (e.g. EGFR inhibitor treatment in EGFR-mutant lung cancers). However, genetically-driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1122 EGFR-mutant lung cancer cell-free DNA samples and whole exome analysis of seven longitudinally collected tumor samples from an EGFR-mutant lung cancer patient, we identify critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We define new pathways limiting EGFR inhibitor response, including WNT/β-catenin and cell cycle gene (e.g. CDK4, CDK6) alterations. Tumor genomic complexity increases with EGFR inhibitor treatment and co-occurring alterations in CTNNB1, and PIK3CA exhibit non-redundant functions that cooperatively promote tumor metastasis or limit EGFR inhibitor response. This study challenges the prevailing single-gene driver oncogene view and links clinical outcomes to co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancer patients. PMID:29106415

Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer.

PubMed

Pawar, Aishwarya; Gollavilli, Paradesi Naidu; Wang, Shaomeng; Asangani, Irfan A

2018-02-27

BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration-resistant prostate cancer (CRPC). Several BET inhibitors (BETi) that displace BRD4 from chromatin are being evaluated in clinical trials for CRPC. Here, we describe mechanisms of acquired resistance to BETi that are amenable to targeted therapies in CRPC. BETi-resistant CRPC cells displayed cross-resistance to a variety of BETi in the absence of gatekeeper mutations, exhibited reduced chromatin-bound BRD4, and were less sensitive to BRD4 degraders/knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR signaling due to CDK9-mediated phosphorylation of AR, resulting in sensitivity to CDK9 inhibitors and enzalutamide. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DDR genes was observed, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy; however, our BETi resistance data suggest unique opportunities for combination therapies in treating CRPC. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Fatigue, alopecia and stomatitis among patients with breast cancer receiving cyclin-dependent kinase 4 and 6 inhibitors: a systematic review and meta-analysis.

PubMed

Lasheen, Shaimaa; Shohdy, Kyrillus S; Kassem, Loay; Abdel-Rahman, Omar

2017-09-01

Cyclin-dependent kinase (CDK) inhibitors emerge as efficacious agents in hormone positive metastatic breast cancer with more acceptable toxicity profiles than cytotoxic chemotherapy. However, some adverse effects such as fatigue, alopecia and stomatitis, vastly concern patients. The search was conducted in PubMed, American Society of Clinical Oncology meeting library, European Society for Medical Oncology meeting abstract, and the San Antonio meeting abstract databases. We identified phase 2 or 3 trials recruiting patients with breast cancer, randomized to receive hormonal treatment plus either CDK4/6 inhibitors or placebo. We considered studies providing incidence of fatigue, alopecia and stomatitis relevant. One thousand records were screened. 34 studies were considered relevant. Four studies were found to be eligible for meta-analysis with a total of 2007 patients. The relative risk for all grade fatigue was 1.34 [95% CI: 1.17-1.54, p < 0.0001], for all grade alopecia was 2.14 [95% CI: 1.23-3.73, p = 0.007], and for all grade stomatitis 4.87 [95% CI: 2.11-11.24, p = 0.0002]. In addition, the relative risk for high grade fatigue was 2.40 [95% CI: 1.10-5.26, p = 0.03]. CDK4/6 inhibitors were associated with an increased risk of fatigue, alopecia and stomatitis. Further studies with self-reported questionnaires may elucidate the impact of the increased risk of these selected adverse effects on the patients' quality of life.

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

PubMed

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

2017-12-01

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

The CDK4/6 inhibitor LY2835219 overcomes vemurafenib resistance resulting from MAPK reactivation and cyclin D1 upregulation.

PubMed

Yadav, Vipin; Burke, Teresa F; Huber, Lysiane; Van Horn, Robert D; Zhang, Youyan; Buchanan, Sean G; Chan, Edward M; Starling, James J; Beckmann, Richard P; Peng, Sheng-Bin

2014-10-01

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma. ©2014 American Association for Cancer Research.

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

PubMed

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

2018-03-30

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

Targeted overexpression of Tumor Necrosis Factor-α increases Cyclin-dependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons

PubMed Central

Rozas, Pablo; Lazcano, Pablo; Piña, Ricardo; Cho, Andrew; Terse, Anita; Pertusa, Maria; Madrid, Rodolfo; Gonzalez-Billault, Christian; Kulkarni, Ashok B.; Utreras, Elias

2016-01-01

We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain increases Cdk5 activity, a key kinase involved in brain development and function and recently in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TG), we engineered a transgenic mouse model (TNFglo) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNFglo mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca2+ influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, suggesting that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity, therefore this mouse model would be valuable for investigating mechanism involved TNF-α in orofacial pain. PMID:26894912

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

PubMed

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

2008-06-01

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

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

PubMed

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

2009-02-15

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

Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

DOE PAGES

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

2014-11-25

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here in this paper, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation ofmore » CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.« less

The point of no return: The poly(A)-associated elongation checkpoint.

PubMed

Tellier, Michael; Ferrer-Vicens, Ivan; Murphy, Shona

2016-01-01

Cyclin-dependent kinases play critical roles in transcription by RNA polymerase II (pol II) and processing of the transcripts. For example, CDK9 regulates transcription of protein-coding genes, splicing, and 3' end formation of the transcripts. Accordingly, CDK9 inhibitors have a drastic effect on the production of mRNA in human cells. Recent analyses indicate that CDK9 regulates transcription at the early-elongation checkpoint of the vast majority of pol II-transcribed genes. Our recent discovery of an additional CDK9-regulated elongation checkpoint close to poly(A) sites adds a new layer to the control of transcription by this critical cellular kinase. This novel poly(A)-associated checkpoint has the potential to powerfully regulate gene expression just before a functional polyadenylated mRNA is produced: the point of no return. However, many questions remain to be answered before the role of this checkpoint becomes clear. Here we speculate on the possible biological significance of this novel mechanism of gene regulation and the players that may be involved.

Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA*

PubMed Central

Burger, Kaspar; Mühl, Bastian; Rohrmoser, Michaela; Coordes, Britta; Heidemann, Martin; Kellner, Markus; Gruber-Eber, Anita; Heissmeyer, Vigo; Strässer, Katja; Eick, Dirk

2013-01-01

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing. PMID:23744076

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

PubMed

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

2017-02-21

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2004-05-01

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

Development of mice without Cip/Kip CDK inhibitors

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

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu

2012-10-19

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

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

PubMed

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

2015-06-30

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

Cell Attachment to the Extracellular Matrix Induces Proteasomal Degradation of p21CIP1 via Cdc42/Rac1 Signaling

PubMed Central

Bao, Wenjie; Thullberg, Minna; Zhang, Hongquan; Onischenko, Anatoli; Strömblad, Staffan

2002-01-01

The cyclin-dependent kinase 2 (Cdk2) inhibitors p21CIP1 and p27KIP1 are negatively regulated by anchorage during cell proliferation, but it is unclear how integrin signaling may affect these Cdk2 inhibitors. Here, we demonstrate that integrin ligation led to rapid reduction of p21CIP1 and p27KIP1 protein levels in three distinct cell types upon attachment to various extracellular matrix (ECM) proteins, including fibronectin (FN), or to immobilized agonistic anti-integrin monoclonal antibodies. Cell attachment to FN did not rapidly influence p21CIP1 mRNA levels, while the protein stability of p21CIP1 was decreased. Importantly, the down-regulation of p21CIP1 and p27KIP1 was completely blocked by three distinct proteasome inhibitors, demonstrating that integrin ligation induced proteasomal degradation of these Cdk2 inhibitors. Interestingly, ECM-induced proteasomal proteolysis of a ubiquitination-deficient p21CIP1 mutant (p21K6R) also occurred, showing that the proteasomal degradation of p21CIP1 was ubiquitin independent. Concomitant with our finding that the small GTPases Cdc42 and Rac1 were activated by attachment to FN, constitutively active (ca) Cdc42 and ca Rac1 promoted down-regulation of p21CIP1. However, dominant negative (dn) Cdc42 and dn Rac1 mutants blocked the anchorage-induced degradation of p21CIP1, suggesting that an integrin-induced Cdc42/Rac1 signaling pathway activates proteasomal degradation of p21CIP1. Our results indicate that integrin-regulated proteasomal proteolysis might contribute to anchorage-dependent cell cycle control. PMID:12052868

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

PubMed Central

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

2016-01-01

Ewing's sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewing's sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewing's sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewing's sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer. PMID:27286459

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

PubMed

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

2016-07-26

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

A Review of Fulvestrant in Breast Cancer.

PubMed

Nathan, Mark R; Schmid, Peter

2017-01-01

Fulvestrant is a selective estrogen receptor degrader that binds, blocks and degrades the estrogen receptor (ER), leading to complete inhibition of estrogen signaling through the ER. This review article further explains the mechanism of action of the drug and goes on to review the trials carried out to optimize its dosing. Multiple trials have been undertaken to compare fulvestrant with other endocrine treatments, and results have shown it to have similar efficacy to anastrozole, tamoxifen and exemestane at 250 mg every 28 days. However, when given at 500 mg every 28 days, with an extra loading dose on day 14, it has demonstrated an improved progression-free survival (PFS) compared to anastrozole. We look at how fulvestrant has been used in combination with CDK4/6 inhibitors such as palbociclib (PALOMA-3) and ribociclib (MONALEESA-3) and drugs targeting the PI3K/AKT/mTOR pathway such as pictilisib (FERGI) and buparlisib (BELLE-2 and BELLE-3). We then go on to describe a selection of the ongoing clinical trials looking at combination therapy involving fulvestrant. Finally, we review the effect of fulvestrant in patients who have developed resistance to aromatase inhibitors via ESR1 mutation, where it has been shown to offer a PFS benefit that is further improved by the addition of the CDK4/6 inhibitor palbociclib. Whilst fulvestrant is clearly an effective drug as monotherapy, we believe that its role in the treatment of ER-positive breast cancer may be best reserved for combination therapy, and whilst there are multiple trials currently in progress, it would appear that the combination with CDK4/6 inhibitors would offer the greatest promise in terms of balancing benefit with toxicity.

Gastrointestinal adverse effects of cyclin-dependent kinase 4 and 6 inhibitors in breast cancer patients: a systematic review and meta-analysis

PubMed Central

Shohdy, Kyrillus S.; Lasheen, Shaimaa; Kassem, Loay; Abdel-Rahman, Omar

2017-01-01

Background: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors show promising results in metastatic breast cancer. However, an increased incidence of adverse events is remarkable. Among others, gastrointestinal (GI) involvement is of momentous impact on patients and their quality of life. Methods: Our search included PubMed, ASCO, ESMO and SABCS databases. Randomized phase II/III trials in metastatic breast cancer receiving CDK4/6 inhibitors were identified and considered relevant based on providing a sufficient safety profile on the incidence of adverse GI effects. Results: Of the 999 records initially screened for relevance, 33 articles were found relevant and 4 studies were finally eligible for meta-analysis with a total of 2007 patients. The relative risk (RR) for all-grade nausea was 1.48 [95% confidence interval (CI): 1.12–1.93, p = 0.005], vomiting was 1.74 (95% CI: 1.09–2.76, p = 0.02), decreased appetite was 1.42 (95% CI: 1.07–1.88, p = 0.02), and for diarrhea it was 1.44 (95% CI: 1.19–1.74, p = 0.0002). Meanwhile, the RR for high-grade nausea was 1.10 (95% CI: 0.29–4.13, p = 0.89), vomiting was 1.38 (95% CI: 0.25–7.75, p = 0.72), decreased appetite was 4.00 (95% CI: 0.87–18.37, p = 0.07), and high-grade diarrhea was 1.19 (95% CI: 0.44–3.21, p = 0.73). Conclusion: Selective CDK4/6 inhibitors were not associated with higher-grade GI toxicities reflecting a well-tolerated safety profile. Regarding the increase in all-grade GI toxicities, it needs further caution with addition of cytotoxic chemotherapy. PMID:29090083

Gastrointestinal adverse effects of cyclin-dependent kinase 4 and 6 inhibitors in breast cancer patients: a systematic review and meta-analysis.

PubMed

Shohdy, Kyrillus S; Lasheen, Shaimaa; Kassem, Loay; Abdel-Rahman, Omar

2017-11-01

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors show promising results in metastatic breast cancer. However, an increased incidence of adverse events is remarkable. Among others, gastrointestinal (GI) involvement is of momentous impact on patients and their quality of life. Our search included PubMed, ASCO, ESMO and SABCS databases. Randomized phase II/III trials in metastatic breast cancer receiving CDK4/6 inhibitors were identified and considered relevant based on providing a sufficient safety profile on the incidence of adverse GI effects. Of the 999 records initially screened for relevance, 33 articles were found relevant and 4 studies were finally eligible for meta-analysis with a total of 2007 patients. The relative risk (RR) for all-grade nausea was 1.48 [95% confidence interval (CI): 1.12-1.93, p = 0.005], vomiting was 1.74 (95% CI: 1.09-2.76, p = 0.02), decreased appetite was 1.42 (95% CI: 1.07-1.88, p = 0.02), and for diarrhea it was 1.44 (95% CI: 1.19-1.74, p = 0.0002). Meanwhile, the RR for high-grade nausea was 1.10 (95% CI: 0.29-4.13, p = 0.89), vomiting was 1.38 (95% CI: 0.25-7.75, p = 0.72), decreased appetite was 4.00 (95% CI: 0.87-18.37, p = 0.07), and high-grade diarrhea was 1.19 (95% CI: 0.44-3.21, p = 0.73). Selective CDK4/6 inhibitors were not associated with higher-grade GI toxicities reflecting a well-tolerated safety profile. Regarding the increase in all-grade GI toxicities, it needs further caution with addition of cytotoxic chemotherapy.

Synthetic cyclin dependent kinase inhibitors. New generation of potent anti-cancer drugs.

PubMed

Hajdúch, M; Havlíèek, L; Veselý, J; Novotný, R; Mihál, V; Strnad, M

1999-01-01

The unsatisfactory results of current anti-cancer therapies require the active search for new drugs, new treatment strategies and a deeper understanding of the host-tumour relationship. From this point of view, the drugs with a capacity to substitute the functions of altered tumour suppressor genes are of prominent interest. Since one of the main functions of oncosuppressors is to mediate cell cycle arrest via modification of cyclin dependent kinases (CDKs) activity, the compounds with ability to substitute altered functions of these genes in neoplastic cells are of prominent interest. Synthetic inhibitors of cyclin dependent kinases (CDKIs) are typical representatives of such drugs. Olomoucine (OC), flavopiridol (FP), butyrolactone I (BL) and their derivatives selectively inhibit CDKs and thus constrain tumor cell proliferation under in vitro and/or in vivo conditions. We originally discovered OC and its inhibitory activity toward CDK1 family of CDKs, and recently reported the induction of apoptosis and tumor regression following OC application. Moreover, the OC family of synthetic CDKIs has the capacity of directly inhibit CDK7, the principal enzyme required for activating other CDKs, and thus these compounds are the first known CDK7 inhibitors. Its unique mechanism of action and potent anti-cancer activity under both in vitro and in vivo conditions provide a unique tool to inhibit tumour cell proliferation, and to selectively induce apoptosis in neoplastic tissues. The mechanisms of anti-cancer activities of FP, BL, OC and related synthetic CDKIs are compared and discussed in this paper.

Expression of cdk4 and p16 in Oral Lichen Planus.

PubMed

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

2015-01-01

The purpose of this study was to evaluate the expression of cdk4 and p16, the proteins implicated in hyperproliferation and arrest in oral lichen planus and to compare their expression in erosive and non-erosive oral lichen planus and with normal mucosa and oral squamous cell carcinoma. Analysis of cdk4 and p16 expression was done in 43 erosive oral lichen planus (EOLP) and 17 non-erosive oral lichen planus (NOLP) cases, 10 normal mucosa and 10 oral squamous cell carcinoma (OSCC) cases with immunohistochemistry. This study demonstrated a significantly increased expression of cytoplasmic cdk4 (80% cases, cells stained - 19.6%), and cytoplasmic p16 (68.3% cases, cells stained - 16.4%) in oral lichen planus (OLP) compared to normal mucosa. cdk4 was much higher in OSCC in both cytoplasm and nuclei compared to normal mucosa. Also, while comparing OLP with positive control, significant difference was noted for cdk4 and p16, with expression being more in OSCC. While comparing EOLP with NOLP; significant differences were seen for cdk4 cytoplasmic staining only, for number of cases with positive staining as well as number of cells stained. Overexpression of cytoplasmic cdk4 and p16 was registered in oral lichen planus, however considerably lower than in squamous cell carcinoma. Erosive oral lichen planus demonstrated overexpression of cytoplasmic cdk4 and premalignant nature compared to non-erosive lesion. Therefore there is an obvious possibility for cytoplasmic expression of cdk4 and p16 to predict malignant potential of oral lichen planus lesions.

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

PubMed

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

2010-12-15

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2015-12-01

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

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

PubMed

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

2015-12-01

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

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

PubMed

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

2018-01-01

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

Class I HDACs Regulate Angiotensin II-Dependent Cardiac Fibrosis via Fibroblasts and Circulating Fibrocytes

PubMed Central

Williams, Sarah M.; Golden-Mason, Lucy; Ferguson, Bradley S.; Douglas, Katherine B.; Cavasin, Maria A.; Demos-Davies, Kim; Yeager, Michael E.; Stenmark, Kurt R.; McKinsey, Timothy A.

2014-01-01

Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells. PMID:24374140

Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

PubMed

Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden

2015-01-01

Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and the establishment of metaphase II arrest.

The point of no return: The poly(A)-associated elongation checkpoint

PubMed Central

Tellier, Michael; Ferrer-Vicens, Ivan; Murphy, Shona

2016-01-01

abstract Cyclin-dependent kinases play critical roles in transcription by RNA polymerase II (pol II) and processing of the transcripts. For example, CDK9 regulates transcription of protein-coding genes, splicing, and 3′ end formation of the transcripts. Accordingly, CDK9 inhibitors have a drastic effect on the production of mRNA in human cells. Recent analyses indicate that CDK9 regulates transcription at the early-elongation checkpoint of the vast majority of pol II-transcribed genes. Our recent discovery of an additional CDK9-regulated elongation checkpoint close to poly(A) sites adds a new layer to the control of transcription by this critical cellular kinase. This novel poly(A)-associated checkpoint has the potential to powerfully regulate gene expression just before a functional polyadenylated mRNA is produced: the point of no return. However, many questions remain to be answered before the role of this checkpoint becomes clear. Here we speculate on the possible biological significance of this novel mechanism of gene regulation and the players that may be involved. PMID:26853452

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

PubMed

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

2015-11-10

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

Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

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

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

2008-01-05

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

A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

PubMed

Motomura, Kazuki; Kawashima, Tomokazu; Berger, Frédéric; Kinoshita, Tetsu; Higashiyama, Tetsuya; Maruyama, Daisuke

2018-01-29

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control. © 2018. Published by The Company of Biologists Ltd.

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

PubMed Central

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

2013-01-01

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

Combined venetoclax and alvocidib in acute myeloid leukemia.

PubMed

Bogenberger, James; Whatcott, Clifford; Hansen, Nanna; Delman, Devora; Shi, Chang-Xin; Kim, Wontak; Haws, Hillary; Soh, Katherine; Lee, Ye Sol; Peterson, Peter; Siddiqui-Jain, Adam; Weitman, Steven; Stewart, Keith; Bearss, David; Mesa, Ruben; Warner, Steven; Tibes, Raoul

2017-12-05

More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25-50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models in vitro , ex vivo and in vivo . Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS.

Combined venetoclax and alvocidib in acute myeloid leukemia

PubMed Central

Bogenberger, James; Whatcott, Clifford; Hansen, Nanna; Delman, Devora; Shi, Chang-Xin; Kim, Wontak; Haws, Hillary; Soh, Katherine; Lee, Ye Sol; Peterson, Peter; Siddiqui-Jain, Adam; Weitman, Steven; Stewart, Keith; Bearss, David; Mesa, Ruben; Warner, Steven; Tibes, Raoul

2017-01-01

More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25–50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models in vitro, ex vivo and in vivo. Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS. PMID:29291023

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

PubMed Central

Bernabò, Nicola; Valbonetti, Luca; Greco, Luana; Capacchietti, Giulia; Ramal Sanchez, Marina; Palestini, Paola; Botto, Laura; Mattioli, Mauro; Barboni, Barbara

2017-01-01

The adoption of high-througput technologies demonstrated that in mature spermatozoa are present proteins that are thought to be not present or active in sperm cells, such as those involved in control of cell cycle. Here, by using an in silico approach based on the application of networks theory, we found that Cyclins/Cdk complexes could play a central role in signal transduction active during capacitation. Then, we tested this hypothesis in the vitro model. With this approach, spermatozoa were incubated under capacitating conditions in control conditions (CTRL) or in the presence of Aminopurvalanol A a potent, selective and cell permeable inhibitor of Cyclins/Cdk complexes at different concentrations (2, 10, and 20 μM). We found that this treatment caused dose-dependent inhibition of sperm fertilizing ability. We attribute this event to the loss of acrosome integrity due to the inhibition of physiological capacitation-dependent actin polymerization, rather than to a detrimental effect on membrane lipid remodeling or on other signaling pathways such as tubulin reorganization or MAPKs activation. In our opinion, these data could revamp the knowledge on biochemistry of sperm capacitation and could suggest new perspectives in studying male infertility. PMID:29312003

Computation Molecular Kinetics Model of HZE Induced Cell Cycle Arrest

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Ren, Lei

2004-01-01

Cell culture models play an important role in understanding the biological effectiveness of space radiation. High energy and charge (HZE) ions produce prolonged cell cycle arrests at the G1/S and G2/M transition points in the cell cycle. A detailed description of these phenomena is needed to integrate knowledge of the expression of DNA damage in surviving cells, including the determination of relative effectiveness factors between different types of radiation that produce differential types of DNA damage and arrest durations. We have developed a hierarchical kinetics model that tracks the distribution of cells in various cell phase compartments (early G1, late G1, S, G2, and M), however with transition rates that are controlled by rate-limiting steps in the kinetics of cyclin-cdk's interactions with their families of transcription factors and inhibitor molecules. The coupling of damaged DNA molecules to the downstream cyclin-cdk inhibitors is achieved through a description of the DNA-PK and ATM signaling pathways. For HZE irradiations we describe preliminary results, which introduce simulation of the stochastic nature of the number of direct particle traversals per cell in the modulation of cyclin-cdk and cell cycle population kinetics. Comparison of the model to data for fibroblast cells irradiated photons or HZE ions are described.

HER2 signaling drives DNA anabolism and proliferation through SRC-3 phosphorylation and E2F1-regulated genes

PubMed Central

Nikolai, Bryan C.; Lanz, Rainer B.; York, Brian; Dasgupta, Subhamoy; Mitsiades, Nicholas; Creighton, Chad J.; Tsimelzon, Anna; Hilsenbeck, Susan G.; Lonard, David M.; Smith, Carolyn L.; O’Malley, Bert W.

2016-01-01

Approximately 20% of early-stage breast cancers display amplification or overexpression of the ErbB2/HER2 oncogene, conferring poor prognosis and resistance to endocrine therapy. Targeting HER2+ tumors with trastuzumab or the receptor tyrosine kinase (RTK) inhibitor lapatinib significantly improves survival, yet tumor resistance and progression of metastatic disease still develop over time. While the mechanisms of cytosolic HER2 signaling are well studied, nuclear signaling components and gene regulatory networks that bestow therapeutic resistance and limitless proliferative potential are incompletely understood. Here, we use biochemical and bioinformatic approaches to identify effectors and targets of HER2 transcriptional signaling in human breast cancer. Phosphorylation and activity of the Steroid Receptor Coactivator-3 (SRC-3) is reduced upon HER2 inhibition, and recruitment of SRC-3 to regulatory elements of endogenous genes is impaired. Transcripts regulated by HER2 signaling are highly enriched with E2F1 binding sites and define a gene signature associated with proliferative breast tumor subtypes, cell cycle progression, and DNA replication. We show that HER2 signaling promotes breast cancer cell proliferation through regulation of E2F1-driven DNA metabolism and replication genes together with phosphorylation and activity of the transcriptional coactivator SRC-3. Furthermore, our analyses identified a cyclin dependent kinase (CDK) signaling node that, when targeted using the CDK4/6 inhibitor Palbociclib, defines overlap and divergence of adjuvant pharmacological targeting. Importantly, lapatinib and palbociclib strictly block de novo synthesis of DNA, mostly through disruption of E2F1 and its target genes. These results have implications for rational discovery of pharmacological combinations in pre-clinical models of adjuvant treatment and therapeutic resistance. PMID:26833126

Endocrine resistance in breast cancer – an overview and update

PubMed Central

Clarke, Robert; Tyson, John J.; Dixon, J. Michael

2015-01-01

Tumors that express detectable levels of the product of the ESR1 gene (estrogen receptor-α; ERα) represent the single largest molecular subtype of breast cancer. More women eventually die from ERα+ breast cancer than from either HER2+ disease (almost half of which also express ERα) and/or from triple negative breast cancer (ERα-negative, progesterone receptor-negative, and HER2-negative). Antiestrogens and aromatase inhibitors are largely indistinguishable from each other in their abilities to improve overall survival and almost 50% of ERα+ breast cancers will eventually fail one or more of these endocrine interventions. The precise reasons why these therapies fail in ERα+ breast cancer remain largely unknown. Pharmacogenetic explanations for Tamoxifen resistance are controversial. The role of ERα mutations in endocrine resistance remains unclear. Targeting the growth factors and oncogenes most strongly correlated with endocrine resistance has proven mostly disappointing in their abilities to improve overall survival substantially, particularly in the metastatic setting. Nonetheless, there are new concepts in endocrine resistance that integrate molecular signaling, cellular metabolism, and stress responses including endoplasmic reticulum stress and the unfolded protein response (UPR) that provide novel insights and suggest innovative therapeutic targets. Encouraging evidence that drug combinations with CDK4/CDK6 inhibitors can extend recurrence free survival may yet translate to improvements in overall survival. Whether the improvements seen with immunotherapy in other cancers can be achieved in breast cancer remains to be determined, particularly for ERα+ breast cancers. This review explores the basic mechanisms of resistance to endocrine therapies, concluding with some new insights from systems biology approaches further implicating autophagy and the UPR in detail, and a brief discussion of exciting new avenues and future prospects. PMID:26455641

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

PubMed

Narayanan, Bhagavathi A

2006-12-01

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

Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

PubMed Central

Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

2009-01-01

Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

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

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

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

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

CDK activity provides temporal and quantitative cues for organizing genome duplication

PubMed Central

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

2018-01-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

1998-07-10

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

2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase.

PubMed

Kurasawa, Osamu; Homma, Misaki; Oguro, Yuya; Miyazaki, Tohru; Mori, Kouji; Uchiyama, Noriko; Iwai, Kenichi; Ohashi, Akihiro; Hara, Hideto; Yoshida, Sei; Cho, Nobuo

2017-07-15

In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC 50 =0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

PubMed

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

[The mechanism of phenoptosis: 2. Hayflick limit is caused by the programmed attenuation of bioenergetics].

PubMed

Trubitsin, A G

2010-01-01

This article continues earlier started theme on a substantiation of the programmed aging mechanism (phenoptosis). The concept underlying this mechanism is that the life represents a lot of the interconnected physical and chemical processes moving by the bioenergetics. The gradual programmed decrease of the level of bioenergetics causes the slow and coordinated attenuation of all physiological functions, i.e. aging. For a convincing substantiation of such mechanism it is necessary to show, how attenuation of bioenergetics causes the basic nocuous processes accompanying aging. It is shown earlier that the age dependent decrease in level of bioenergetics causes increase in production of reactive oxygen species by mitochondria and decrease in overall level of protein synthesis. The proof that Hayflick limit is also caused by the decrease in level of bioenergetics is presented in this article. Decrease in level of bioenergetics below certain critical level deprives a cell the ability to pass the restriction point of G1-phase of proliferative cycle. The inhibitor of cyclin-dependent kinase, p27, prevents the passage through this critical point in all normal cells. During division of normal somatic cells p27 is removed by cyclin E-Cdk2 complex. Interaction p27 with cyclin E-Cdk2 complex can have two consequences. At the normal physiological level of bioenergetics the cyclin E-Cdk2 phosphorylates p27, then the latter is destroyed by proteolytic enzymes--the cell enters in S-phase. When the programme decreases the bioenergetics level below certain value the cyclin E-Cdk2 becomes the target for p27. As a result the inhibitor evacuation stops and restriction point becomes closed--a cell enters irreversible proliferative rest.

Virtual screening of selective multitarget kinase inhibitors by combinatorial support vector machines.

PubMed

Ma, X H; Wang, R; Tan, C Y; Jiang, Y Y; Lu, T; Rao, H B; Li, X Y; Go, M L; Low, B C; Chen, Y Z

2010-10-04

Multitarget agents have been increasingly explored for enhancing efficacy and reducing countertarget activities and toxicities. Efficient virtual screening (VS) tools for searching selective multitarget agents are desired. Combinatorial support vector machines (C-SVM) were tested as VS tools for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). C-SVM trained on 233-1,316 non-dual-inhibitors correctly identified 26.8%-57.3% (majority >36%) of the 56-230 intra-kinase-group dual-inhibitors (equivalent to the 50-70% yields of two independent individual target VS tools), and 12.2% of the 41 inter-kinase-group dual-inhibitors. C-SVM were fairly selective in misidentifying as dual-inhibitors 3.7%-48.1% (majority <20%) of the 233-1,316 non-dual-inhibitors of the same kinase pairs and 0.98%-4.77% of the 3,971-5,180 inhibitors of other kinases. C-SVM produced low false-hit rates in misidentifying as dual-inhibitors 1,746-4,817 (0.013%-0.036%) of the 13.56 M PubChem compounds, 12-175 (0.007%-0.104%) of the 168 K MDDR compounds, and 0-84 (0.0%-2.9%) of the 19,495-38,483 MDDR compounds similar to the known dual-inhibitors. C-SVM was compared to other VS methods Surflex-Dock, DOCK Blaster, kNN and PNN against the same sets of kinase inhibitors and the full set or subset of the 1.02 M Zinc clean-leads data set. C-SVM produced comparable dual-inhibitor yields, slightly better false-hit rates for kinase inhibitors, and significantly lower false-hit rates for the Zinc clean-leads data set. Combinatorial SVM showed promising potential for searching selective multitarget agents against intra-kinase-group kinases without explicit knowledge of multitarget agents.

Drug synergy screen and network modeling in dedifferentiated liposarcoma identifies CDK4 and IGF1R as synergistic drug targets.

PubMed

Miller, Martin L; Molinelli, Evan J; Nair, Jayasree S; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M; Singer, Samuel; Schwartz, Gary K; Sander, Chris

2013-09-24

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depends on the activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.

Drug Synergy Screen and Network Modeling in Dedifferentiated Liposarcoma Identifies CDK4 and IGF1R as Synergistic Drug Targets

PubMed Central

Miller, Martin L.; Molinelli, Evan J.; Nair, Jayasree S.; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M.; Singer, Samuel; Schwartz, Gary K.; Sander, Chris

2014-01-01

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depend on activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies. PMID:24065146

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation.

PubMed

Ikegami, Yosuke; Goto, Hidemasa; Kiyono, Tohru; Enomoto, Masato; Kasahara, Kousuke; Tomono, Yasuko; Tozawa, Keiichi; Morita, Akimichi; Kohri, Kenjiro; Inagaki, Masaki

2008-12-26

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.

The death effector domain-containing DEDD forms a complex with Akt and Hsp90, and supports their stability

PubMed Central

Kurabe, Nobuya; Mori, Mayumi; Kurokawa, Jun; Taniguchi, Kaori; Aoyama, Hisatoshi; Atsuda, Kazuhiro; Nishijima, Akemi; Odawara, Nariaki; Harada, Saori; Nakashima, Katsuhiko; Arai, Satoko; Miyazaki, Toru

2010-01-01

Insulin secretion and glucose transport are the major mechanisms to balance glucose homeostasis. Recently, we found that the death effector domain-containing DEDD inhibits cyclin-dependent kinase 1 (Cdk1) function, thereby preventing Cdk1-dependent inhibitory phosphorylation of S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase (PI3K), which overall results in maintenance of S6K1 activity. Here we newly show that DEDD forms a complex with Akt and heat-shock protein 90 (Hsp90), and supports the stability of both proteins. Hence, in DEDD−/− mice, Akt protein levels are diminished in skeletal muscles and adipose tissues, which interferes with the translocation of glucose transporter 4 (GLUT4) upon insulin stimulation, leading to inefficient incorporation of glucose in these organs. Interestingly, as for the activation of S6K1, suppression of Cdk1 is involved in the stabilization of Akt protein by DEDD, since diminishment of Cdk1 in DEDD−/− cells via siRNA expression or treatment with a Cdk1-inhibitor, increases both Akt and Hsp90 protein levels. Such multifaceted involvement of DEDD in glucose homeostasis by supporting both insulin secretion (via maintenance of S6K1 activity) and glucose uptake (via stabilizing Akt protein), may suggest an association of DEDD-deficiency with the pathogenesis of type 2 diabetes mellitus. PMID:20043882

Exploration of cell cycle regulation and modulation of the DNA methylation mechanism of pelargonidin: Insights from the molecular modeling approach.

PubMed

Karthi, Natesan; Karthiga, Arumugasamy; Kalaiyarasu, Thangaraj; Stalin, Antony; Manju, Vaiyapuri; Singh, Sanjeev Kumar; Cyril, Ravi; Lee, Sang-Myeong

2017-10-01

Pelargonidin is an anthocyanidin isolated from plant resources. It shows strong cytotoxicity toward various cancer cell lines, even though the carcinogenesis-modulating pathway of pelargonidin is not yet known. One of our previous reports showed that pelargonidin arrests the cell cycle and induces apoptosis in HT29 cells. Flowcytometry and immunoblot analysis confirmed that pelargonidin specifically inhibits the activation of CDK1 and blocks the G2-M transition of the cell cycle. In addition, DNA fragmentation was observed along with induction of cytochrome c release-mediated apoptosis. Hence, the aim of the present study was to investigate the molecular mechanism of pelargonidin's action on cell cycle regulators CDK1, CDK4, and CDK6 as well as the substrate-binding domain of DNMT1 and DNMT3A, which regulate the epigenetic signals related to DNA methylation. The results of docking analysis, binding free energy calculation, and molecular dynamics simulation correlated with the experimental results, and pelargonidin showed a specific interaction with CDK1. In this context, pelargonidin may also inhibit the recognition of DNA and catalytic binding by DNMT1 and DNMT3A. The HOMO-LUMO analysis mapped the functional groups of pelargonidin. Prediction of pharmacological descriptors suggested that pelargonidin can serve as a multitarget inhibitor for cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-07-02

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

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

PubMed

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

2014-03-01

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

Frequent disruption of the RB pathway in indolent follicular lymphoma suggests a new combination therapy

PubMed Central

Oricchio, Elisa; Ciriello, Giovanni; Jiang, Man; Boice, Michael H.; Schatz, Jonathan H.; Heguy, Adriana; Viale, Agnes; de Stanchina, Elisa; Teruya-Feldstein, Julie; Bouska, Alyssa; McKeithan, Tim; Sander, Chris; Tam, Wayne; Seshan, Venkatraman E.; Chan, Wing-Chung; Chaganti, R.S.K.

2014-01-01

Loss of cell cycle controls is a hallmark of cancer and has a well-established role in aggressive B cell malignancies. However, the role of such lesions in indolent follicular lymphoma (FL) is unclear and individual lesions have been observed with low frequency. By analyzing genomic data from two large cohorts of indolent FLs, we identify a pattern of mutually exclusive (P = 0.003) genomic lesions that impair the retinoblastoma (RB) pathway in nearly 50% of FLs. These alterations include homozygous and heterozygous deletions of the p16/CDKN2a/b (7%) and RB1 (12%) loci, and more frequent gains of chromosome 12 that include CDK4 (29%). These aberrations are associated with high-risk disease by the FL prognostic index (FLIPI), and studies in a murine FL model confirm their pathogenic role in indolent FL. Increased CDK4 kinase activity toward RB1 is readily measured in tumor samples and indicates an opportunity for CDK4 inhibition. We find that dual CDK4 and BCL2 inhibitor treatment is safe and effective against available models of FL. In summary, frequent RB pathway lesions in indolent, high-risk FLs indicate an untapped therapeutic opportunity. PMID:24913233

Discovery and development of pyrazole-scaffold Hsp90 inhibitors.

PubMed

McDonald, Edward; Jones, Keith; Brough, Paul A; Drysdale, Martin J; Workman, Paul

2006-01-01

This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin.

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Transforming Growth Factor-B Receptors in Human Breast Cancer.

DTIC Science & Technology

1998-05-01

I., Polyak, K., Iavarone, A., and Massagud, J. Kip/ Cip and Ink4 cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-ß. Genes Dev...specimens. Thirdly, we have developped transient transfection assays to determine how specific TßR mutations affect affect receptor function. Using...Growth Factor-ß (TGFß) is the most potent known inhibitor of cell cycle progression of normal mammary epithelial cells; in addition, it causes cells

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

PubMed

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

2014-11-01

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

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

PubMed Central

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

2014-01-01

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

CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma.

PubMed

Zhou, Jin; Wu, Zhong; Wong, Gabrielle; Pectasides, Eirini; Nagaraja, Ankur; Stachler, Matthew; Zhang, Haikuo; Chen, Ting; Zhang, Haisheng; Liu, Jie Bin; Xu, Xinsen; Sicinska, Ewa; Sanchez-Vega, Francisco; Rustgi, Anil K; Diehl, J Alan; Wong, Kwok-Kin; Bass, Adam J

2017-01-06

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial-mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically.

Cyclin D2 induces proliferation of cardiac myocytes and represses hypertrophy

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

Busk, Peter K.; Hinrichsen, Rebecca; Bartkova, Jirina

2005-03-10

The myocytes of the adult mammalian heart are considered unable to divide. Instead, mitogens induce cardiomyocyte hypertrophy. We have investigated the effect of adenoviral overexpression of cyclin D2 on myocyte proliferation and morphology. Cardiomyocytes in culture were identified by established markers. Cyclin D2 induced DNA synthesis and proliferation of cardiomyocytes and impaired hypertrophy induced by angiotensin II and serum. At the molecular level, cyclin D2 activated CDK4/6 and lead to pRB phosphorylation and downregulation of the cell cycle inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}. Expression of the CDK4/6 inhibitor p16 inhibited proliferation and cyclin D2 overexpressing myocytes became hypertrophic undermore » such conditions. Inhibition of hypertrophy by cyclin D2 correlated with downregulation of p27{sup Kip1}. These data show that hypertrophy and proliferation are highly related processes and suggest that cardiomyocyte hypertrophy is due to low amounts of cell cycle activators unable to overcome the block imposed by cell cycle inhibitors. Cell cycle entry upon hypertrophy may be converted to cell division by increased expression of activators such as cyclin D2.« less

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

PubMed

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

2015-08-01

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

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

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

Zaja, Ivan; Bai, Xiaowen, E-mail: xibai@mcw.edu; Liu, Yanan

Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1more » (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.« less

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

PubMed

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

1995-07-01

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

The CDK inhibitor p21 is a novel target gene of ATF4 and contributes to cell survival under ER stress.

PubMed

Inoue, Yasumichi; Kawachi, Shiori; Ohkubo, Tsubasa; Nagasaka, Mai; Ito, Shogo; Fukuura, Keishi; Itoh, Yuka; Ohoka, Nobumichi; Morishita, Daisuke; Hayashi, Hidetoshi

2017-11-01

Activating transcription factor 4 (ATF4) is well known for its role in the endoplasmic reticulum (ER) stress response. ATF4 also transcriptionally induces multiple effectors that determine cell fate depending on cellular context. In addition, ATF4 can communicate both pro-apoptotic and pro-survival signals. How ATF4 mediates its prosurvival roles, however, requires further investigation. Here, we report that the CDK inhibitor p21 is a novel target gene of ATF4. We identified two ATF4-responsive elements, one of which directly binds ATF4, within the first intron of the p21 gene. Importantly, overexpression of p21 enhances cell survival following ER stress induction, while p21 knockdown increases cell death. These results suggest that p21 induction plays a vital role in the cellular response to ER stress and indicate that p21 is a prosurvival effector of ATF4. © 2017 Federation of European Biochemical Societies.

Localisation of the gene for cylindromatosis (turban tumor syndrome) to chromosome 9p12-13

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

Wooster, R.; Mangion, J.; Quirk, Y.

Cylindromatosis (multiple cylindromas, tomato syndrome syndrome, turban tumor syndrome) is a rare autosomal dominant disease characterized by the development of multiple, slow growing neoplasms of the skin appendages. The tumors, known as dermal cylindromas, exhibit histological features of eccrine or apocrine sweat glands and occur most commonly in the scalp area. Genetic linkage analysis of two families yielded a maximum two point LOD score of 3.2 at D9S169. Critical recombinants place the gene between D9S161 and IFN, a distance of approximately 9 cM. This region of chromosome 9 harbors a gene that encodes a 16 kD protein which is anmore » inhibitor of cyclin dependent kinase 4 (CDK-4) and which is somatically mutated in many classes of cancer. However, the observation of recombinants between the disease and a polymorphic microsatellite repeat CT29 close to this gene, suggests that the CDK-4 inhibitor gene is unlikely to be responsible for cylindromatosis.« less

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

PubMed Central

Stetz, Gabrielle; Tse, Amanda

2017-01-01

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

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

NASA Astrophysics Data System (ADS)

Poulsen, Anders; William, Anthony; Blanchard, Stéphanie; Lee, Angeline; Nagaraj, Harish; Wang, Haishan; Teo, Eeling; Tan, Evelyn; Goh, Kee Chuan; Dymock, Brian

2012-04-01

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

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

PubMed Central

Bolton, Eric C.

2015-01-01

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

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

PubMed

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

2006-11-24

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

Use of ATP analogs to inhibit HIV-1 transcription

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2016-01-01

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

Structural and dynamic determinants of ligand binding and regulation of cyclin-dependent kinase 5 by pathological activator p25 and inhibitory peptide CIP.

PubMed

Cardone, A; Hassan, S A; Albers, R W; Sriram, R D; Pant, H C

2010-08-20

The crystal structure of the cdk5/p25 complex has provided information on possible molecular mechanisms of the ligand binding, specificity, and regulation of the kinase. Comparative molecular dynamics simulations are reported here for physiological conditions. This study provides new insight on the mechanisms that modulate such processes, which may be exploited to control pathological activation by p25. The structural changes observed in the kinase are stabilized by a network of interactions involving highly conserved residues within the cyclin-dependent kinase (cdk) family. Collective motions of the proteins (cdk5, p25, and CIP) and their complexes are identified by principal component analysis, revealing two conformational states of the activation loop upon p25 complexation, which are absent in the uncomplexed kinase and not apparent from the crystal. Simulations of the uncomplexed inhibitor CIP show structural rearrangements and increased flexibility of the interfacial loop containing the critical residue E240, which becomes fully hydrated and available for interactions with one of several positively charged residues in the kinase. These changes provide a rationale for the observed high affinity and enhanced inhibitory action of CIP when compared to either p25 or the physiological activators of cdk5. Published by Elsevier Ltd.

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

Lovastatin inhibits proliferation of anaplastic thyroid cancer cells through up-regulation of p27 by interfering with the Rho/ROCK-mediated pathway.

PubMed

Zhong, Wen-Bin; Hsu, Sung-Po; Ho, Pei-Yin; Liang, Yu-Chih; Chang, Tien-Chun; Lee, Wen-Sen

2011-12-01

Previously, we demonstrated that lovastatin, a HMG-CoA reductase inhibitor, induced apoptosis, differentiation, and inhibition of invasiveness of human anaplastic thyroid carcinoma cells (ATCs). Here, we further examined the effect of lovastatin on the growth of ARO cells. Lovastatin (0-20μM) concentration-dependently decreased cell number in cultured ATC and arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that lovastatin caused an increase of the protein level of p27 and cyclin-dependent kinase (CDK)4 and a decrease of the protein level of cyclin A2, cyclin D3, and phosphorylated Rb (pRb), but did not significantly change the protein levels of p21, cyclins D1 and E, and CDK2, in ARO cells. The formation of the CDK2-p27 complex was increased and the CDK2 activity was decreased in the lovastatin-treated ARO cells. Pretreatment of ARO cells with a p27, but not p21, antisense oligonucleotide prevented the lovastatin-induced G0/G1 arrest in ARO cells. The lovastatin-induced growth inhibition and translocation of RhoA and Rac1 in ARO cells were completely prevented by mevalonate and partially by geranylgeranyl pyrophosphate. Treatment of ARO cells with Y27632, an inhibitor of Rho-associated kinase, abolished the GGPP-mediated prevention of lovastatin-induced anti-proliferation and up-regulation and prolonged degradation of p27. Taken together, these data suggest that lovastatin treatment caused a reduction of Rho geranylgeranylation, which in turn increased the expression and stability of p27, and then inhibited ARO cell proliferation. These data suggest that lovastatin merits further investigation as multipotent therapy for treatment ATC. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2005-01-01

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

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

PubMed

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

2005-01-01

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

AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21.

PubMed

Gongpan, Pianchou; Lu, Yanting; Wang, Fang; Xu, Yuhui; Xiong, Wenyong

2016-07-02

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.

Structure-based Design of Cdk4/6-Specific Inhibitors

DTIC Science & Technology

2005-10-01

were selected to facilitate interactions with residues in neighboring repeats. Moreover,buried hydrophilic residues were mutated to hydrophobic res- Fio ... Russo , A. A., Tong, L., Lee, J.-O., Jeffrey, P. D., and Pavletich, N. P. (1998) tion, although we do not yet have structures for the p18"NK4c Nature 395

γ-Secretase inhibitor-resistant glioblastoma stem cells require RBPJ to propagate.

PubMed

Fan, Xing

2016-07-01

Targeting glioblastoma stem cells with γ-secretase inhibitors (GSIs) disrupts the Notch pathway and has shown some benefit in both pre-clinical models and in patients during phase I/II clinical trials. However, it is largely unknown why some glioblastoma (GBM) does not respond to GSI treatment. In this issue of the JCI, Xie et al. determined that GSI-resistant brain tumor-initiating cells (BTICs) from GBM express a higher level of the gene RBPJ, which encodes a mediator of canonical Notch signaling, compared to non-BTICs. Knockdown of RBPJ in BTICs decreased propagation in vitro and in vivo by inducing apoptosis. Interestingly, RBPJ was shown to regulate a different transcription program than Notch in BTICs by binding CDK9, thereby affecting Pol II-regulated transcript elongation. Targeting CDK9 or c-MYC, an upstream regulator of RBPJ, with small molecules also decreased BTIC propagation, and prolonged survival in mice bearing orthotopic GBM xenografts. This study not only provides a mechanism for GSI treatment resistance, but also identifies two potential therapeutic strategies to target GSI-resistant BTICs.

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

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

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

2007-07-15

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

Chk1 promotes replication fork progression by controlling replication initiation

PubMed Central

Petermann, Eva; Woodcock, Mick; Helleday, Thomas

2010-01-01

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity. PMID:20805465

Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

PubMed Central

Mansilla, Sabrina F; Bertolin, Agustina P; Bergoglio, Valérie; Pillaire, Marie-Jeanne; González Besteiro, Marina A; Luzzani, Carlos; Miriuka, Santiago G; Hoffmann, Jean-Sébastien; Gottifredi, Vanesa

2016-01-01

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis. DOI: http://dx.doi.org/10.7554/eLife.18020.001 PMID:27740454

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

PubMed

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

2015-11-01

To identify the mechanisms of cisplatin resistance, global microRNA (miR) expression was tested. The expression of miR-145 was consistently higher in resistant cells. The expression of cyclin-dependent kinase 6 (CDK6), a potential target of miR-145, was lower in resistant cells, and inhibition of CDK4/6 protected cells from cisplatin. Cell cycle inhibition, currently being tested in clinical trials, might be antagonistic to cisplatin and other cytotoxic drugs. Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Platinum-based chemotherapeutic drugs are the most active agents in treating advanced disease. Resistance to these drugs is common and multifactorial; insight into the molecular mechanisms involved will likely enhance efficacy. A set of NSCLC platinum-resistant sublines was created from the Calu6 cell line. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentially expressed microRNAs (miRs) in these lines were identified using Affymetrix miR arrays. The potential genes targeted by these miRs were searched using the TargetScan algorithm. The expression levels of miRs and mRNA were tested using real-time polymerase chain reaction. miR-145 was reproducibly elevated in all the resistant sublines tested; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin-dependent kinase 6 (CDK6), an important regulator of cell proliferation. The mRNA and protein levels of CDK6 were both downregulated in the resistant sublines. An inhibitor of CDK4/6 (PD0332991) protected parental NSCLC cells from cisplatin cytotoxicity. In the present study, we identified miRs differentially expressed in cisplatin-resistant cell lines, including miR-145. A predicted target of miR-145 is CDK6, and its expression was found to be downregulated in the resistant sublines, although not directly by miR-145. Inhibition of CDK6 antagonizes cisplatin-induced NSCLC cell cytotoxicity, suggesting that agents that inhibit CDK6 should be avoided during cisplatin therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

Kim, Eun-Kyung; Cho, Jae Hee; Kim, EuiJoo; Kim, Yoon Jae

2017-01-01

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

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

PubMed Central

Kim, EuiJoo

2017-01-01

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

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

PubMed

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

2015-11-15

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

ABCB1 as predominant resistance mechanism in cells with acquired SNS-032 resistance

PubMed Central

Rothweiler, Florian; Voges, Yvonne; Balónová, Barbora; Blight, Barry A.; Cinatl, Jindrich

2016-01-01

The CDK inhibitor SNS-032 had previously exerted promising anti-neuroblastoma activity via CDK7 and 9 inhibition. ABCB1 expression was identified as major determinant of SNS-032 resistance. Here, we investigated the role of ABCB1 in acquired SNS-032 resistance. In contrast to ABCB1-expressing UKF-NB-3 sub-lines resistant to other ABCB1 substrates, SNS-032-adapted UKF-NB-3 (UKF-NB-3rSNS- 032300nM) cells remained sensitive to the non-ABCB1 substrate cisplatin and were completely re-sensitized to cytotoxic ABCB1 substrates by ABCB1 inhibition. Moreover, UKF-NB-3rSNS-032300nM cells remained similarly sensitive to CDK7 and 9 inhibition as UKF-NB-3 cells. In contrast, SHEPrSNS-0322000nM, the SNS-032-resistant sub-line of the neuroblastoma cell line SHEP, displayed low level SNS-032 resistance also when ABCB1 was inhibited. This discrepancy may be explained by the higher SNS-032 concentrations that were used to establish SHEPrSNS-0322000nM cells, since SHEP cells intrinsically express ABCB1 and are less sensitive to SNS-032 (IC50 912 nM) than UKF-NB-3 cells (IC50 153 nM). In conclusion, we show that ABCB1 expression represents the primary (sometimes exclusive) resistance mechanism in neuroblastoma cells with acquired resistance to SNS-032. Thus, ABCB1 inhibitors may increase the SNS-032 efficacy in ABCB1-expressing cells and prolong or avoid resistance formation. PMID:27517323

Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells

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

Horiuchi, Shinji; Kato, Kiyoko; Suga, Shin

2005-05-01

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phasemore » of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.« less

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.

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

PubMed

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

2013-06-01

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

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

PubMed Central

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

2011-01-01

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

Progression-Free Survival Among Patients With Well-Differentiated or Dedifferentiated Liposarcoma Treated With CDK4 Inhibitor Palbociclib: A Phase 2 Clinical Trial.

PubMed

Dickson, Mark A; Schwartz, Gary K; Keohan, Mary Louise; D'Angelo, Sandra P; Gounder, Mrinal M; Chi, Ping; Antonescu, Cristina R; Landa, Jonathan; Qin, Li-Xuan; Crago, Aimee M; Singer, Samuel; Koff, Andrew; Tap, William D

2016-07-01

More than 90% of well-differentiated or dedifferentiated liposarcomas (WD/DDLS) have CDK4 amplification. The selective CDK4 and CDK6 inhibitor palbociclib inhibits growth and induces senescence in liposarcoma cell lines and xenografts. Our prior phase 2 study demonstrated that treatment with palbociclib (200 mg daily for 14 days every 21 days) resulted in clinical benefit in WD/DDLS but moderate hematologic toxic effects. It is important to understand whether palbociclib at a new dose and schedule-125 mg daily for 21 days every 28 days-results in clinical benefit and manageable toxic effects. To determine the progression-free survival (PFS) at 12 weeks of patients with WD/DDLS treated with palbociclib (PD0332991). In this phase 2, nonrandomized, open-label clinical trial conducted at the Memorial Sloan Kettering Cancer Center, 60 patients 18 years and older with advanced WD/DDLS and measurable disease by RECIST 1.1 were enrolled from December 2011 to January 2014 and followed to March 2015. Patients received oral palbociclib at 125 mg daily for 21 days in 28-day cycles. Primary end point was PFS. Secondary end points included response rate and toxic effects. Overall, 30 patients were enrolled in the initial cohort and 30 more in an expansion cohort. Median (range) age was 61.5 (35-87) years; 31 patients (52%) were male; median (range) Eastern Cooperative Oncology Group score was 0 (0-1). Progression-free survival at 12 weeks was 57.2% (2-sided 95% CI, 42.4%-68.8%), and the median PFS was 17.9 weeks (2-sided 95% CI, 11.9-24.0 weeks). There was 1 complete response. Toxic effects were primarily hematologic and included neutropenia (grade 3, n = 20 [33%]; grade 4, n = 2 [3%]) but no neutropenic fever. In patients with advanced WD/DDLS, treatment with palbociclib was associated with a favorable PFS and occasional tumor response. This dose and schedule appears active and may have less toxic effects than 200 mg for 14 days. clinicaltrials.gov Identifier: NCT01209598.

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

PubMed

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

2017-01-01

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

The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability.

PubMed

Grey, William; Ivey, Adam; Milne, Thomas A; Haferlach, Torsten; Grimwade, David; Uhlmann, Frank; Voisset, Edwige; Yu, Veronica

2018-01-01

The Cdc28 protein kinase subunits, Cks1 and Cks2, play dual roles in Cdk-substrate specificity and Cdk-independent protein degradation, in concert with the E3 ubiquitin ligase complexes SCF Skp2 and APC Cdc20 . Notable targets controlled by Cks include p27 and Cyclin A. Here, we demonstrate that Cks1 and Cks2 proteins interact with both the Mll N and Mll C subunits of Mll1 (Mixed-lineage leukaemia 1), and together, the Cks proteins define Mll1 levels throughout the cell cycle. Overexpression of CKS1B and CKS2 is observed in multiple human cancers, including various MLL-rearranged (MLLr) AML subtypes. To explore the importance of MLL-Fusion Protein regulation by CKS1/2, we used small molecule inhibitors (MLN4924 and C1) to modulate their protein degradation functions. These inhibitors specifically reduced the proliferation of MLLr cell lines compared to primary controls. Altogether, this study uncovers a novel regulatory pathway for MLL1, which may open a new therapeutic approach to MLLr leukaemia. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2016-01-27

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

Cell Cycle Reprogramming for PI3K Inhibition Overrides Relapse-Specific C481S BTK Mutation Revealed by Longitudinal Functional Genomics in Mantle Cell Lymphoma

PubMed Central

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

2014-01-01

Despite the unprecedented clinical activity of the Bruton’s tyrosine kinase inhibitor ibrutinib in MCL, acquired-resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib-binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary-resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired-ibrutinib resistance in MCL and suggest a strategy to override both primary- and acquired-ibrutinib resistance. PMID:25082755

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

PubMed

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

2017-09-01

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

Synergistic effect of eribulin and CDK inhibition for the treatment of triple negative breast cancer.

PubMed

Rao, Shreyas S; Stoehr, Jenna; Dokic, Danijela; Wan, Lei; Decker, Joseph T; Konopka, Kristine; Thomas, Alexandra L; Wu, Jia; Kaklamani, Virginia G; Shea, Lonnie D; Jeruss, Jacqueline S

2017-10-13

Activation of CDK2 in triple negative breast cancer (TNBC) can contribute to non-canonical phosphorylation of a TGFβ signaling component, Smad3, promoting cell proliferation and migration. Inhibition of CDK2 was shown to decrease breast cancer oncogenesis. Eribulin chemotherapy was used effectively in the treatment of TNBC. To this end, we tested therapeutic efficacy of a novel CDK2/9 inhibitor, CYC065, eribulin, and the combination of CYC065 and eribulin in 3 different TNBC cell lines, and an in vivo xenograft model. Specifically, we characterized cell proliferation, apoptosis, migration, cell cycle associated protein expression, treatment-related transcription factor activity, and tumor growth in TNBC. Treatment with CYC065 and eribulin in combination had a superior effect on decreasing cell proliferation, inducing apoptosis, and inhibiting migration in TNBC cell lines in vitro . Combination therapy inhibited non-canonical Smad3 phosphorylation at the T179 site in the protein linker region, and resulted in increased p15 and decreased c-myc expression. In a transcription factor array, combination treatment significantly increased activity of AP1 and decreased activity of factors including NFκB, SP1, E2F, and SMAD3. In an in vivo xenograft model of TNBC, individual and combination treatments resulted in a decrease in both tumor volume and mitotic indices. Taken together, these studies highlight the potential of this novel drug combination, CYC065 and eribulin, to suppress the growth of TNBC cells in vitro and in vivo, warranting further clinical investigation.

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2012-01-01

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

Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin.

PubMed

Liu, Kang; Lin, Fang-Tsyr; Graves, Joshua D; Lee, Yu-Ju; Lin, Weei-Chin

2017-05-09

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: ( i ) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and ( ii ) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

PubMed Central

Loeffler-Wirth, Henry; Hopp, Lydia; Schadendorf, Dirk; Schartl, Manfred; Anderegg, Ulf; Camp, Gray; Treutlein, Barbara; Binder, Hans; Kunz, Manfred

2017-01-01

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy. PMID:27903987

Comparing sixteen scoring functions for predicting biological activities of ligands for protein targets.

PubMed

Xu, Weijun; Lucke, Andrew J; Fairlie, David P

2015-04-01

Accurately predicting relative binding affinities and biological potencies for ligands that interact with proteins remains a significant challenge for computational chemists. Most evaluations of docking and scoring algorithms have focused on enhancing ligand affinity for a protein by optimizing docking poses and enrichment factors during virtual screening. However, there is still relatively limited information on the accuracy of commercially available docking and scoring software programs for correctly predicting binding affinities and biological activities of structurally related inhibitors of different enzyme classes. Presented here is a comparative evaluation of eight molecular docking programs (Autodock Vina, Fitted, FlexX, Fred, Glide, GOLD, LibDock, MolDock) using sixteen docking and scoring functions to predict the rank-order activity of different ligand series for six pharmacologically important protein and enzyme targets (Factor Xa, Cdk2 kinase, Aurora A kinase, COX-2, pla2g2a, β Estrogen receptor). Use of Fitted gave an excellent correlation (Pearson 0.86, Spearman 0.91) between predicted and experimental binding only for Cdk2 kinase inhibitors. FlexX and GOLDScore produced good correlations (Pearson>0.6) for hydrophilic targets such as Factor Xa, Cdk2 kinase and Aurora A kinase. By contrast, pla2g2a and COX-2 emerged as difficult targets for scoring functions to predict ligand activities. Although possessing a high hydrophobicity in its binding site, β Estrogen receptor produced reasonable correlations using LibDock (Pearson 0.75, Spearman 0.68). These findings can assist medicinal chemists to better match scoring functions with ligand-target systems for hit-to-lead optimization using computer-aided drug design approaches. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2017-01-01

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

Preclinical Study of AUY922, a Novel Hsp90 Inhibitor, in the Treatment of Esophageal Adenocarcinoma.

PubMed

Kosovec, Juliann E; Zaidi, Ali H; Kelly, Lori A; Rotoloni, Christina L; Vytlacil, Christopher; DiCarlo, Christina; Matsui, Daisuke; Komatsu, Yoshihiro; Boyd, Natalie H; Omstead, Ashten; Kolano, Elena L; Biederman, Robert W W; Finley, Gene; Silverman, Jan F; Landreneau, Rodney J; Jobe, Blair A

2016-08-01

To assess the efficacy of heat-shock protein 90 (Hsp90) inhibitor, NVP-AUY922-AG (AUY922), in the treatment of esophageal adenocarcinoma (EAC) in vitro and in vivo. EAC is a leading cause of cancer death, and current treatment options are limited. Hsp90, a chaperone protein that regulates several oncoproteins, is upregulated in EAC, and may be a novel target for therapy. In vitro, EAC cell lines were utilized to evaluate AUY922, alone and in combination with 5-fluorouracil (5-FU) and cisplatin. BrdU ELISA and flow cytometry were used to assess proliferation and measure apoptosis, respectively. Western blot and RT-PCR were performed to quantitate Hsp90 pathway expression. In vivo, esophagojejunostomy was performed on rats and treatment animals received AUY922 32 to 40 weeks postoperatively. Drug efficacy was evaluated with magnetic resonance imaging (MRI), endoscopic biopsy, gross histological evaluation, and Hsp90 pathway expression. In vitro, AUY922 demonstrated antiproliferative activity in both cell lines and showed enhanced efficacy with cisplatin and 5-FU. Western Blot and RT-PCR demonstrated downregulation of CDK1 and CDK4 and upregulation of Hsp72. In vivo, AUY922 showed decrease in tumor volume in 36.4% of rats (control = 9.4%), increase in 9.1% (control = 37.5%), and stable disease in 54.5% (control = 43.7%). Necropsy confirmed the presence of EAC in 50% of treatment animals and 75% of control animals. mRNA expression, pre- and posttreatment, demonstrated significant downregulation of MIF, Hsp70, Hsp90β, and CDK4, and upregulation of Hsp72. AUY922 exhibits antitumor efficacy in vitro and in vivo for EAC, suggesting the need for human clinical trials.

CRMP2 Phosphorylation Drives Glioblastoma Cell Proliferation.

PubMed

Moutal, Aubin; Villa, Lex Salas; Yeon, Seul Ki; Householder, Kyle T; Park, Ki Duk; Sirianni, Rachael W; Khanna, Rajesh

2018-05-01

Glioblastoma (GBM) is an aggressive primary brain tumor. The rapid growth and the privileged provenance of the tumor within the brain contribute to its aggressivity and poor therapeutic targeting. A poor prognostic factor in glioblastoma is the deletion or mutation of the Nf1 gene. This gene codes for the protein neurofibromin, a tumor suppressor gene that is known to interact with the collapsin response mediator protein 2 (CRMP2). CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 have recently been implicated in cancer progression. The CRMP2-neurofibromin interaction protects CRMP2 from its phosphorylation by cyclin-dependent kinase 5 (Cdk5), an event linked to cancer progression. In three human glioblastoma cell lines (GL15, A172, and U87), we observed an inverse correlation between neurofibromin expression and CRMP2 phosphorylation levels. Glioblastoma cell proliferation was dependent on CRMP2 expression and phosphorylation by Cdk5 and glycogen synthase kinase 3 beta (GSK3β). The CRMP2 phosphorylation inhibitor (S)-lacosamide reduces, in a concentration-dependent manner, glioblastoma cell proliferation and induced apoptosis in all three GBM cell lines tested. Since (S)-lacosamide is bioavailable in the brain, we tested its utility in an in vivo orthotopic model of GBM using GL261-LucNeo glioma cells. (S)-lacosamide decreased tumor size, as measured via in vivo bioluminescence imaging, by ~54% compared to vehicle control. Our results introduce CRMP2 expression and phosphorylation as a novel player in GBM proliferation and survival, which is enhanced by loss of Nf1.

Novel Pieces for the Emerging Picture of Sulfoximines in Drug Discovery: Synthesis and Evaluation of Sulfoximine Analogues of Marketed Drugs and Advanced Clinical Candidates.

PubMed

Sirvent, Juan Alberto; Lücking, Ulrich

2017-04-06

Sulfoximines have gained considerable recognition as an important structural motif in drug discovery of late. In particular, the clinical kinase inhibitors for the treatment of cancer, roniciclib (pan-CDK inhibitor), BAY 1143572 (P-TEFb inhibitor), and AZD 6738 (ATR inhibitor), have recently drawn considerable attention. Whilst the interest in this underrepresented functional group in drug discovery is clearly on the rise, there remains an incomplete understanding of the medicinal-chemistry-relevant properties of sulfoximines. Herein we report the synthesis and in vitro characterization of a variety of sulfoximine analogues of marketed drugs and advanced clinical candidates to gain a better understanding of this neglected functional group and its potential in drug discovery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2017-02-01

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

Early Intervention with Cdk9 Inhibitors to Prevent Post-Traumatic Osteoarthritis

DTIC Science & Technology

2015-10-01

Manuscript published in Biochem Biophys Res Commun, title “High abundant protein removal from rodent blood for biomarker discovery”. Included as...Vivo Drug Deliv- ery via the TAT Protein Transduction Domain. Poster Presentation, Osteoarthritis Research Society International (OARSI), Seattle, WA o...COL2A, aggrecan, and cartilage oligomeric matrix protein ) and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity, as assessed by

P276-00, a cyclin-dependent kinase inhibitor, modulates cell cycle and induces apoptosis in vitro and in vivo in mantle cell lymphoma cell lines

PubMed Central

2012-01-01

Background Mantle cell lymphoma (MCL) is a well-defined aggressive lymphoid neoplasm characterized by proliferation of mature B-lymphocytes that have a remarkable tendency to disseminate. This tumor is considered as one of the most aggressive lymphoid neoplasms with poor responses to conventional chemotherapy and relatively short survival. Since cyclin D1 and cell cycle control appears as a natural target, small-molecule inhibitors of cyclin-dependent kinases (Cdks) and cyclins may play important role in the therapy of this disorder. We explored P276-00, a novel selective potent Cdk4-D1, Cdk1-B and Cdk9-T1 inhibitor discovered by us against MCL and elucidated its potential mechanism of action. Methods The cytotoxic effect of P276-00 in three human MCL cell lines was evaluated in vitro. The effect of P276-00 on the regulation of cell cycle, apoptosis and transcription was assessed, which are implied in the pathogenesis of MCL. Flow cytometry, western blot, immunoflourescence and siRNA studies were performed. The in vivo efficacy and effect on survival of P276-00 was evaluated in a Jeko-1 xenograft model developed in SCID mice. PK/PD analysis of tumors were performed using LC-MS and western blot analysis. Results P276-00 showed a potent cytotoxic effect against MCL cell lines. Mechanistic studies confirmed down regulation of cell cycle regulatory proteins with apoptosis. P276-00 causes time and dose dependent increase in the sub G1 population as early as from 24 h. Reverse transcription PCR studies provide evidence that P276-00 treatment down regulated transcription of antiapoptotic protein Mcl-1 which is a potential pathogenic protein for MCL. Most importantly, in vivo studies have revealed significant efficacy as a single agent with increased survival period compared to vehicle treated. Further, preliminary combination studies of P276-00 with doxorubicin and bortezomib showed in vitro synergism. Conclusion Our studies thus provide evidence and rational that P276-00 alone or in combination is a potential therapeutic molecule to improve patients’ outcome in mantle cell lymphoma. PMID:23075291

Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation.

PubMed

Zhao, Xuan; Hirota, Tsuyoshi; Han, Xuemei; Cho, Han; Chong, Ling-Wa; Lamia, Katja; Liu, Sihao; Atkins, Annette R; Banayo, Ester; Liddle, Christopher; Yu, Ruth T; Yates, John R; Kay, Steve A; Downes, Michael; Evans, Ronald M

2016-06-16

Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

A gene expression signature of retinoblastoma loss-of-function is a predictive biomarker of resistance to palbociclib in breast cancer cell lines and is prognostic in patients with ER positive early breast cancer.

PubMed

Malorni, Luca; Piazza, Silvano; Ciani, Yari; Guarducci, Cristina; Bonechi, Martina; Biagioni, Chiara; Hart, Christopher D; Verardo, Roberto; Di Leo, Angelo; Migliaccio, Ilenia

2016-09-13

Palbociclib is a CDK4/6 inhibitor that received FDA approval for treatment of hormone receptor positive (HR+) HER2 negative (HER2neg) advanced breast cancer. To better personalize patients treatment it is critical to identify subgroups that would mostly benefit from it. We hypothesize that complex alterations of the Retinoblastoma (Rb) pathway might be implicated in resistance to CDK4/6 inhibitors and aim to investigate whether signatures of Rb loss-of-function would identify breast cancer cell lines resistant to palbociclib. We established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in breast cancers within The Cancer Genome Atlas. We assessed the RBsig prognostic role in the METABRIC and in a comprehensive breast cancer meta-dataset. Finally, we analyzed whether RBsig would discriminate palbociclib-sensitive and -resistant breast cancer cells in a large RNA sequencing-based dataset. The RBsig was associated with RB1 genetic status in all tumors (p <7e-32) and in luminal or basal subtypes (p < 7e-11 and p < 0.002, respectively). The RBsig was prognostic in the METABRIC dataset (discovery: HR = 1.93 [1.5-2.4] p = 1.4e-08; validation: HR = 2.01 [1.6-2.5] p = 1.3e-09). Untreated and endocrine treated patients with estrogen receptor positive breast cancer expressing high RBsig had significantly worse recurrence free survival compared to those with low RBsig (HR = 2.37 [1.8 - 3.2] p = 1.87e-08 and HR = 2.62 [1.9- 3.5] p = 8.6e-11, respectively). The RBsig was able to identify palbociclib resistant and sensitive breast cancer cells (ROC AUC = 0,7778). Signatures of RB loss might be helpful in personalizing treatment of patients with HR+/HER2neg breast cancer. Further validation in patients receiving palbociclib is warranted.

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

PubMed Central

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

2014-01-01

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

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

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

Li Chenchen; Xing Tairan; Tang Mingliang

2008-06-15

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

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

PubMed

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

2009-05-29

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

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

PubMed

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

2017-10-28

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

Role of Cyclin D1 and cdk Inhibitors in Breast Cancer Pathogenesis

DTIC Science & Technology

2001-10-01

0.1 [tg of a Renilla luciferase expression plasmid (pRL-SV40 Promega) to normalize for transfection efficiency. After an overnight recovery, the...saline, collected, lysed, and analyzed for luciferase and Renilla luciferase activity by using the Dual-Luciferase reporter assay system (Promega...Cyclin A promoter-driven luciferase activity was then normalized to a constant activity of Renilla luciferase. 7 Results In our previous reports we

Novel CDK inhibition profiles of structurally varied 1-aza-9-oxafluorenes.

PubMed

Voigt, Burkhardt; Meijer, Laurent; Lozach, Olivier; Schächtele, Christoph; Totzke, Frank; Hilgeroth, Andreas

2005-02-01

A series of 1-aza-9-oxafluorenes with functionally varied 3-substituents have been prepared from N-phenoxycarbonyl-4-phenyl-1,4-dihydropyridines and p-benzoquinone and biologically evaluated as inhibitors of various cyclin-dependant kinases. The absence of a 3-hydrogen bond acceptor function leads to a complete loss of inhibitory activity. Differing hydrogen bond acceptor functions surprisingly cause significant shifts in the selectivity of inhibition profiles.

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

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

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

DOE PAGES

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan; ...

2017-05-17

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

17β-Estradiol Induces Overproliferation in Adenomyotic Human Uterine Smooth Muscle Cells of the Junctional Zone Through Hyperactivation of the Estrogen Receptor-Enhanced RhoA/ROCK Signaling Pathway.

PubMed

Sun, Fu-Qing; Duan, Hua; Wang, Sha; Li, Jin-Jiao

2015-11-01

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. Recent models favor abnormal thickening of the junctional zone (JZ) may be the causative factor in the development of ADS. RhoA, a small guanosine triphosphatase which controls multiple cellular processes, is involved in the control of cell proliferation. Here we demonstrate that treatment of human uterine smooth muscle cells (SMCs) of the JZ with 17β-estradiol (E2) increased expression of RhoA and its downstream effectors (-associated coiled coil containing protein kinase [ROCK] 1 and ROCK2). Compared with non-ADS cells, RhoA, ROCK1, and ROCK2 were overexpressed and hyperactivated in ADS cells. These effects were suppressed in the presence of ICI 182,780, supporting an estrogen receptor (ER)-dependent mechanism. Hyperactivation of ER-enhanced RhoA/ROCK signaling was associated with overproliferation in ADS human uterine SMCs of the JZ. Moreover, E2-induced overproliferation was accompanied by downregulation of cyclin-dependent kinases inhibitors (CKIs; p21(Waf1/Cip1) and p27(Kip1)) and upregulation of cyclin-dependent kinases (CDKs) and cyclins (cyclin D1, cyclin E1, CDK2, CDK4, and CDK6). © The Author(s) 2015.

Protein Phosphatase 2A Antagonizes ATM and ATR in a Cdk2- and Cdc7-Independent DNA Damage Checkpoint

PubMed Central

Petersen, Paris; Chou, Danny M.; You, Zhongsheng; Hunter, Tony; Walter, Johannes C.; Walter, Gernot

2006-01-01

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts. PMID:16479016

Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases.

PubMed

Abraham, R T; Acquarone, M; Andersen, A; Asensi, A; Bellé, R; Berger, F; Bergounioux, C; Brunn, G; Buquet-Fagot, C; Fagot, D

1995-01-01

Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.

Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors.

PubMed

Zhu, Jin-Yi; Cuellar, Rebecca A; Berndt, Norbert; Lee, Hee Eun; Olesen, Sanne H; Martin, Mathew P; Jensen, Jeffrey T; Georg, Gunda I; Schönbrunn, Ernst

2017-09-28

Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

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

Qiao, Lan; Department of Pharmaceutical Sciences, Jilin University, Changchun 130021; Paul, Pritha

2013-05-31

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated thatmore » GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.« less

The stomatin-like protein SLP-1 and Cdk2 interact with the F-Box protein Fbw7-γ.

PubMed

Zhang, Wei; MacDonald, Elizabeth M; Koepp, Deanna M

2012-01-01

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-γ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-γ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-γ. Overexpression of SLP-1 inhibits the degradation of Fbw7-γ, suggesting that this interaction can happen in vivo. When Fbw7-γ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF(Fbw7-γ) complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-γ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-γ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions.

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

PubMed

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

2007-06-15

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

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

PubMed Central

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

2007-01-01

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

Biochemical Characterization of Complexes with p21, a CDK Inhibitor

DTIC Science & Technology

1998-08-01

of kinases at different stages of the cell cycle or at different times during development . Since p107 is highly related to the retinoblastoma tumor...and Materiel Command, 504 Scott Street, Fort Detrick, Maryland 21702-5012. 13. ABSTRACT (Maximum 200 Cell cycle progression and proliferation are...cells. 14. SUBJECT TERMS Breast Cancer , cell cycle , cyclin-dependent 15. NUMBER OF PAGES cycli-depedent66 kinases (Cdks), p21, p107, growth control

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

PubMed

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

2000-11-01

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

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

PubMed

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

2015-11-24

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

Role of CDK4 in Breast Development and Cancer

DTIC Science & Technology

2008-04-01

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

G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

PubMed

Giráldez, Servando; Galindo-Moreno, María; Limón-Mortés, M Cristina; Rivas, A Cristina; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2017-07-01

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCF βTrCP /proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G 1 /S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCF βTrCP /proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G 1 -to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G 1 /S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis. © FASEB.

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

PubMed

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2014-11-04

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

PubMed Central

2015-01-01

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

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

PubMed

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

2001-03-01

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

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

PubMed

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

2016-06-01

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

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

PubMed

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

2015-05-07

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

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

PubMed

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

2017-10-01

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

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

PubMed

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

2017-04-01

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

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

PubMed Central

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

2011-01-01

Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1. PMID:22163316

MicroRNA-193 Pro-Proliferation Effects for Bone Mesenchymal Stem Cells After Low-Level Laser Irradiation Treatment Through Inhibitor of Growth Family, Member 5

PubMed Central

Wang, Jue; Huang, Weicong; Wu, Yihe; Hou, Jianfeng; Nie, Yu; Gu, Haiyong; Li, Jun; Hu, Shengshou

2012-01-01

The enhanced proliferation of mesenchymal stem cells (MSCs) can be helpful for the clinical translation of cell therapy. Low-level laser irradiation (LLLI) has been demonstrated as regulating MSC proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in stem cells, but the role of miRNAs in the LLLI-based promotion of MSC proliferation remains unclear. We found that the proliferation level and cell cycle-associated genes in MSCs were increased after LLLI treatment in a time-dependent manner. Microarray assays revealed subsets of miRNAs to be differentially regulated, and these dynamic changes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) after LLLI. miR-193 was the most highly up-regulated miRNA, and the change in it was related with the proliferation level. Gain-loss function experiments demonstrated that miR-193 could regulate the proliferation of MSCs, including human's and rat's, but could not affect the apoptosis and differentiation level. Blockade of miR-193 repressed the MSC proliferation induced by LLLI. By qRT-PCR, we found that miR-193, in particular, regulated cyclin-dependent kinase 2 (CDK2) expression. Bioinformatic analyses and luciferase reporter assays revealed that inhibitor of growth family, member 5 (ING5) could be the best target of miR-193 to functionally regulate proliferation and CDK2 activity, and the mRNA and protein level of ING5 was regulated by miR-193. Furthermore, the ING5 inhibited by small interfering RNA (siRNA) could up-regulate the proliferation of MSCs and the expression of CDK2. Taken together, these results strongly suggest that miR-193 plays a critical part in MSC proliferation in response to LLLI stimulation, which is potentially amenable to therapeutic manipulation for clinical application. PMID:22384930

Pyrazolo[1,5-a]-1,3,5-triazine as a purine bioisostere: access to potent cyclin-dependent kinase inhibitor (R)-roscovitine analogue.

PubMed

Popowycz, Florence; Fournet, Guy; Schneider, Cédric; Bettayeb, Karima; Ferandin, Yoan; Lamigeon, Cyrile; Tirado, Oscar M; Mateo-Lozano, Silvia; Notario, Vicente; Colas, Pierre; Bernard, Philippe; Meijer, Laurent; Joseph, Benoît

2009-02-12

Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure, we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine 7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1-f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced biomedical potential.

The circadian molecular clock regulates adult hippocampal neurogenesis by controlling the timing of cell-cycle entry and exit.

PubMed

Bouchard-Cannon, Pascale; Mendoza-Viveros, Lucia; Yuen, Andrew; Kærn, Mads; Cheng, Hai-Ying M

2013-11-27

The subgranular zone (SGZ) of the adult hippocampus contains a pool of quiescent neural progenitor cells (QNPs) that are capable of entering the cell cycle and producing newborn neurons. The mechanisms that control the timing and extent of adult neurogenesis are not well understood. Here, we show that QNPs of the adult SGZ express molecular-clock components and proliferate in a rhythmic fashion. The clock proteins PERIOD2 and BMAL1 are critical for proper control of neurogenesis. The absence of PERIOD2 abolishes the gating of cell-cycle entrance of QNPs, whereas genetic ablation of bmal1 results in constitutively high levels of proliferation and delayed cell-cycle exit. We use mathematical model simulations to show that these observations may arise from clock-driven expression of a cell-cycle inhibitor that targets the cyclin D/Cdk4-6 complex. Our findings may have broad implications for the circadian clock in timing cell-cycle events of other stem cell populations throughout the body. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition.

PubMed

Szaniawski, Matthew A; Spivak, Adam M; Cox, James E; Catrow, Jonathan L; Hanley, Timothy; Williams, Elizabeth S C P; Tremblay, Michel J; Bosque, Alberto; Planelles, Vicente

2018-05-15

Macrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted. IMPORTANCE Our experimental results demonstrate that SAMHD1 dephosphorylation at threonine-592 represents a central mechanism of HIV-1 restriction that is common to the three known families of IFNs. While IFN types I and II were potent inhibitors of HIV-1, type III IFN showed modest to undetectable activity. Regulation of SAMHD1 by IFNs involved changes in phosphorylation status but not in protein levels. Phosphorylation of SAMHD1 in macrophages occurred at least in part via CDK1. Tyrosine kinase inhibitors similarly induced SAMHD1 dephosphorylation, which protects macrophages from HIV-1 in a SAMHD1-dependent manner. SAMHD1 is a critical restriction factor regulating HIV-1 infection of macrophages. Copyright © 2018 Szaniawski et al.

Early Intervention with Cdk9 Inhibitors to Prevent Post-Traumatic Osteoarthritis

DTIC Science & Technology

2013-10-01

media from day 3 to day 6 was determined by the colorimetric dimethylmethylene blue dye-assay, with chondroitin sulfate as standard (11). The...Res Ther 2013; 15: R223. 9. Pecchi E, Priam S, Mladenovic Z, Gosset M, Saurel AS, Aguilar L, et al. A potential role of chondroitin sulfate on bone...100 mM dithiothreitol; 4% 2-mercaptoethanol; 2% sodium dodecyl sulfate ; 10% glycerol). Lysates were resolved by 4-12% SDS- polyacrylamide gels and

Role of p53 in cdk Inhibitor VMY-1-103-Induced Apoptosis in Prostate Cancer

DTIC Science & Technology

2012-09-01

trioxide inhibits human cancer cell growth and tumor development in mice by blocking Hedgehog /GLI pathway. J Clin Invest. 2011 Jan 4;121(1):148- 60...subclassified the tumors based on gene expression patterns and chromosomal abnormalities.4-6 Dysregulation of Hedgehog (Hh) signaling, defined as the c3...Eberhart CG. Hedgehog signaling promotes medulloblastoma survival via Bc/II. Am J Pathol 2007; 170:347-55; PMID:17200206; DOI:10.2353/ajpath

Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells.

PubMed

Carlson, Morgan E; Hsu, Michael; Conboy, Irina M

2008-07-24

Adult skeletal muscle robustly regenerates throughout an organism's life, but as the muscle ages, its ability to repair diminishes and eventually fails. Previous work suggests that the regenerative potential of muscle stem cells (satellite cells) is not triggered in the old muscle because of a decline in Notch activation, and that it can be rejuvenated by forced local activation of Notch. Here we report that, in addition to the loss of Notch activation, old muscle produces excessive transforming growth factor (TGF)-beta (but not myostatin), which induces unusually high levels of TGF-beta pSmad3 in resident satellite cells and interferes with their regenerative capacity. Importantly, endogenous Notch and pSmad3 antagonize each other in the control of satellite-cell proliferation, such that activation of Notch blocks the TGF-beta-dependent upregulation of the cyclin-dependent kinase (CDK) inhibitors p15, p16, p21 and p27, whereas inhibition of Notch induces them. Furthermore, in muscle stem cells, Notch activity determines the binding of pSmad3 to the promoters of these negative regulators of cell-cycle progression. Attenuation of TGF-beta/pSmad3 in old, injured muscle restores regeneration to satellite cells in vivo. Thus a balance between endogenous pSmad3 and active Notch controls the regenerative competence of muscle stem cells, and deregulation of this balance in the old muscle microniche interferes with regeneration.

Interaction energies for the purine inhibitor roscovitine with cyclin-dependent kinase 2: correlated ab initio quantum-chemical, DFT and empirical calculations.

PubMed

Dobes, Petr; Otyepka, Michal; Strnad, Miroslav; Hobza, Pavel

2006-05-24

The interaction between roscovitine and cyclin-dependent kinase 2 (cdk2) was investigated by performing correlated ab initio quantum-chemical calculations. The whole protein was fragmented into smaller systems consisting of one or a few amino acids, and the interaction energies of these fragments with roscovitine were determined by using the MP2 method with the extended aug-cc-pVDZ basis set. For selected complexes, the complete basis set limit MP2 interaction energies, as well as the coupled-cluster corrections with inclusion of single, double and noninteractive triples contributions [CCSD(T)], were also evaluated. The energies of interaction between roscovitine and small fragments and between roscovitine and substantial sections of protein (722 atoms) were also computed by using density-functional tight-binding methods covering dispersion energy (DFTB-D) and the Cornell empirical potential. Total stabilisation energy originates predominantly from dispersion energy and methods that do not account for the dispersion energy cannot, therefore, be recommended for the study of protein-inhibitor interactions. The Cornell empirical potential describes reasonably well the interaction between roscovitine and protein; therefore, this method can be applied in future thermodynamic calculations. A limited number of amino acid residues contribute significantly to the binding of roscovitine and cdk2, whereas a rather large number of amino acids make a negligible contribution.

FT-IR, FT-Raman and NMR characterization of 2-isopropyl-5-methylcyclohexyl quinoline-2-carboxylate and investigation of its reactive and optoelectronic properties by molecular dynamics simulations and DFT calculations

NASA Astrophysics Data System (ADS)

Menon, Vidya V.; Fazal, Edakot; Mary, Y. Sheena; Panicker, C. Yohannan; Armaković, Stevan; Armaković, Sanja J.; Nagarajan, Subban; Van Alsenoy, C.

2017-01-01

The FT-IR and FT-Raman spectra of the synthesized compound, 2-isopropyl-5-methylcyclohexyl quinoline-2-carboxylate is recorded and analyzed. Optimized molecular structure, wave numbers, corresponding assignments regarding 2-isopropyl-5-methylcyclohexyl quinoline-2-carboxylate has become screened tentatively as well as hypothetically using Gaussian09 program package. Natural bonding orbital assessment has been completed with a reason to clarify charge transfer or conjugative interaction, the intra-molecular re-hybridization and delocalization of electron density within the molecule. The NMR spectral assessment had been made choosing structure property relationship by chemical shifts along with the magnetic shielding effects regarding the title compound. The first and second hyperpolarizabilities were calculated. The calculated first order hyperpolarizability is commensurate with the documented worth of very similar derivatives and could be an interesting object for more experiments on nonlinear optics. Local reactivity properties have been investigated using average local ionization energies and Fukui functions. Investigation of optoelectronic properties encompassed calculations of reorganization energies and hopping rates of charge carriers within the framework of Marcus semi-empiric approach. The docked ligand title compound forms a stable complex with CDK inhibitors and gives a binding affinity value of -9.7 kcal/mol and molecular docking results suggest that the compound might exhibit inhibitory activity against CDK inhibitors.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

PubMed

Minemoto, Yuzuru; Uchida, Sanae; Ohtsubo, Motoaki; Shimura, Mari; Sasagawa, Toshiyuki; Hirata, Masato; Nakagama, Hitoshi; Ishizaka, Yukihito; Yamashita, Katsumi

2003-04-01

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

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

PubMed

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

2009-11-01

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

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

PubMed

Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

2015-01-01

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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

PubMed

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

2017-05-03

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

Active Cdk5 Immunoprecipitation and Kinase Assay

PubMed Central

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

2017-01-01

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

A novel coumarin-quinone derivative SV37 inhibits CDC25 phosphatases, impairs proliferation, and induces cell death.

PubMed

Bana, Emilie; Sibille, Estelle; Valente, Sergio; Cerella, Claudia; Chaimbault, Patrick; Kirsch, Gilbert; Dicato, Mario; Diederich, Marc; Bagrel, Denyse

2015-03-01

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation by regulating CDK/cyclin complexes. Overexpression of these enzymes is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, targeting CDC25 by compounds, able to inhibit their activity, appears a good therapeutic approach. Here, we describe the synthesis of a new inhibitor (SV37) whose structure is based on both coumarin and quinone moieties. An analytical in vitro approach shows that this compound efficiently inhibits all three purified human CDC25 isoforms (IC50 1-9 µM) in a mixed-type mode. Moreover, SV37 inhibits growth of breast cancer cell lines. In MDA-MB-231 cells, reactive oxygen species generation is followed by pCDK accumulation, a mark of CDC25 dysfunction. Eventually, SV37 treatment leads to activation of apoptosis and DNA cleavage, underlining the potential of this new type of coumarin-quinone structure. © 2013 Wiley Periodicals, Inc.

Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation

PubMed Central

Whalley, Helen J.; Porter, Andrew P.; Diamantopoulou, Zoi; White, Gavin R. M.; Castañeda-Saucedo, Eduardo; Malliri, Angeliki

2015-01-01

Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the development of Eg5 inhibitors as cancer therapeutics. PMID:26078008

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia-lymphoma cells.

PubMed

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-12-01

Adult T-cell leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia–lymphoma cells

PubMed Central

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-01-01

Adult T-cell leukemia–lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. PMID:25263741

Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma.

PubMed

Ponder, Katelyn G; Matulis, Shannon M; Hitosugi, Sadae; Gupta, Vikas A; Sharp, Cathy; Burrows, Francis; Nooka, Ajay K; Kaufman, Jonathan L; Lonial, Sagar; Boise, Lawrence H

2016-07-02

Carfilzomib (Kyprolis®), a second generation proteasome inhibitor, is FDA approved for single-agent use among relapsed/refractory multiple myeloma (MM). To enhance the therapeutic efficacy of carfilzomib, we sought to combine carfilzomib with other novel agents. TG02, a multi-kinase inhibitor, targets JAK2 and CDK9. The rationale for co-treatment with carfilzomib and TG02 is that both independently target Mcl-1 and most myeloma cells are dependent on this anti-apoptotic protein for survival. We observed at least additive effects using the combination treatment in MM cell lines and patient samples. To determine how the bone marrow environment affects the efficacy of the combination we conducted co-culture experiments with Hs-5 stromal cells. We also examined the mechanism of increased apoptosis by determining the affect on expression of the Bcl-2 family of proteins. We found that carfilzomib increases NOXA mRNA expression, as expected, and TG02 treatment caused a decrease in Mcl-1 protein but not mRNA levels. Consistent with this possibility, we find silencing CDK9 does not change carfilzomib sensitivity in the same manner as addition of TG02. Since changes in Mcl-1 protein occur in the presence of a proteasome inhibitor we hypothesize that regulation of Mcl-1 translation is the most likely mechanism. Taken together our data suggest that dual inhibition of Mcl-1 via decreased expression and the induction of its antagonist NOXA by the combination of carfilzomib and TG02 is active in myeloma and warrants further testing preclinically and in clinical trials. Moreover, regulation of Mcl-1 by TG02 is more complex than initially appreciated.

Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells.

PubMed

Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Gjörloff Wingren, Anette; Mongan, Nigel P; Heery, David M; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L

2016-09-27

One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies.

Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells

PubMed Central

Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Wingren, Anette Gjörloff; Mongan, Nigel P.; Heery, David M.; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L.

2016-01-01

One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies. PMID:27588408

CDK4/6 Inhibitors Sensitize Rb-positive Sarcoma Cells to Wee1 Kinase Inhibition through Reversible Cell-Cycle Arrest.

PubMed

Francis, Ashleigh M; Alexander, Angela; Liu, Yanna; Vijayaraghavan, Smruthi; Low, Kwang Hui; Yang, Dong; Bui, Tuyen; Somaiah, Neeta; Ravi, Vinod; Keyomarsi, Khandan; Hunt, Kelly K

2017-09-01

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G 1 phase of the cell cycle, and upon drug removal cells progress through the cell cycle as expected within 6-24 hours. Using a long-term high-throughput assay that allows us to examine drugs in different sequences or concurrently, we found that palbociclib-induced cell-cycle arrest poises Rb-positive sarcoma cells (SK-LMS1 and HT-1080) to be more sensitive to agents that work preferentially in S-G 2 phase such as doxorubicin and Wee1 kinase inhibitors (AZD1775). The synergy between palbociclib and AZD1775 was also validated in vivo using SK-LMS1 xenografts as well as Rb-positive patient-derived xenografts (PDX) developed from leiomyosarcoma patients. This work provides the necessary preclinical data in support of a clinical trial utilizing this treatment strategy. Mol Cancer Ther; 16(9); 1751-64. ©2017 AACR . ©2017 American Association for Cancer Research.

Palbociclib (PD0332991)-a Selective and Potent Cyclin-Dependent Kinase Inhibitor: A Review of Pharmacodynamics and Clinical Development.

PubMed

Clark, Amy S; Karasic, Thomas B; DeMichele, Angela; Vaughn, David J; O'Hara, Mark; Perini, Rodolfo; Zhang, Paul; Lal, Priti; Feldman, Michael; Gallagher, Maryann; O'Dwyer, Peter J

2016-02-01

Palbociclib (PD0332991) is a newly developed drug that received breakthrough designation and recent US Food and Drug Administration approval in combination with endocrine therapy in the treatment of hormone receptor positive, ERBB2-negative (formerly HER2 or HER2/neu) breast cancer in the first-line metastatic setting. Herein we describe the preclinical and translational data and early- and late-phase clinical trials in which palbociclib has been investigated in a broad array of tumor types. We discuss the pharmacodynamics, pharmacokinetics, toxic effects, and clinical response rates. On March 1, 2015, we conducted a review of the literature describing the development of palbociclib. We used the PubMed search terms "PD0332991," "palbociclib," and "CDK4/6 inhibitor" to find all published articles of interest, without limitation as to publication date. Palbociclib is a potent and specific oral cyclin-dependent kinase (CDK) 4/6 inhibitor that has strong preclinical data to support its activity in retinoblastoma protein-expressing tumors. Phase 1 trials have demonstrated safety, and phase 2 trials have shown single-agent activity in mantle-cell lymphoma, breast cancer, liposarcoma, and teratoma with reversible neutropenia as the main toxic effect. Addition of palbociclib to endocrine therapy improves progression-free survival in endocrine therapy-naïve and endocrine therapy-resistant metastatic settings. Palbociclib is well tolerated and has therapeutic potential for multiple cancers, including breast cancer, where its efficacy has been demonstrated alone and in combination with endocrine therapy. Additional combinations of palbociclib with endocrine therapy, chemotherapy, and targeted therapy have potential in various tumors, and phase 3 trials are under way.

Sequential inductions of the ZEB1 transcription factor caused by mutation of Rb and then Ras proteins are required for tumor initiation and progression.

PubMed

Liu, Yongqing; Sánchez-Tilló, Ester; Lu, Xiaoqin; Huang, Li; Clem, Brian; Telang, Sucheta; Jenson, Alfred B; Cuatrecasas, Miriam; Chesney, Jason; Postigo, Antonio; Dean, Douglas C

2013-04-19

Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2018-03-23

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

Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

PubMed

Rao, Mala V; Campbell, Jabbar; Palaniappan, Arti; Kumar, Asok; Nixon, Ralph A

2016-04-01

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly, CAST over-expression in neurons attenuated pathological effects of TDP-43, α-synuclein and Huntingtin. These results suggest a potential value of specific small molecule inhibitors of calpains in delaying the development of ALS. Read the Editorial Highlight for this article on page 140. © 2016 International Society for Neurochemistry.

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

PubMed Central

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

2005-01-01

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

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

PubMed

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

2017-08-30

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

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

PubMed Central

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

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

PubMed

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

2007-11-21

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

Cyclin-dependent kinase 2 protects podocytes from apoptosis

PubMed Central

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

2016-01-01

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

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

PubMed Central

Fu, Amy KY

2007-01-01

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

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

PubMed

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

2016-08-22

Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. Copyright © 2016 Elsevier Inc. All rights reserved.

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. ©2016 American Association for Cancer Research.

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

PubMed

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

2017-11-21

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

BCR-ABL1 promotes leukemia by converting p27 into a cytoplasmic oncoprotein

PubMed Central

Mackenzie, Ryan J.; Besson, Arnaud; Jeng, Sophia; Carey, Alyssa; LaTocha, Dorian H.; Fleischman, Angela G.; Duquesnes, Nicolas; Eide, Christopher A.; Vasudevan, Kavin B.; Loriaux, Marc M.; Firpo, Eduardo; Cortes, Jorge E.; McWeeney, Shannon; O’Hare, Thomas; Roberts, James M.; Druker, Brian J.; Deininger, Michael W.

2014-01-01

Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27CK−) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27T187A) or nuclear retention (p27S10A) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization. PMID:25293778

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

PubMed Central

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

2013-01-01

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

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

PubMed Central

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

2015-01-01

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

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

PubMed Central

Shackelford, R E; Kaufmann, W K; Paules, R S

1999-01-01

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling. Images Figure 3 PMID:10229703

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways

PubMed Central

Watanabe, Nobumoto; Arai, Harumi; Iwasaki, Jun-ichi; Shiina, Masaaki; Ogata, Kazuhiro; Hunter, Tony; Osada, Hiroyuki

2005-01-01

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCFβ-TrCP. The F-box protein β-TrCP (β-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to β-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved β-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in β-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to β-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of β-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second β-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis. PMID:16085715

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2002-02-01

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

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

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

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

2012-08-15

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

Molecular mechanism of APC/C activation by mitotic phosphorylation.

PubMed

Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-05-12

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

MEK Inhibitors in the Treatment of Metastatic Melanoma and Solid Tumors.

PubMed

Grimaldi, Antonio M; Simeone, Ester; Festino, Lucia; Vanella, Vito; Strudel, Martina; Ascierto, Paolo A

2017-12-01

The mitogen-activated protein kinase (MAPK) cascade is an intracellular signaling pathway involved in the regulation of cellular proliferation and the survival of tumor cells. Several different mutations, involving BRAF or NRAS, exert an oncogenic effect by activating the MAPK pathway, resulting in an increase in cellular proliferation. These mutations have become targets for new therapeutic strategies in melanoma and other cancers. Selective MEK inhibitors have the ability to inhibit growth and induce cell death in BRAF- and NRAS-mutant melanoma cell lines. MEK inhibitor therapy in combination with a BRAF inhibitor is more effective and less toxic than treatment with a BRAF inhibitor alone, and has become the standard of care for patients with BRAF-mutated melanoma. Trametinib was the first MEK inhibitor approved for the treatment of BRAF-mutated metastatic melanoma not previously treated with BRAF inhibitors, and is also approved in combination with the BRAF inhibitor dabrafenib. Furthermore, cobimetinib is another MEK inhibitor approved for the treatment of BRAF-mutated metastatic melanoma in combination with a BRAF inhibitor, vemurafenib. The MEK inhibitor binimetinib in combination with the BRAF inhibitor encorafenib is in clinical development. The addition of an anti-PD-1/PD-L1 agent, such as pembrolizumab, durvalumab or atezolizumab, to combined BRAF and MEK inhibition has shown considerable promise, with several trials ongoing in metastatic melanoma. Binimetinib has also shown efficacy in NRAS-mutated melanoma patients. Future possibilities for MEK inhibitors in advanced melanoma, as well as other solid tumors, include their use in combination with other targeted therapies (e.g. anti-CDK4/6 inhibitors) and/or various immune-modulating antibodies.

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

PubMed

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

2018-06-15

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

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

PubMed

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

2018-06-15

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

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

PubMed

Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

2013-01-01

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model.

PubMed

Rajasekaran, Devaraja; Siddiq, Ayesha; Willoughby, Jennifer L S; Biagi, Jessica M; Christadore, Lisa M; Yunes, Sarah A; Gredler, Rachel; Jariwala, Nidhi; Robertson, Chadia L; Akiel, Maaged A; Shen, Xue-Ning; Subler, Mark A; Windle, Jolene J; Schaus, Scott E; Fisher, Paul B; Hansen, Ulla; Sarkar, Devanand

2015-09-22

Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

PubMed Central

Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

2016-01-01

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

PubMed

Doucette, Carolyn D; Rodgers, Gemma; Liwski, Robert S; Hoskin, David W

2015-11-01

Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders. © 2015 Wiley Periodicals, Inc.

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

PubMed

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

2013-09-01

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

γ-Secretase inhibitor–resistant glioblastoma stem cells require RBPJ to propagate

PubMed Central

Fan, Xing

2016-01-01

Targeting glioblastoma stem cells with γ-secretase inhibitors (GSIs) disrupts the Notch pathway and has shown some benefit in both pre-clinical models and in patients during phase I/II clinical trials. However, it is largely unknown why some glioblastoma (GBM) does not respond to GSI treatment. In this issue of the JCI, Xie et al. determined that GSI-resistant brain tumor–initiating cells (BTICs) from GBM express a higher level of the gene RBPJ, which encodes a mediator of canonical Notch signaling, compared to non-BTICs. Knockdown of RBPJ in BTICs decreased propagation in vitro and in vivo by inducing apoptosis. Interestingly, RBPJ was shown to regulate a different transcription program than Notch in BTICs by binding CDK9, thereby affecting Pol II–regulated transcript elongation. Targeting CDK9 or c-MYC, an upstream regulator of RBPJ, with small molecules also decreased BTIC propagation, and prolonged survival in mice bearing orthotopic GBM xenografts. This study not only provides a mechanism for GSI treatment resistance, but also identifies two potential therapeutic strategies to target GSI-resistant BTICs. PMID:27322058

Liposarcoma: multimodality management and future targeted therapies

PubMed Central

Crago, Aimee M.; Dickson, Mark A.

2016-01-01

SYNOPSIS There are three biologic groups of liposarcoma: well- and dedifferentiated liposarcoma (WD/DDLS), myxoid/round cell liposarcoma (M/RCLS) and pleomorphic liposarcoma. WD/DDLS is characterized by amplification of 12q13-15 (including the oncogenes MDM2 and CDK4), M/RCLS by FUS-DDIT3 translocations, and pleomorphic liposarcoma by loss of p53 and Rb. In all three groups, complete surgical resection is central in treatment aimed at cure and is based on grade. Radiation can reduce risk of local recurrence in high-grade lesions or minimize surgical morbidity in the highly radiosensitive M/RCLS group. The biologic groups differ greatly in their chemosensitivity, so adjuvant chemotherapy is selectively utilized in chemosensitive histologies with metastatic potential (i.e. round cell and pleomorphic liposarcomas) but not in the relatively resistant subtype DDLS. An improved understanding of the genetic aberrations that lead to liposarcoma initiation is also allowing for the rapid development of targeted therapies for liposarcoma. Among such therapies are CDK4 inhibitors in WD/DDLS and trabectedin, which prevents FUS-DDIT3 binding to DNA, in M/RCLS. PMID:27591497

Genetic Testing in Pancreatic Ductal Adenocarcinoma: Implications for Prevention and Treatment.

PubMed

Peters, Mary Linton B; Tseng, Jennifer F; Miksad, Rebecca A

2016-07-01

This article reviews the progress to date and future directions for investigation of germline and somatic genetic testing to inform pancreatic adenocarcinoma (PDAC) treatment, screening, and prevention strategies. We searched PubMed to identify recent articles regarding genetic testing in pancreatic cancer, including both germline and somatic testing, and recent genome-wide association studies. References were specifically hand searched as relevant. Guidelines for testing and screening high-risk individuals were included. We searched clinicaltrials.gov to review the current landscape of active clinical trials. Approximately 10% of PDACs are associated with an identified germline mutation. Although germline mutations may inform treatment options and identify high-risk individuals for screening in other cancers, the data on PDAC are only now emerging. For example, poly adenosine diphosphate ribose polymerase (PARP) inhibitors are under investigation for BRCA-associated PDAC. Somatic mutations have also been identified in PDAC. However, current data are limited regarding treatment for potential PDAC somatic driver mutations. Although erlotinib is used in PDAC, its use is not targeted based on a tumor marker. Many tyrosine kinase inhibitors targeted toward potential driver mutations and critical pathways are in development, including BRAF/MEK, ALK, and CDK4/6. A consensus on screening strategies for individuals at high risk for PDAC is still evolving because of the relatively low prevalence of the disease, the relative invasiveness of endoscopic procedures often used as part of screening, and the lack of a clear survival benefit. Pancreatic cancer has been slower to move toward genomic testing, partially because of a lower prevalence of mutations and partially because of a limited effect of results on treatment choices outside a clinical trial. This is an area of active investigation, and we anticipate that there will be both preventive and therapeutic implications of driver mutations in the coming decade. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

Cdk1-dependent control of membrane-trafficking dynamics

PubMed Central

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

2012-01-01

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

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

PubMed

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

2017-09-07

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

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

PubMed

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

2008-04-01

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

Next-Generation Sequencing of Circulating Tumor DNA Reveals Frequent Alterations in Advanced Hepatocellular Carcinoma.

PubMed

Ikeda, Sadakatsu; Tsigelny, Igor F; Skjevik, Åge A; Kono, Yuko; Mendler, Michel; Kuo, Alexander; Sicklick, Jason K; Heestand, Gregory; Banks, Kimberly C; Talasaz, AmirAli; Lanman, Richard B; Lippman, Scott; Kurzrock, Razelle

2018-05-01

Because imaging has a high sensitivity to diagnose hepatocellular carcinoma (HCC) and tissue biopsies carry risks such as bleeding, the latter are often not performed in HCC. Blood-derived circulating tumor DNA (ctDNA) analysis can identify somatic alterations, but its utility has not been characterized in HCC. We evaluated 14 patients with advanced HCC (digital ctDNA sequencing [68 genes]). Mutant relative to wild-type allele fraction was calculated. All patients (100%) had somatic alterations (median = 3 alterations/patient [range, 1-8]); median mutant allele fraction, 0.29% (range, 0.1%-37.77%). Mutations were identified in several genes: TP53 (57% of patients), CTNNB1 (29%), PTEN (7%), CDKN2A (7%), ARID1A (7%), and MET (7%); amplifications, in CDK6 (14%), EGFR (14%), MYC (14%), BRAF (7%), RAF1 (7%), FGFR1 (7%), CCNE1 (7%), PIK3CA (7%), and ERBB2/HER2 (7%). Eleven patients (79%) had ≥1 theoretically actionable alteration. No two patients had identical genomic portfolios, suggesting the need for customized treatment. A patient with a CDKN2A -inactivating and a CTNNB1 -activating mutation received matched treatment: palbociclib (CDK4/6 inhibitor) and celecoxib (COX-2/Wnt inhibitor); des-gamma-carboxy prothrombin level decreased by 84% at 2 months (1,410 to 242 ng/mL [normal: ≤7.4 ng/mL]; alpha fetoprotein [AFP] low at baseline). A patient with a PTEN -inactivating and a MET -activating mutation (an effect suggested by in silico molecular dynamic simulations) received sirolimus (mechanistic target of rapamycin inhibitor) and cabozantinib (MET inhibitor); AFP declined by 63% (8,320 to 3,045 ng/mL [normal: 0-15 ng/mL]). ctDNA derived from noninvasive blood tests can provide exploitable genomic profiles in patients with HCC. This study reports that blood-derived circulating tumor DNA can provide therapeutically exploitable genomic profiles in hepatocellular cancer, a malignancy that is known to be difficult to biopsy. © AlphaMed Press 2018.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

PubMed

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

2014-04-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

PubMed Central

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

2014-01-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the protein data bank against curcumin. Cyclin dependent kinase 2 (CDK2), a major cell cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of Rb, a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine if CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

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

PubMed Central

Gérard, Claude; Goldbeter, Albert

2012-01-01

We recently proposed a detailed model describing the dynamics of the network of cyclin-dependent kinases (Cdks) driving the mammalian cell cycle (Gérard and Goldbeter, 2009). The model contains four modules, each centered around one cyclin/Cdk complex. Cyclin D/Cdk4–6 and cyclin E/Cdk2 promote progression in G1 and elicit the G1/S transition, respectively; cyclin A/Cdk2 ensures progression in S and the transition S/G2, while the activity of cyclin B/Cdk1 brings about the G2/M transition. This model shows that in the presence of sufficient amounts of growth factor the Cdk network is capable of temporal self-organization in the form of sustained oscillations, which correspond to the ordered, sequential activation of the various cyclin/Cdk complexes that control the successive phases of the cell cycle. The results suggest that the switch from cellular quiescence to cell proliferation corresponds to the transition from a stable steady state to sustained oscillations in the Cdk network. The transition depends on a finely tuned balance between factors that promote or hinder progression in the cell cycle. We show that the transition from quiescence to proliferation can occur in multiple ways that alter this balance. By resorting to bifurcation diagrams, we analyze the mechanism of oscillations in the Cdk network. Finally, we show that the complexity of the detailed model can be greatly reduced, without losing its key dynamical properties, by considering a skeleton model for the Cdk network. Using such a skeleton model for the mammalian cell cycle we show that positive feedback (PF) loops enhance the amplitude and the robustness of Cdk oscillations with respect to molecular noise. We compare the relative merits of the detailed and skeleton versions of the model for the Cdk network driving the mammalian cell cycle. PMID:23130001

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

PubMed

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

2017-04-01

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

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

PubMed

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

2017-12-11

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

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

PubMed Central

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

2014-01-01

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

Curcumin synergizes the growth inhibitory properties of Indian toad (Bufo melanostictus Schneider) skin-derived factor (BM-ANF1) in HCT-116 colon cancer cells.

PubMed

Giri, Biplab; Gomes, Antony; Sengupta, Radha; Banerjee, Sanjeev; Nautiyal, Jyoti; Sarkar, Fazlul H; Majumdar, Adhip P N

2009-01-01

Curcumin, an active ingredient of turmeric with no discernable toxicity, inhibits the growth of transformed cells and the development and progression of colon carcinogenesis in experimental animals. Recent data from one of our laboratories demonstrated that a crude skin extract or a purified crystalline compound (Bufo melanostictus-antineoplastic factor 1, BM-ANF1) from Indian common toad (Bufo melanostictus, Schneider) skin inhibits the growth of human leukemic cells. The present investigation was undertaken to determine whether combining BM-ANF1 with curcumin would be a better therapeutic strategy for colon cancer. Colon cancer HCT-116 cells were used. Changes in growth, apoptosis, growth factor receptor signaling and events of the cell cycle were analyzed. Curcumin together with BM-ANF1 produced a greater inhibition of HCT-116 cells growth than either agent alone, attributable to the inhibition of proliferation and stimulation of apoptosis, as evidenced by suppression of proliferating cell nuclear antigen (PCNA) expression, cell cycle arrest at the G2/M-phase and caspase-3 activation. There was also a marked reduction of cyclin-dependent kinase (CDK)2, CDK4 and cyclin B expression and up-regulation of CDK inhibitors (p21, p27) and p53, accompanied by attenuation of Akt signaling and nuclear factor-kappa B (NF-kappaB) activation. BM-ANF1 in combination with curcumin causes a marked inhibition of growth of colon cancer cells and could be an effective therapeutic strategy for colon cancer.

Modulatory effect of phytoglycoprotein (38 kDa) on cyclin D1/CDK4 in BNL CL.2 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine.

PubMed

Lee, Jin; Lim, Kye-Taek

2012-02-01

In the developmental stages of cancer, cell transformation occurs after the promotion stage and is a marker of cancer progression. This cell transformation is related to abnormal proliferation during the cancer initiation stage. The purpose of this study was to evaluate the effect of Styrax japonica Siebold et al. Zuccarin (SJSZ) glycoprotein on cell transformation in murine embryonic liver cells (BNL CL.2) following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. To determine abnormal proliferation during the initiation stage, intracellular reactive oxygen species (ROS), phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), activities of cell cycle-related factors [cyclin D1/cyclin dependent kinase (CDK) 4], cell cycle inhibitors (p53, p21, and p27), nuclear factor (NF)-κB, and proliferating cell nuclear antigen (PCNA) were evaluated using Western blot analysis and real-time PCR. Our study demonstrated that SJSZ glycoprotein (50 μg/ml) reduces foci formation with combined treatment [MNNG and 12-O-tetradecanoyl phorbol-13-acetate] of BNL CL.2 cells. With regard to proliferation-related signals, our finding indicated that SJSZ glycoprotein (50 μg/ml) diminished the production of intracellular ROS, activity of phosphorylated ERK, p38 MAPK, NF-κB (p50 and p65), PCNA, and cyclin D1/CDK4 in MNNG-induced BNL CL.2 cells. Taken together, these results lead us to speculate that SJSZ glycoprotein can inhibit abnormal cell proliferation at the initiation stage of hepatocarcinogenesis.

A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

NASA Technical Reports Server (NTRS)

Ratner, N.; Bloom, G. S.; Brady, S. T.

1998-01-01

Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

Phospho-T356RB1 predicts survival in HPV-negative squamous cell carcinoma of the head and neck

PubMed Central

Handorf, Elizabeth; Nikonova, Anna; Dubyk, Cara; Peri, Suraj; Lango, Miriam; Ridge, John A.; Serebriiskii, Ilya G.; Burtness, Barbara; Golemis, Erica A.; Mehra, Ranee

2015-01-01

Locally advanced squamous cell carcinoma of the head and neck (SCCHN) that is not associated with human papillomavirus (HPV) has a poor prognosis in contrast to HPV-positive disease. To better understand the importance of RB1 activity in HPV-negative SCCHN, we investigated the prognostic value of inhibitory CDK4/6 phosphorylation of RB1 on threonine 356 (T356) in archival HPV-negative tumor specimens from patients who underwent surgical resection and adjuvant radiation. We benchmarked pT356RB1 to total RB1, Ki67, pT202/Y204ERK1/2, and TP53, as quantified by automatic quantitative analysis (AQUA), and correlated protein expression with tumor stage and grade. High expression of pT356RB1 but not total RB1 predicted reduced overall survival (OS; P = 0.0295), indicating the potential relevance of post-translational phosphorylation. Paired analysis of The Cancer Genome Atlas (TCGA) data for regulators of this RB1 phosphorylation identified loss or truncating mutation of negative regulator CDKN2A (p16) and elevated expression of the CDK4/6 activator CCND1 (cyclin D) as also predicting poor survival. Given that CDK4/6 inhibitors have been most effective in the context of functional RB1 and low expression or deletion of p16 in other tumor types, these data suggest such agents may merit evaluation in HPV-negative SCCHN, specifically in cases associated with high pT356RB1. PMID:26265441

Phosphorylation of amyloid precursor protein at threonine 668 is essential for its copper-responsive trafficking in SH-SY5Y neuroblastoma cells.

PubMed

Acevedo, Karla M; Opazo, Carlos M; Norrish, David; Challis, Leesa M; Li, Qiao-Xin; White, Anthony R; Bush, Ashley I; Camakaris, James

2014-04-18

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.

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

PubMed

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

2006-10-01

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

Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells*

PubMed Central

Acevedo, Karla M.; Opazo, Carlos M.; Norrish, David; Challis, Leesa M.; Li, Qiao-Xin; White, Anthony R.; Bush, Ashley I.; Camakaris, James

2014-01-01

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells. PMID:24610780

A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.

PubMed

Arai, Kazuya; Eguchi, Takanori; Rahman, M Mamunur; Sakamoto, Ruriko; Masuda, Norio; Nakatsura, Tetsuya; Calderwood, Stuart K; Kozaki, Ken-Ichi; Itoh, Manabu

2016-01-01

Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

Reversal of collapsing glomerulopathy in mice with the cyclin-dependent kinase inhibitor CYC202.

PubMed

Gherardi, Dana; D'Agati, Vivette; Chu, Te-Hua Tearina; Barnett, Anna; Gianella-Borradori, Athos; Gelman, Irwin H; Nelson, Peter J

2004-05-01

Collapsing glomerulopathy (CG) has become an important cause of end-stage renal disease. Whether associated with HIV-1 or other potential etiologies, the pathogenesis of CG converges to induce aberrant proliferation of renal epithelium along the entire nephron. This raises the possibility that targeting cell-cycle progression may be an effective therapeutic strategy for CG. Here, we ask whether the cyclin-dependent kinase (CDK) inhibitor, CYC202 (R-roscovitine), could attenuate or reverse existing renal disease in Tg26 mice, a well characterized HIV-1 transgenic mouse model of CG. Tg26 mice were age and disease matched through analysis of urine (protein/creatinine) to generate 12 treatment pairs covering a range of mild to severe CG. One mouse from each pair received either vehicle or 75 mg/kg of CYC202 every 12 h for 20 d, a dose 20% above that needed to prevent the development of CG. After treatment, urinary, serologic, and histopathologic indices of nephrosis showed reversal of CG in 8 of 12 CYC202-treated mice compared with progression of CG in 10 of 12 vehicle-treated mice, demonstrating a significant therapeutic benefit from CYC202 (P < 0.05). Pharmacokinetic profiles showed that concentrations of CYC202 known to inhibit cell-cycle and transcriptional CDK in vitro were achieved in plasma at efficacious doses. However, amelioration of CG by CYC202 did not correlate with decreases in kidney HIV-1 transgene expression, indicating that suppression of HIV-1 transcription was not a prerequisite for the antiproliferative activity of CYC202. These results demonstrate a novel therapeutic strategy for CG.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

PubMed Central

Ravi, R K; Weber, E; McMahon, M; Williams, J R; Baylin, S; Mal, A; Harter, M L; Dillehay, L E; Claudio, P P; Giordano, A; Nelkin, B D; Mabry, M

1998-01-01

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors. PMID:9421477

Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

PubMed Central

Long, John J.; Leresche, Anne; Kriwacki, Richard W.; Gottesfeld, Joel M.

1998-01-01

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis. PMID:9488463

The cyclin-dependent kinase inhibitor p57Kip2 regulates cell cycle exit, differentiation, and migration of embryonic cerebral cortical precursors.

PubMed

Tury, Anna; Mairet-Coello, Georges; DiCicco-Bloom, Emanuel

2011-08-01

Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57(Kip2) and p27(Kip1), control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57(Kip2) remain poorly defined. Using in vivo and culture approaches, we show p57(Kip2) overexpression at E14.5-15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57(Kip2)-deficient precursors. Studies at later ages indicate p57(Kip2) overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57(Kip2) overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57(Kip2) was twice as effective as p27(Kip1) in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57(Kip2) alone inhibits in vivo migration. Furthermore, p57(Kip2) effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27(Kip1) studies report cell cycle-independent functions. These observations suggest p57(Kip2) coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27(Kip1).

Targeting ornithine decarboxylase in Myc-induced lymphomagenesis prevents tumor formation.

PubMed

Nilsson, Jonas A; Keller, Ulrich B; Baudino, Troy A; Yang, Chunying; Norton, Sara; Old, Jennifer A; Nilsson, Lisa M; Neale, Geoffrey; Kramer, Debora L; Porter, Carl W; Cleveland, John L

2005-05-01

Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention.

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

PubMed Central

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

2014-01-01

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

Cyclin C influences the timing of mitosis in fission yeast

PubMed Central

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

2017-01-01

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

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

PubMed

Klinman, Eva; Holzbaur, Erika L F

2015-07-21

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

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

PubMed

Huber, Robert J; O'Day, Danton H

2011-08-01

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

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

PubMed

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

2018-04-23

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

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

PubMed

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

2013-01-01

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

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

PubMed

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

2012-01-03

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2003-06-10

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

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

PubMed Central

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

2014-01-01

Background The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas. Methods From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR). Results There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05). Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041). High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012) and multivariate analyses (P = 0.020). Conclusions Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection. PMID:25121597

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

PubMed

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

2015-02-01

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-08-01

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

Synthesis and biological evaluation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors.

PubMed

Chioua, Mourad; Samadi, Abdelouahid; Soriano, Elena; Lozach, Olivier; Meijer, Laurent; Marco-Contelles, José

2009-08-15

The synthesis and biological evaluation of a number of differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives are reported. From the inhibition results on a selection of disease-relevant protein kinases [IC(50) (microM) DYRK1A=11; CDK5=0.41; GSK-3=1.5] we have observed that 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4) constitutes a potential new and simple lead compound in the search of drugs for the treatment of Alzheimer's disease.

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

PubMed Central

Merrick, Karl A.; Fisher, Robert P.

2010-01-01

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

Expression of CDK6 in Oral Squamous Cell Carcinomas

PubMed

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

2018-04-25

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

Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma.

PubMed

Faria, Claudia C; Agnihotri, Sameer; Mack, Stephen C; Golbourn, Brian J; Diaz, Roberto J; Olsen, Samantha; Bryant, Melissa; Bebenek, Matthew; Wang, Xin; Bertrand, Kelsey C; Kushida, Michelle; Head, Renee; Clark, Ian; Dirks, Peter; Smith, Christian A; Taylor, Michael D; Rutka, James T

2015-08-28

Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma.

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

PubMed Central

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

2016-01-01

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

Palbociclib in Hormone-Receptor-Positive Advanced Breast Cancer.

PubMed

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

2015-07-16

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

AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

PubMed Central

Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S.; Olson, Arthur J.; Sanner, Michel F.

2015-01-01

Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down-weighting the receptor internal energy improves the ranking of correctly docked poses and that runtime for AutoDockFR scales linearly when side-chain flexibility is added. PMID:26629955

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

PubMed

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

2015-10-01

Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1 chromatin target was further demonstrated using chromatin immunoprecipitation (ChIP). These data indicate that CDK9 is a dynamic multifunctional enzyme complex mediating not only transcriptional elongation, but also alternative RNA splicing and potentially translational control. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

PubMed Central

Barr, Alexis R.; Cooper, Samuel; Heldt, Frank S.; Butera, Francesca; Stoy, Henriette; Mansfeld, Jörg; Novák, Béla; Bakal, Chris

2017-01-01

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability. PMID:28317845

Robust mitotic entry is ensured by a latching switch.

PubMed

Tuck, Chloe; Zhang, Tongli; Potapova, Tamara; Malumbres, Marcos; Novák, Béla

2013-01-01

Cell cycle events are driven by Cyclin dependent kinases (CDKs) and by their counter-acting phosphatases. Activation of the Cdk1:Cyclin B complex during mitotic entry is controlled by the Wee1/Myt1 inhibitory kinases and by Cdc25 activatory phosphatase, which are themselves regulated by Cdk1:Cyclin B within two positive circuits. Impairing these two feedbacks with chemical inhibitors induces a transient entry into M phase referred to as mitotic collapse. The pathology of mitotic collapse reveals that the positive circuits play a significant role in maintaining the M phase state. To better understand the function of these feedback loops during G2/M transition, we propose a simple model for mitotic entry in mammalian cells including spatial control over Greatwall kinase phosphorylation. After parameter calibration, the model is able to recapture the complex and non-intuitive molecular dynamics reported by Potapova et al. (Potapova et al., 2011). Moreover, it predicts the temporal patterns of other mitotic regulators which have not yet been experimentally tested and suggests a general design principle of cell cycle control: latching switches buffer the cellular stresses which accompany cell cycle processes to ensure that the transitions are smooth and robust.

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

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

Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa

2015-04-17

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

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

PubMed

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

2017-12-12

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

Functional characterization of the 19q12 amplicon in grade III breast cancers

PubMed Central

2012-01-01

Introduction The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. Methods We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. Results 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. Conclusions The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a rationale for the testing of these chemical inhibitors in a subgroup of patients with ER-negative grade III breast cancers. PMID:22433433

Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid

PubMed Central

Tsai, Shang-Yi A.; Pokrass, Michael J.; Klauer, Neal R.; Nohara, Hiroshi; Su, Tsung-Ping

2015-01-01

Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor’s interaction with myristic acid. In Sig-1R–KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R–KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R–KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25. PMID:25964330

Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid.

PubMed

Tsai, Shang-Yi A; Pokrass, Michael J; Klauer, Neal R; Nohara, Hiroshi; Su, Tsung-Ping

2015-05-26

Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor's interaction with myristic acid. In Sig-1R-KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R-KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R-KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25.

Nephrotoxicity of Epigenetic Inhibitors Used for the Treatment of Cancer

PubMed Central

Scholpa, N.E.; Kolli, R.T.; Moore, M.; Arnold, R.D.; Glenn, T.C.; Cummings, B.S.

2016-01-01

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 hr. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 hr. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury. PMID:27543423

Cdk5 Regulates Activity-Dependent Gene Expression and Dendrite Development.

PubMed

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

2015-11-11

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

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

PubMed

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

2018-03-06

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

Kinase inhibitors for CNS diseases: an analysis of the recent patent literature.

PubMed

Amigoni, Federica; Legnaghi, Elena; Pevarello, Paolo

2012-05-01

Protein kinases (PKs), as members of an important target class in current pharmaceutical research, have been mostly exploited so far in therapeutic areas such as oncology and inflammation. However, basic research on some PKs as key components of molecular mechanisms underlying neurodegeneration and neuroprotection may translate into new medicines for CNS diseases in the next few years. This review is an account of recent patents dealing with kinase inhibitors primarily designed for CNS indications. CNS-directed patents on kinase modulators published after 2008 were surveyed using SciFinder(®) and public patent search engines. Some PK targets, such as GSK-3β, CDK5, ROCK and p38α MAPK, continue to attract interest even though a clinical proof-of-concept is yet to be attained in a CNS setting. Less established PKs such as LRRK2, MLK, PAK and DAPK-1 hold promise as valuable targets of the future.

Activator-inhibitor coupling between Rho signaling and actin assembly make the cell cortex an excitable medium

PubMed Central

Bement, William M.; Leda, Marcin; Moe, Alison M.; Kita, Angela M.; Larson, Matthew E.; Golding, Adriana E.; Pfeuti, Courtney; Su, Kuan-Chung; Miller, Ann L.; Goryachev, Andrew B.; von Dassow, George

2016-01-01

Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, while Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modeling results show that waves represent excitable dynamics of a reaction diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation. PMID:26479320

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

PubMed

Ruegsegger, Gregory N; Toedebusch, Ryan G; Childs, Thomas E; Grigsby, Kolter B; Booth, Frank W

2017-01-01

Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases. The nucleus accumbens (the pleasure and reward 'hub' in the brain) influences wheel running behaviour in rodents. RNA-sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age-related reductions in wheel running. Upon completion of follow-up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age-related changes in voluntary running. Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running. The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age-dependent reductions in the motivation to be physically active. Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA-sequencing was used to interrogate transcriptomic changes between 8- and 14-week-old wheel running rats, and select transcripts were later analysed by quantitative RT-PCR in age-matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP-mediated signalling, dopamine- and cAMP-regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8- vs. 14-week-old rats. In depth analysis of these networks showed significant (∼20-30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin-dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14- vs. 8-week-old sedentary rats (P = 0.03). Intriguingly, intra-NAc injection of the Cdk5 inhibitor roscovitine, dose-dependently decreased wheel running. Collectively, these experiments suggest that an age-dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age-related declines in voluntary running behaviour. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

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

PubMed Central

Ruegsegger, Gregory N.; Toedebusch, Ryan G.; Childs, Thomas E.; Grigsby, Kolter B.

2016-01-01

Key points Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases.The nucleus accumbens (the pleasure and reward ‘hub’ in the brain) influences wheel running behaviour in rodents.RNA‐sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age‐related reductions in wheel running. Upon completion of follow‐up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age‐related changes in voluntary running.Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running.The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age‐dependent reductions in the motivation to be physically active. Abstract Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA‐sequencing was used to interrogate transcriptomic changes between 8‐ and 14‐week‐old wheel running rats, and select transcripts were later analysed by quantitative RT‐PCR in age‐matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP‐mediated signalling, dopamine‐ and cAMP‐regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8‐ vs. 14‐week‐old rats. In depth analysis of these networks showed significant (∼20–30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin‐dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14‐ vs. 8‐week‐old sedentary rats (P = 0.03). Intriguingly, intra‐NAc injection of the Cdk5 inhibitor roscovitine, dose‐dependently decreased wheel running. Collectively, these experiments suggest that an age‐dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age‐related declines in voluntary running behaviour. PMID:27461471

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

PubMed

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

2015-03-01

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

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

PubMed Central

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

1998-01-01

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

The Molecular Chaperone Hsp90 Is Required for Cell Cycle Exit in Drosophila melanogaster

PubMed Central

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

2013-01-01

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

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

PubMed

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

2013-01-01

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

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

PubMed

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

2017-11-01

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

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Bag, Indira; Hunt, Clayton R; Ramaiah, M Janaki; Pandita, Tej K; Bhadra, Utpal; Pal-Bhadra, Manika

2014-02-01

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

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

PubMed

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

2018-05-02

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

OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo.

PubMed

Zhi, Xiao; Chen, Wei; Xue, Fei; Liang, Chao; Chen, Bryan Wei; Zhou, Yue; Wen, Liang; Hu, Liqiang; Shen, Jian; Bai, Xueli; Liang, Tingbo

2015-09-22

Despite its relative rarity, pancreatic ductal adenocarcinoma (PDAC) accounts for a large percentage of cancer deaths. In this study, we investigated the in vitro efficacy of OSI-027, a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, to treat PDAC cell lines alone, and in combination with gemcitabine (GEM). Similarly, we tested the efficacy of these two compounds in a xenograft mouse model of PDAC. OSI-027 significantly arrested cell cycle in G0/G1 phase, inhibited the proliferation of Panc-1, BxPC-3, and CFPAC-1 cells, and downregulated mTORC1, mTORC2, phospho-Akt, phospho-p70S6K, phospho-4E-BP1, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in these cells. Moreover, OSI-027 also downregulated multidrug resistance (MDR)-1, which has been implicated in chemotherapy resistance in PDAC cells and enhanced apoptosis induced by GEM in the three PDAC cell lines. When combined, OSI-027 with GEM showed synergistic cytotoxic effects both in vitro and in vivo. This is the first evidence of the efficacy of OSI-027 in PDAC and may provide the groundwork for a new clinical PDAC therapy.

OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo

PubMed Central

Xue, Fei; Liang, Chao; Chen, Bryan Wei; Zhou, Yue; Wen, Liang; Hu, Liqiang; Shen, Jian; Bai, Xueli; Liang, Tingbo

2015-01-01

Despite its relative rarity, pancreatic ductal adenocarcinoma (PDAC) accounts for a large percentage of cancer deaths. In this study, we investigated the in vitro efficacy of OSI-027, a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, to treat PDAC cell lines alone, and in combination with gemcitabine (GEM). Similarly, we tested the efficacy of these two compounds in a xenograft mouse model of PDAC. OSI-027 significantly arrested cell cycle in G0/G1 phase, inhibited the proliferation of Panc-1, BxPC-3, and CFPAC-1 cells, and downregulated mTORC1, mTORC2, phospho-Akt, phospho-p70S6K, phospho-4E-BP1, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in these cells. Moreover, OSI-027 also downregulated multidrug resistance (MDR)-1, which has been implicated in chemotherapy resistance in PDAC cells and enhanced apoptosis induced by GEM in the three PDAC cell lines. When combined, OSI-027 with GEM showed synergistic cytotoxic effects both in vitro and in vivo. This is the first evidence of the efficacy of OSI-027 in PDAC and may provide the groundwork for a new clinical PDAC therapy. PMID:26213847

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

PubMed

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

2018-06-04

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

Climatic droplet keratopathy, exfoliation syndrome, and cataract.

PubMed Central

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

1991-01-01

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

Therapeutically targeting cyclin D1 in primary tumors arising from loss of Ini1

PubMed Central

Smith, Melissa E.; Cimica, Velasco; Chinni, Srinivasa; Jana, Suman; Koba, Wade; Yang, Zhixia; Fine, Eugene; Zagzag, David; Montagna, Cristina; Kalpana, Ganjam V.

2011-01-01

Rhabdoid tumors (RTs) are rare, highly aggressive pediatric malignancies with poor prognosis and with no standard or effective treatment strategies. RTs are characterized by biallelic inactivation of the INI1 tumor suppressor gene. INI1 directly represses CCND1 and activates cyclin-dependent kinase (cdk) inhibitors p16Ink4a and p21CIP. RTs are exquisitely dependent on cyclin D1 for genesis and survival. To facilitate translation of unique therapeutic strategies, we have used genetically engineered, Ini1+/− mice for therapeutic testing. We found that PET can be used to noninvasively and accurately detect primary tumors in Ini1+/− mice. In a PET-guided longitudinal study, we found that treating Ini1+/− mice bearing primary tumors with the pan-cdk inhibitor flavopiridol resulted in complete and stable regression of some tumors. Other tumors showed resistance to flavopiridol, and one of the resistant tumors overexpressed cyclin D1, more than flavopiridol-sensitive cells. The concentration of flavopiridol used was not sufficient to down-modulate the high level of cyclin D1 and failed to induce cell death in the resistant cells. Furthermore, FISH and PCR analyses indicated that there is aneuploidy and increased CCND1 copy number in resistant cells. These studies indicate that resistance to flavopiridol may be correlated to elevated cyclin D1 levels. Our studies also indicate that Ini1+/− mice are valuable tools for testing unique therapeutic strategies and for understanding mechanisms of drug resistance in tumors that arise owing to loss of Ini1, which is essential for developing effective treatment strategies against these aggressive tumors. PMID:21173237

Rb1 loss modifies but does not initiate alveolar rhabdomyosarcoma

PubMed Central

2013-01-01

Background Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. Methods We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. Results Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. Conclusions Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function. PMID:24274149

Selective Effects of PD-1 on Akt and Ras Pathways Regulate Molecular Components of the Cell Cycle and Inhibit T Cell Proliferation

PubMed Central

Patsoukis, Nikolaos; Brown, Julia; Petkova, Victoria; Liu, Fang; Li, Lequn; Boussiotis, Vassiliki A.

2017-01-01

The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4+ T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCFSkp2 degrades p27kip1, an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G1 phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G1 phase inhibitor p15INK4 and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase–Akt and Ras–mitogen-activated and extracellular signal–regulated kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle. PMID:22740686

A phase 1 clinical trial of flavopiridol consolidation in chronic lymphocytic leukemia patients following chemoimmunotherapy

PubMed Central

Awan, Farrukh T.; Jones, Jeffrey A.; Maddocks, Kami; Poi, Ming; Grever, Michael R.; Johnson, Amy; Byrd, John C.; Andritsos, Leslie A.

2016-01-01

Patients with chronic lymphocytic leukemia (CLL) who receive chemoimmunotherapy and do not achieve complete remission experience significantly shortened progression-free interval (PFS). Additionally, the majority of patients treated for relapsed disease demonstrate evidence of measurable disease. Eradication of minimal residual disease (MRD) results in improved PFS and overall survival. Maintenance therapy might result in eradication of MRD and improve response duration but might be associated with an increase in incidence of infectious complications. Flavopiridol is a broad cyclin-dependent kinase (CDK) inhibitor with established safety and efficacy in patients with relapsed CLL, particularly patients with high-risk cytogenetic features. A pharmacologically derived schedule was utilized as consolidation therapy in this phase I study to assess the safety and feasibility of outpatient therapy with flavopiridol in patients with low tumor burden. Flavopiridol was administered as a 30-min loading dose of 30 mg/m2 followed by a 4-h infusion of 30 mg/ m2 once weekly for 3 weeks every 5 weeks (1 cycle) for planned 2 cycles in ten patients. Therapy was extremely well tolerated and no patient developed acute tumor lysis syndrome. The most common toxicities were gastrointestinal. Of the patients, 22 % improved their response from a PR to CR. Eighty-eight percent experienced a reduction in tumor burden as measured by extent of bone marrow involvement including patients with del17p and complex karyotype. The study establishes the safety and efficacy of flavopiridol as consolidation therapy after chemoimmunotherapy for patients with CLL. Further evaluation is required in larger trials for the utility of CDK inhibitors as consolidation or maintenance strategies. Registration number at ClinicalTrials.gov: NCT00377104. PMID:27118540

The Cdk5 inhibitor olomoucine promotes corneal debridement wound closure in vivo

PubMed Central

Tripathi, Brajendra K.; Stepp, Mary A.; Gao, Chun Y.

2008-01-01

Purpose To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo. Methods Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 μl drop of 15 µM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson’s dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting. Results Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization. Conclusions Topical application of olomoucine in 1% DMSO significantly enhances closure of small epithelial debridement wounds without increasing inflammation or impairing reepithelialization. PMID:18385789

Comprehensive profiling of DNA repair defects in breast cancer identifies a novel class of endocrine therapy resistance drivers.

PubMed

Anurag, Meenakshi; Punturi, Nindo; Hoog, Jeremy; Bainbridge, Matthew N; Ellis, Matthew J; Haricharan, Svasti

2018-05-23

This study was undertaken to conduct a comprehensive investigation of the role of DNA damage repair (DDR) defects in poor outcome ER+ disease. Expression and mutational status of DDR genes in ER+ breast tumors were correlated with proliferative response in neoadjuvant aromatase inhibitor therapy trials (discovery data set), with outcomes in METABRIC, TCGA and Loi data sets (validation data sets), and in patient derived xenografts. A causal relationship between candidate DDR genes and endocrine treatment response, and the underlying mechanism, was then tested in ER+ breast cancer cell lines. Correlations between loss of expression of three genes: CETN2 (p<0.001) and ERCC1 (p=0.01) from the nucleotide excision repair (NER) and NEIL2 (p=0.04) from the base excision repair (BER) pathways were associated with endocrine treatment resistance in discovery data sets, and subsequently validated in independent patient cohorts. Complementary mutation analysis supported associations between mutations in NER and BER pathways and reduced endocrine treatment response. A causal role for CETN2, NEIL2 and ERCC1 loss in intrinsic endocrine resistance was experimentally validated in ER+ breast cancer cell lines, and in ER+ patient-derived xenograft models. Loss of CETN2, NEIL2 or ERCC1 induced endocrine treatment response by dysregulating G1/S transition, and therefore, increased sensitivity to CDK4/6 inhibitors. A combined DDR signature score was developed that predicted poor outcome in multiple patient cohorts. This report identifies DDR defects as a new class of endocrine treatment resistance drivers and indicates new avenues for predicting efficacy of CDK4/6 inhibition in the adjuvant treatment setting. Copyright ©2018, American Association for Cancer Research.

Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

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

Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonarymore » hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.« less

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing.

PubMed

Sakurai, Takashi; Kaneko, Kumi; Okuno, Misako; Wada, Koji; Kashiyama, Taku; Shimizu, Hideaki; Akagi, Takumi; Hashikawa, Tsutomu; Nukina, Nobuyuki

2008-10-20

Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (Abeta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Abeta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing

PubMed Central

Sakurai, Takashi; Kaneko, Kumi; Okuno, Misako; Wada, Koji; Kashiyama, Taku; Shimizu, Hideaki; Akagi, Takumi; Hashikawa, Tsutomu; Nukina, Nobuyuki

2008-01-01

Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons. PMID:18936252

Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E.

PubMed

Antonello, Zeus A; Hsu, Nancy; Bhasin, Manoj; Roti, Giovanni; Joshi, Mukta; Van Hummelen, Paul; Ye, Emily; Lo, Agnes S; Karumanchi, S Ananth; Bryke, Christine R; Nucera, Carmelo

2017-10-17

Papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAF V600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAF WT/V600E -positive PTC patient-derived cells with P16 -/- (CDKN2A -/- ). Following treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples. Vemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAF WT/V600E -PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAF WT/V600E mutation. Critically, our findings suggest for the first time that targeting BRAF WT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAF WT/V600E -PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAF V600E inhibitor.

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

PubMed

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

2017-12-29

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

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

PubMed

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

2018-05-03

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

Two Bistable Switches Govern M Phase Entry.

PubMed

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

2016-12-19

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

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

PubMed

Hossain, Manzar; Stillman, Bruce

2012-08-15

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

τ kinases in the rat heat shock model: Possible implications for Alzheimer disease

PubMed Central

Shanavas, Alikunju; Papasozomenos, Sozos Ch.

2000-01-01

We have previously shown, by using the phosphate-dependent anti-τ antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of τ followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague–Dawley rats the activities of extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) at 0 (n = 5), 3 (n = 4), 6 (n = 5), and 12 (n = 5) h after heat shock and in non-heat-shocked controls (n = 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3β, which showed increased activity. At 3–6 h, the activities of ERK1/2, JNK, Cdk5, and GSK-3β toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3β but not ERK1/2 were overactivated toward purified bovine τ. At 3–6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either τ or selective substrates. All of eight anti-τ antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3–6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1/2, JNK, and GSK-3β confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because τ is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3β, and Cdk5 may play a role in its pathogenesis. PMID:11121021

Mutations in CDK5RAP2 cause Seckel syndrome.

PubMed

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

2015-09-01

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

Mutations in CDK5RAP2 cause Seckel syndrome

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-05-02

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

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

PubMed

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

2014-10-28

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

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

PubMed

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

1996-06-01

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

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

PubMed

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

2017-01-01

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

Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

PubMed

Yothaisong, Supak; Dokduang, Hasaya; Anzai, Naohiko; Hayashi, Keitaro; Namwat, Nisana; Yongvanit, Puangrat; Sangkhamanon, Sakkarn; Jutabha, Promsuk; Endou, Hitoshi; Loilome, Watcharin

2017-03-01

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na + -independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [ 14 C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

cDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells

PubMed Central

Shi, Ting; Mazumdar, Tapati; DeVecchio, Jennifer; Duan, Zhong-Hui; Agyeman, Akwasi; Aziz, Mohammad; Houghton, Janet A.

2010-01-01

Background Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited. Methodology/Principal Findings To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling. Conclusions/Significance This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary. PMID:20957031

Runx1t1 (Runt-Related Transcription Factor 1; Translocated to, 1) Epigenetically Regulates the Proliferation and Nitric Oxide Production of Microglia

PubMed Central

Baby, Nimmi; Li, Yali; Ling, Eng-Ang; Lu, Jia; Dheen, S. Thameem

2014-01-01

Background Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions. Methods and Findings siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2. Conclusion/Interpretation In conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs. PMID:24586690

Drug sensitivity profiling identifies potential therapies for lymphoproliferative disorders with overactive JAK/STAT3 signaling

PubMed Central

Kuusanmäki, Heikki; Dufva, Olli; Parri, Elina; van Adrichem, Arjan J.; Rajala, Hanna; Majumder, Muntasir M.; Yadav, Bhagwan; Parsons, Alun; Chan, Wing C.; Wennerberg, Krister; Mustjoki, Satu; Heckman, Caroline A.

2017-01-01

Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3. PMID:29228628

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

PubMed Central

Kim, So Yoon; Rane, Sushil G.

2011-01-01

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

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

PubMed

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

2016-07-15

The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. © 2016 Höckner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199)

PubMed Central

Phillips, D C; Xiao, Y; Lam, L T; Litvinovich, E; Roberts-Rapp, L; Souers, A J; Leverson, J D

2015-01-01

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2High) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2High cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2High NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2Low NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2Low) that could benefit from BCL-XL (navitoclax)-driven combination therapy. PMID:26565405

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199).

PubMed

Phillips, D C; Xiao, Y; Lam, L T; Litvinovich, E; Roberts-Rapp, L; Souers, A J; Leverson, J D

2015-11-13

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2(High)) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2(High) cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2(High) NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2(Low) NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2(Low)) that could benefit from BCL-XL (navitoclax)-driven combination therapy.

Panobinostat induces apoptosis via production of reactive oxygen species and synergizes with topoisomerase inhibitors in cervical cancer cells.

PubMed

Wasim, Lubna; Chopra, Madhu

2016-12-01

Cervical cancer is the fourth major cause of cancer-related deaths in women worldwide and is the most common cancer in developing countries. Therefore, a search for novel treatment modalities is warranted. The present study is designed to investigate the effect of pan histone deacetylase inhibitor, 'panobinostat', on cervical cancer cells alone and in combination with topoisomerase inhibitors. We assessed the effect of panobinostat on two cervical cancer cell lines, HeLa and SiHa, for cell viability, apoptosis, oxidative stress and mitochondrial function using various assays. The results indicate that panobinostat reduces the viability of cervical cancer cells in a dose- and time-dependent manner; it arrests HeLa cells in G0/G1 and SiHa cells in G2/M phase of the cell cycle. Panobinostat induced apoptosis through an increase in the ROS production and the disruption of mitochondrial membrane potential. Concomitantly the expression of anti-apoptotic gene Bcl-xL was reduced, while levels of CDK inhibitor p21 and caspase-9 were increased. Panobinostat increased the acetylation of histone H3 indicating HDAC inhibition. In addition, panobinostat also showed synergistic effect with topoisomerase inhibitors mediated by increased activation of caspase-3/7 activity compared to that in cells treated with panobinostat alone. These results suggest a combination therapy using inhibitors of histone deacetylase and topoisomerase together could hold the promise for an effective targeted therapeutic strategy. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

PubMed Central

Gutiérrez-Escribano, Pilar; Nurse, Paul

2015-01-01

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

Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation

PubMed Central

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

2015-01-01

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

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, sensitizes lung cancer cells to treatment with epidermal growth factor receptor tyrosine kinase inhibitors

PubMed Central

Li, Ying; Li, Yongwen; Zhang, Hongbing; Liu, Hongyu; Chen, Jun

2016-01-01

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge to targeted therapy for non-small cell lung cancer (NSCLC). We investigated whether a cyclin D kinase 4/6 (CDK4/6) inhibitor, PD 0332991, could reverse EGFR-TKI resistance in human lung cancer cells and explored the underlying mechanisms. We found that PD 0332991 potentiated gefitinib-induced growth inhibition in both EGFR-TKI-sensitive (PC-9) and EGFR-TKI-resistant (PC-9/AB2) cells by down-regulating proliferation and inducing apoptosis and G0/G1 cell cycle arrest. Tumor xenografts were then used to verify the effects of PD 0332991 in vivo. Mice treated with a combination of PD 0332991 and gefitinib had the fastest tumor regression and delayed relapse. Tumors from mice receiving the combination treatment exhibited down-regulated proliferation, up-regulated apoptosis, and less angiogenesis. Finally, lung adenocarcinoma patients with acquired resistance to EGFR-TKIs were given an exploratory treatment of PD 0332991. One patient with gefitinib resistance exhibited clinical remission after treatment with PD 0332991. These findings suggest PD 0332991 reverses acquired EGFR-TKI-resistance in NSCLC cells, and may provide a novel treatment strategy for NSLSC patients with EGFR-TKI resistance. PMID:27825114

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

PubMed

Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

2017-01-01

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

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

PubMed

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

2011-12-27

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

Cyclin C influences the timing of mitosis in fission yeast.

PubMed

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

2017-07-01

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

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

PubMed

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

2010-10-01

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