Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

PubMed

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-07-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster

PubMed Central

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-01-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390

Regulation of steroid hormone receptors and coregulators during the cell cycle highlights potential novel function in addition to roles as transcription factors

PubMed Central

Zheng, Yingfeng; Murphy, Leigh C.

2016-01-01

Cell cycle progression is tightly controlled by several kinase families including Cyclin-Dependent Kinases, Polo-Like Kinases, and Aurora Kinases. A large amount of data show that steroid hormone receptors and various components of the cell cycle, including cell cycle regulated kinases, interact, and this often results in altered transcriptional activity of the receptor. Furthermore, steroid hormones, through their receptors, can also regulate the transcriptional expression of genes that are required for cell cycle regulation. However, emerging data suggest that steroid hormone receptors may have roles in cell cycle progression independent of their transcriptional activity. The following is a review of how steroid receptors and their coregulators can regulate or be regulated by the cell cycle machinery, with a particular focus on roles independent of transcription in G2/M. PMID:26778927

Checkpoints couple transcription network oscillator dynamics to cell-cycle progression.

PubMed

Bristow, Sara L; Leman, Adam R; Simmons Kovacs, Laura A; Deckard, Anastasia; Harer, John; Haase, Steven B

2014-09-05

The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior. Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression. Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.

Capacity decline of ambient temperature secondary lithium battery

NASA Technical Reports Server (NTRS)

Shen, D. H.; Subbarao, S.; Nakamura, B. J.; Yen, S. P. S.; Bankston, C. P.

1988-01-01

The use of ambient temperature secondary lithium cells is limited primarily because of the poor cycle life performance. Much of the cell capacity is irreversibly lost upon cycling. Studies have been undertaken to understand the problem of capacity decline. Experimental Li-TiS2 cells were fabricated and tested for their cycle life performance. Cells were disassembled at different stages of cycle life, and cell active components were analyzed by various analytical techniques. The results of this study indicate that all the cell's active components/materials are undergoing degradation. Details of the experiments carried out and the results obtained are described.

The therapeutic potential of cell cycle targeting in multiple myeloma.

PubMed

Maes, Anke; Menu, Eline; Veirman, Kim De; Maes, Ken; Vand Erkerken, Karin; De Bruyne, Elke

2017-10-27

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.

The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

PubMed

Ewald, Jennifer C; Kuehne, Andreas; Zamboni, Nicola; Skotheim, Jan M

2016-05-19

Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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

Outside-in control -Does plant cell wall integrity regulate cell cycle progression?

PubMed

Gigli-Bisceglia, Nora; Hamann, Thorsten

2018-04-13

During recent years it has become accepted that plant cell walls are not inert objects surrounding all plant cells but are instead highly dynamic, plastic structures. They are involved in a large number of cell biological processes and contribute actively to plant growth, development and interaction with environment. Therefore, it is not surprising that cellular processes can control plant cell wall integrity while, simultaneously, cell wall integrity can influence cellular processes. In yeast and animal cells such a bi-directional relationship also exists between the yeast/animal extra-cellular matrices and the cell cycle. In yeast, the cell wall integrity maintenance mechanism and a dedicated plasmamembrane integrity checkpoint are mediating this relationship. Recent research has yielded insights into the mechanism controlling plant cell wall metabolism during cytokinesis. However, knowledge regarding putative regulatory pathways controlling adaptive modifications in plant cell cycle activity in response to changes in the state of the plant cell wall are not yet identified. In this review, we summarize similarities and differences in regulatory mechanisms coordinating extra cellular matrices and cell cycle activity in animal and yeast cells, discuss the available evidence supporting the existence of such a mechanism in plants and suggest that the plant cell wall integrity maintenance mechanism might also control cell cycle activity in plant cells. This article is protected by copyright. All rights reserved.

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach.

PubMed

Heerma van Voss, Marise R; Kammers, Kai; Vesuna, Farhad; Brilliant, Justin; Bergman, Yehudit; Tantravedi, Saritha; Wu, Xinyan; Cole, Robert N; Holland, Andrew; van Diest, Paul J; Raman, Venu

2018-06-01

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

PubMed

Bartlett, W C; McCann, J; Shepherd, D M; Roy, M; Noelle, R J

1990-12-15

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.

Induction of Apoptosis and Antiproliferative Activity of Naringenin in Human Epidermoid Carcinoma Cell through ROS Generation and Cell Cycle Arrest

PubMed Central

Jafri, Asif; Ahmad, Sheeba; Afzal, Mohammad; Arshad, Md

2014-01-01

A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation. PMID:25330158

Cell cycle gene expression under clinorotation

NASA Astrophysics Data System (ADS)

Artemenko, Olga

2016-07-01

Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

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

Bonifati, Serena; Daly, Michele B.; St Gelais, Corine

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G{sub 1}/G{sub 0} phase and reduces apoptosis. These alterationsmore » correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.« less

Equilibrium between cell division and apoptosis in immortal cells as an alternative to the G1 restriction mechanism in mammalian cells.

PubMed

Dedov, Vadim N; Dedova, Irina V; Nicholson, Garth A

2004-04-01

Starvation arrests cultured mammalian cells in the G(1) restriction point of the cell cycle, whereas cancer cells generally lose the regulatory control of the cell cycle. Human lymphocytes, infected with Epstein-Barr virus (EBV), also lose their cell cycle control and produce immortal lymphoblastoid cell lines. We show that during starvation, EBV-lymphoblasts override the cell cycle arrest in the G(1) restriction point and continue cell division. Simultaneously, starvation activates apoptosis in an approximately half of the daughter cells in each cell generation. Continuos cell division and partial removal of cells by apoptosis results in stabilization of viable cell numbers, where a majority of viable cells are in the G(1) phase of the cell cycle. In contrast to starvation, anticancer drug etoposide activates apoptosis indiscriminately in all EBV-lymphoblasts and convertes all the viable cells into apoptotic. We conclude that the removal of surplus cells by apoptosis may represent a survival mechanism of transformed (i.e., cancer) cell population in nutrient restricted conditions, whereas nontransformed mammalian cells are arrested in the G(1) restriction point of the cell cycle.

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.

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

PubMed Central

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

2017-01-01

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

Parkin New Cargos: a New ROS Independent Role for Parkin in Regulating Cell Division.

PubMed

Stieg, David C; Cooper, Katrina F

2016-01-01

Cell cycle progression requires the destruction of key cell cycle regulators by the multi-subunit E3 ligase called the anaphase promoting complex (APC/C). As the cell progresses through the cell cycle, the APC/C is sequentially activated by two highly conserved co-activators called Cdc20 and Cdh1. Importantly, APC/C Cdc20 is required to degrade substrates in G2/M whereas APC Cdh1 drives the cells into G1. Recently, Parkin, a monomeric E3 ligase that is required for ubiquitin-mediated mitophagy following mitochondrial stress, was shown to both bind and be activated by Cdc20 or Cdh1 during the cell cycle. This mitotic role for Parkin does not require an activating phosphorylation by its usual kinase partner PINK. Rather, mitotic Parkin activity requires phosphorylation on a different serine by the polo-like kinase Plk1. Interestingly, although Parkin Cdc20 and Parkin Cdh1 activity is independent of the APC/C, it mediates degradation of an overlapping subset of substrates. However, unlike the APC/C, Parkin is not necessary for cell cycle progression. Despite this, loss of Parkin activity accelerates genome instability and tumor growth in xenograft models. These findings provide a mechanism behind the previously described, but poorly understood, tumor suppressor role for Parkin. Taken together, studies suggest that the APC/C and Parkin have similar and unique roles to play in cell division, possibly being dependent upon the different subcellular address of these two ligases.

Destructive physical analysis results of Ni/H2 cells cycled in LEO regime

NASA Technical Reports Server (NTRS)

Lim, Hong S.; Zelter, Gabriela R.; Smithrick, John J.; Hall, Stephen W.

1991-01-01

Six 48-Ah individual pressure vessel (IPV) Ni/H2 cells containing 26 and 31 percent KOH electrolyte were life cycle tested in low Earth orbit. All three cells containing 31 percent KOH failed (3729, 4165, and 11,355 cycles), while those with 26 percent KOH were cycled over 14,000 times in the continuing test. Destructive physical analysis (DPA) of the failed cells included visual inspections, measurements of electrode thickness, scanning electron microscopy, chemical analysis, and measurements of nickel electrode capacity in an electrolyte flooded cell. The cycling failure was due to a decrease of nickel electrode capacity. As possible causes of the capacity decrease, researchers observed electrode expansion, rupture, and corrosion of the nickel electrode substrate, active material redistribution, and accumulation of electrochemically undischargeable active material with cycling.

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

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.

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

PubMed Central

Chauhan, Neha; Visram, Myriam; Cristobal-Sarramian, Alvaro; Sarkleti, Florian

2015-01-01

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2ACdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle. PMID:25713391

5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint.

PubMed

Luciani, M Gloria; Campregher, Christoph; Fortune, John M; Kunkel, Thomas A; Gasche, Christoph

2007-01-01

Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116(p53-/-), HCT116+chr3, and LoVo were treated with 5-ASA for 2-96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.

Intermittent Stem Cell Cycling Balances Self-Renewal and Senescence of the C. elegans Germ Line.

PubMed

Cinquin, Amanda; Chiang, Michael; Paz, Adrian; Hallman, Sam; Yuan, Oliver; Vysniauskaite, Indre; Fowlkes, Charless C; Cinquin, Olivier

2016-04-01

Self-renewing organs often experience a decline in function in the course of aging. It is unclear whether chronological age or external factors control this decline, or whether it is driven by stem cell self-renewal-for example, because cycling cells exhaust their replicative capacity and become senescent. Here we assay the relationship between stem cell cycling and senescence in the Caenorhabditis elegans reproductive system, defining this senescence as the progressive decline in "reproductive capacity," i.e. in the number of progeny that can be produced until cessation of reproduction. We show that stem cell cycling diminishes remaining reproductive capacity, at least in part through the DNA damage response. Paradoxically, gonads kept under conditions that preclude reproduction keep cycling and producing cells that undergo apoptosis or are laid as unfertilized gametes, thus squandering reproductive capacity. We show that continued activity is in fact beneficial inasmuch as gonads that are active when reproduction is initiated have more sustained early progeny production. Intriguingly, continued cycling is intermittent-gonads switch between active and dormant states-and in all likelihood stochastic. Other organs face tradeoffs whereby stem cell cycling has the beneficial effect of providing freshly-differentiated cells and the detrimental effect of increasing the likelihood of cancer or senescence; stochastic stem cell cycling may allow for a subset of cells to preserve proliferative potential in old age, which may implement a strategy to deal with uncertainty as to the total amount of proliferation to be undergone over an organism's lifespan.

ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress*

PubMed Central

Kemp, Michael G.; Sancar, Aziz

2016-01-01

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase that maintains genome stability and halts cell cycle phase transitions in response to DNA lesions that block DNA polymerase movement. These DNA replication-associated features of ATR function have led to the emergence of ATR kinase inhibitors as potential adjuvants for DNA-damaging cancer chemotherapeutics. However, whether ATR affects the genotoxic stress response in non-replicating, non-cycling cells is currently unknown. We therefore used chemical inhibition of ATR kinase activity to examine the role of ATR in quiescent human cells. Although ATR inhibition had no obvious effects on the viability of non-cycling cells, inhibition of ATR partially protected non-replicating cells from the lethal effects of UV and UV mimetics. Analyses of various DNA damage response signaling pathways demonstrated that ATR inhibition reduced the activation of apoptotic signaling by these agents in non-cycling cells. The pro-apoptosis/cell death function of ATR is likely due to transcription stress because the lethal effects of compounds that block RNA polymerase movement were reduced in the presence of an ATR inhibitor. These results therefore suggest that whereas DNA polymerase stalling at DNA lesions activates ATR to protect cell viability and prevent apoptosis, the stalling of RNA polymerases instead activates ATR to induce an apoptotic form of cell death in non-cycling cells. These results have important implications regarding the use of ATR inhibitors in cancer chemotherapy regimens. PMID:26940878

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.

Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

PubMed Central

Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

2015-01-01

Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114

Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

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

Arcangeletti, Maria-Cristina, E-mail: mariacristina.arcangeletti@unipr.it; Germini, Diego; Rodighiero, Isabella

2013-05-25

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promotingmore » cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.« less

RGC-32 is a novel regulator of the T-lymphocyte cell cycle.

PubMed

Tegla, Cosmin A; Cudrici, Cornelia D; Nguyen, Vinh; Danoff, Jacob; Kruszewski, Adam M; Boodhoo, Dallas; Mekala, Armugam P; Vlaicu, Sonia I; Chen, Ching; Rus, Violeta; Badea, Tudor C; Rus, Horea

2015-06-01

We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice developed normally and did not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells, we determined the proliferative rates of CD4(+) and CD8(+) T cells from the spleens of RGC-32(-/-) mice, as compared to wild-type (WT, RGC-32(+/+)) control mice. After stimulation with anti-CD3/anti-CD28, CD4(+) T cells from RGC-32(-/-) mice displayed a significant increase in [(3)H]-thymidine incorporation when compared to WT mice. In addition, both CD4(+) and CD8(+) T cells from RGC-32(-/-) mice displayed a significant increase in the proportion of proliferating Ki67(+) cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation induced by T-cell receptor/CD28 engagement. Furthermore, Akt and FOXO1 phosphorylation induced in stimulated CD4(+) T-cells from RGC-32(-/-) mice were significantly higher, indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. We also found that IL-2 mRNA and protein expression were significantly increased in RGC-32(-/-) CD4(+) T cells when compared to RGC-32(+/+) CD4(+) T cells. In addition, the effect of RGC-32 on the cell cycle and IL-2 expression was inhibited by pretreatment of the samples with LY294002, indicating a role for phosphatidylinositol 3-kinase (PI3K). Thus, RGC-32 is involved in controlling the cell cycle of T cells in vivo, and this effect is mediated by IL-2 in a PI3K-dependent fashion. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

PubMed

Yeung, A T; Bascomb, N F; Turner, K J; Schmidt, R R

1981-05-01

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins.

PubMed

Bhattacharya, Abhishek; Li, Ke; Quiquand, Manon; Rimesso, Gerard; Baker, Nicholas E

2017-11-15

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. We found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets. Copyright © 2017 Elsevier Inc. All rights reserved.

DCAF1 controls T-cell function via p53-dependent and -independent mechanisms.

PubMed

Guo, Zengli; Kong, Qing; Liu, Cui; Zhang, Song; Zou, Liyun; Yan, Feng; Whitmire, Jason K; Xiong, Yue; Chen, Xian; Wan, Yisong Y

2016-01-05

On activation, naive T cells grow in size and enter cell cycle to mount immune response. How the fundamental processes of T-cell growth and cell cycle entry are regulated is poorly understood. Here we report that DCAF1 (Ddb1-cullin4-associated-factor 1) is essential for these processes. The deletion of DCAF1 in T cells impairs their peripheral homeostasis. DCAF1 is upregulated on T-cell receptor activation and critical for activation-induced T-cell growth, cell cycle entry and proliferation. In addition, DCAF1 is required for T-cell expansion and function during anti-viral and autoimmune responses in vivo. DCAF1 deletion leads to a drastic stabilization of p53 protein, which can be attributed to a requirement of DCAF1 for MDM2-mediated p53 poly-ubiquitination. Importantly, p53 deletion rescues the cell cycle entry defect but not the growth defect of DCAF1-deficient cells. Therefore, DCAF1 is vital for T-cell function through p53-dependent and -independent mechanisms.

DCAF1 controls T-cell function via p53-dependent and -independent mechanisms

PubMed Central

Guo, Zengli; Kong, Qing; Liu, Cui; Zhang, Song; Zou, Liyun; Yan, Feng; Whitmire, Jason K.; Xiong, Yue; Chen, Xian; Wan, Yisong Y.

2016-01-01

On activation, naive T cells grow in size and enter cell cycle to mount immune response. How the fundamental processes of T-cell growth and cell cycle entry are regulated is poorly understood. Here we report that DCAF1 (Ddb1–cullin4-associated-factor 1) is essential for these processes. The deletion of DCAF1 in T cells impairs their peripheral homeostasis. DCAF1 is upregulated on T-cell receptor activation and critical for activation-induced T-cell growth, cell cycle entry and proliferation. In addition, DCAF1 is required for T-cell expansion and function during anti-viral and autoimmune responses in vivo. DCAF1 deletion leads to a drastic stabilization of p53 protein, which can be attributed to a requirement of DCAF1 for MDM2-mediated p53 poly-ubiquitination. Importantly, p53 deletion rescues the cell cycle entry defect but not the growth defect of DCAF1-deficient cells. Therefore, DCAF1 is vital for T-cell function through p53-dependent and -independent mechanisms. PMID:26728942

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2.

PubMed

Mavri-Damelin, Demetra; Eaton, Simon; Damelin, Leonard H; Rees, Myrddin; Hodgson, Humphrey J F; Selden, Clare

2007-01-01

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.

Induction of S phase cell arrest and caspase activation by polysaccharide peptide isolated from Coriolus versicolor enhanced the cell cycle dependent activity and apoptotic cell death of doxorubicin and etoposide, but not cytarabine in HL-60 cells.

PubMed

Hui, Kenrie Pui-Yan; Sit, Wai-Hung; Wan, Jennifer Man-Fan

2005-07-01

Activation of the cell death program (apoptosis) is a strategy for the treatment of human cancer, and unfortunately a large number of drugs identified as cell cycle-specific agents for killing cancer cells are also toxic to normal cells. The present study demonstrates that the polysaccharide peptide (PSP) extracted from the Chinese medicinal mushroom, Coriolus versicolor, used in combination therapy in China, has the ability to lower the cytotoxicity of certain anti-leukemic drugs via their interaction with cell cycle-dependent and apoptotic pathways. Flow cytometry analysis demonstrated that pre-treatment of PSP (25-100 microg/ml) dose-dependently enhanced the cell cycle perturbation and apoptotic activity of doxorubicin (Doxo) and etoposide (VP-16), but not cytarabine (Ara-C) in human promyelocytic leukemia HL-60 cells. The antagonistic result from combined treatment with Ara-C and PSP may be caused by the removal of HL-60 cells in the G1-S boundary by PSP before exposure to Ara-C. A negative correlation between the increase in apoptotic cell population (pre-G1 peak) with the S-phase cell population expression (R2=0.998), the expression of cyclin E expression (R2=0.872) and caspase 3 activity (R2=0.997) suggests that PSP enhanced the apoptotic machinery of Doxo and VP-16 in a cell cycle-dependent manner and is mediated, at least in part, by the PSP-mediated modulation of the regulatory checkpoint cyclin E and caspase 3. This study is the first to describe the cell cycle mechanistic action of PSP and its interaction with other anticancer agents. Our data support the potential development of PSP as an adjuvant for leukemia treatment, but also imply the importance of understanding its interaction with individual anticancer agents.

Day-night cycles and the sleep-promoting factor, Sleepless, affect stem cell activity in the Drosophila testis.

PubMed

Tulina, Natalia M; Chen, Wen-Feng; Chen, Jung Hsuan; Sowcik, Mallory; Sehgal, Amita

2014-02-25

Adult stem cells maintain tissue integrity and function by renewing cellular content of the organism through regulated mitotic divisions. Previous studies showed that stem cell activity is affected by local, systemic, and environmental cues. Here, we explore a role of environmental day-night cycles in modulating cell cycle progression in populations of adult stem cells. Using a classic stem cell system, the Drosophila spermatogonial stem cell niche, we reveal daily rhythms in division frequencies of germ-line and somatic stem cells that act cooperatively to produce male gametes. We also examine whether behavioral sleep-wake cycles, which are driven by the environmental day-night cycles, regulate stem cell function. We find that flies lacking the sleep-promoting factor Sleepless, which maintains normal sleep in Drosophila, have increased germ-line stem cell (GSC) division rates, and this effect is mediated, in part, through a GABAergic signaling pathway. We suggest that alterations in sleep can influence the daily dynamics of GSC divisions.

Cell division cycle 45 promotes papillary thyroid cancer progression via regulating cell cycle.

PubMed

Sun, Jing; Shi, Run; Zhao, Sha; Li, Xiaona; Lu, Shan; Bu, Hemei; Ma, Xianghua

2017-05-01

Cell division cycle 45 was reported to be overexpressed in some cancer-derived cell lines and was predicted to be a candidate oncogene in cervical cancer. However, the clinical and biological significance of cell division cycle 45 in papillary thyroid cancer has never been investigated. We determined the expression level and clinical significance of cell division cycle 45 using The Cancer Genome Atlas, quantitative real-time polymerase chain reaction, and immunohistochemistry. A great upregulation of cell division cycle 45 was observed in papillary thyroid cancer tissues compared with adjacent normal tissues. Furthermore, overexpression of cell division cycle 45 positively correlates with more advanced clinical characteristics. Silence of cell division cycle 45 suppressed proliferation of papillary thyroid cancer cells via G1-phase arrest and inducing apoptosis. The oncogenic activity of cell division cycle 45 was also confirmed in vivo. In conclusion, cell division cycle 45 may serve as a novel biomarker and a potential therapeutic target for papillary thyroid cancer.

"Constructing" the Cell Cycle in 3D

ERIC Educational Resources Information Center

Koc, Isil; Turan, Merve

2012-01-01

The cycle of duplication and division, known as the "cell cycle," is the essential mechanism by which all living organisms reproduce. This activity allows students to develop an understanding of the main events that occur during the typical eukaryotic cell cycle mostly in the process of mitotic phase that divides the duplicated genetic material…

Resveratrol Improves Cell Cycle Arrest in Chronic Prostatitis Rats, by C-kit/SCF Suppression.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Liu, Qi; Yang, Bo

2017-08-01

Chronic prostatitis (CP) with complex pathogenesis is difficult for treatment. c-kit has been associated with the control of cell proliferation of prostate cells. This study aims to evaluate the role of resveratrol, an activator of Sirt1, in regulating the expression of c-kit in CP and investigate the consequent effects on cell cycle. Rat model of CP was established through subcutaneous injections of diphtheria-pertussis-tetanus vaccine and subsequently treated with resveratrol. Hematoxylin and eosin staining was performed to identify the histopathological changes in prostates. Western blotting and immunohistochemical staining examined the expression level of c-kit, stem cell factor (SCF), Sirt1, and cell cycle-associated proteins. The model group exhibited severe diffuse chronic inflammation, characterized by leukocyte infiltration and papillary frond protrusion into the gland cavities, and a notable increase in prostatic epithelial height. Gland lumen diameter was also significantly smaller; the activity of c-kit/SCF in the CP rats was increased significantly compared to the control group. Meanwhile, the cell cycle proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. Dysregulation of cell cycle was involved in the pathological processes of CP, which was improved after resveratrol treatment by the downregulation of c-kit/SCF by activating Sirt1.

Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein.

PubMed

Sheldon, Lynn A

2017-01-01

The regulation of cell cycle progression by steroid hormones and growth factors is important for maintaining normal cellular processes including development and cell proliferation. Deregulated progression through the G1/S and G2/M cell cycle transitions can lead to uncontrolled cell proliferation and cancer. The transcription factor E2F1, a key cell cycle regulator, targets genes encoding proteins that regulate cell cycle progression through the G1/S transition as well as proteins important in DNA repair and apoptosis. E2F1 expression and activity is inhibited by inorganic arsenic (iAs) that has a dual role as a cancer therapeutic and as a toxin that leads to diseases including cancer. An understanding of what underlies this dichotomy will contribute to understanding how to use iAs as a more effective therapeutic and also how to treat cancers that iAs promotes. Here, we show that quiescent breast adenocarcinoma MCF-7 cells treated with 17-β estradiol (E2) progress through the cell cycle, but few cells treated with E2 + iAs progress from G1 into S-phase due to a block in cell cycle progression. Our data support a model in which iAs inhibits the dissociation of E2F1 from the tumor suppressor, retinoblastoma protein (pRB) due to changes in pRB phosphorylation which leads to decreased E2F1 transcriptional activity. These findings present an explanation for how iAs can disrupt cell cycle progression through E2F1-pRB and has implications for how iAs acts as a cancer therapeutic as well as how it may promote tumorigenesis through decreased DNA repair.

Inducing myoblast re-entry into the cell cycle: a potential mechanism for laser-enhanced skeletal muscle regeneration

NASA Astrophysics Data System (ADS)

Liu, T.; Fang, Y.; Zhang, C. P.; Chen, P.; Wang, C. Z.; Kang, H. X.; Shen, B. J.; Liang, J.; Fu, X. B.

2014-09-01

This study investigated the effect of low-level laser irradiation (LLLI) on the cell cycle and proliferative activity of cultured myoblasts, and sought to elucidate the possible cellular mechanism by which LLLI promotes the regeneration of skeletal muscle in vivo. Primary myoblasts isolated from rat hindlegs were irradiated with helium-neon laser light at different energy densities. Distributions of cell-cycle subpopulations and the expression of cell-cycle regulatory proteins in myoblasts were assessed using flow cytometric analysis and western blot assay. It was found that laser irradiation stimulated cell-cycle entry; induced the expression of cyclin A and cyclin D; and increased cell proliferation index and bromodeoxyuridine incorporation as compared to the unirradiated control cells, indicating LLLI augmented the number of proliferative myoblasts in the S phase and G2/M phase of the cell cycle. These results suggest that LLLI at certain fluxes and wavelengths could activate quiescent myoblasts, leading to cell division and facilitating new myofiber formation. This could contribute to the improvement of skeletal muscle regeneration following trauma and myopathic diseases.

ATM and MET kinases are synthetic lethal with non-genotoxic activation of p53

PubMed Central

Sullivan, Kelly D.; Padilla-Just, Nuria; Henry, Ryan E.; Porter, Christopher C.; Kim, Jihye; Tentler, John J.; Eckhardt, S. Gail; Tan, Aik Choon; DeGregori, James; Espinosa, Joaquín M.

2012-01-01

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a ‘Synthetic Lethal with Nutlin-3’ genome-wide shRNA screen, which revealed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors enable Nutlin-3 to kill tumor spheroids. These results identify novel pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies. PMID:22660439

Long life nickel electrodes for a nickel-hydrogen cell: Cycle life tests

NASA Technical Reports Server (NTRS)

Lim, H. S.; Verzwyvelt, S. A.

1985-01-01

In order to develop a long life nickel electrode for a Ni/H2 cell, the cycle life of nickel electrodes was tested in Ni/H2 boiler plate cells. A 19 test cell matrix was made of various nickel electrode designs including three levels each of plaque mechanical strength, median pore size of the plaque, and active material loading. Test cells were cycled to the end of their life (0.5v) in a 45 minute low Earth orbit cycle regime at 80% depth-of-discharge. It is shown that the active material loading level affects the cycle life the most with the optimum loading at 1.6 g/cc void. Mechanical strength does not affect the cycle life noticeably in the bend strength range of 400 to 700 psi. It is found that the best plaque is made of INCO nickel powder type 287 and has median pore size of 13 micron.

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

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

Sumrejkanchanakij, Piyamas; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330; Eto, Kazuhiro

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, amore » process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.« less

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells.

PubMed

Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer.

PubMed

Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

2015-08-01

Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

PubMed

Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

2015-09-08

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

PubMed

Shirazi Fard, Shahrzad; Thyselius, Malin; All-Ericsson, Charlotta; Hallböök, Finn

2014-01-01

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.

Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

PubMed

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

Cell Cycle-Dependent Rho GTPase Activity Dynamically Regulates Cancer Cell Motility and Invasion In Vivo

PubMed Central

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers. PMID:24386239

Effect of berberine on cell cycle arrest and cell survival during cerebral ischemia and reperfusion and correlations with p53/cyclin D1 and PI3K/Akt.

PubMed

Chai, Yu-Shuang; Hu, Jun; Lei, Fan; Wang, Yu-Gang; Yuan, Zhi-Yi; Lu, Xi; Wang, Xin-Pei; Du, Feng; Zhang, Dong; Xing, Dong-Ming; Du, Li-Jun

2013-05-15

Berberine acted as a natural medicine with multiple pharmacological activities. In the present study, we examined the effect of berberine against cerebral ischemia damage from cell cycle arrest and cell survival. Oxygen-glucose deprivation of PC12 cells and primary neurons, and carotid artery ligation in mice were used as in vitro and in vivo cerebral ischemia models. We found that the effect of berberine on cell cycle arrest during ischemia was mediated by decreased p53 and cyclin D1, increased phosphorylation of Bad (higher expression of p-Bad and higher ratio of p-Bad to Bad) and decreased cleavage of caspase 3. Meanwhile, berberine activated the PI3K/Akt pathway during the reperfusion, especially the phosphor-activation of Akt, to promote the cell survival. The neural protective effect of berberine was remained in the presence of inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (MEK), but was suppressed by the inhibitors of PI3K and Akt. We demonstrated that berberine induced cell cycle arrest and cell survival to resist cerebral ischemia injury. Copyright © 2013 Elsevier B.V. All rights reserved.

Nanosecond pulsed electric fields and the cell cycle

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.

Exposure to nanosecond pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. The phase of the cell cycle at the time of exposure is linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Additionally, nsPEFs are capable of activating cell cycle checkpoints, which could lead to apoptosis or slow population growth. NsPEFs are emerging as a method for treating tumors via apoptotic induction; therefore, investigating the relevance of nsPEFs and the cell cycle could translate into improved efficacy in tumor treatment. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate the role of cell cycle phase in survival of nsPEFs. CHO populations appeared similar to sham populations post-nsPEFs but exhibited arrest in the G1 phase at 6h after exposure. Jurkat cells exhibited increased cell death after nsPEFs compared to CHO cells but did not exhibit checkpoint arrest at any observed time point. The G1/S phase checkpoint is partially controlled by the action of p53; the lack of an active p53 response in Jurkat cells could contribute to their ability to pass this checkpoint and resist cell cycle arrest. Both cell lines exhibited increased sensitivity to nsPEFs in G2/M phase. Live imaging of CHO cells after nsPEF exposure supports the theory of G1/S phase arrest, as a reduced number of cells undergo mitosis within 24 h when compared to sham treated cells. CHO cells undergoing mitosis after exposure also exhibit improper separation of chromatids which could indicate loss of function of the mitotic spindle checkpoint. Activation and loss of function of checkpoints in CHO but not Jurkat cells after nsPEF exposure suggests that activation of cell cycle checkpoints could be important in defining the character of cell line specific recovery after nsPEF exposure. Moreover, the increased sensitivity in G2/M phase exhibited by both cell lines indicates that cell cycle phase is an important consideration during nsPEF exposure, particularly when aiming to induce apoptosis.

NONO couples the circadian clock to the cell cycle.

PubMed

Kowalska, Elzbieta; Ripperger, Juergen A; Hoegger, Dominik C; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A

2013-01-29

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation

PubMed Central

Aix, Esther; Gutiérrez-Gutiérrez, Óscar; Sánchez-Ferrer, Carlota; Aguado, Tania

2016-01-01

The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc−/−) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc−/− newborns but rescued in G3 Terc−/−/p21−/− mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts. PMID:27241915

5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

PubMed Central

LUCIANI, M. GLORIA; CAMPREGHER, CHRISTOPH; FORTUNE, JOHN M.; KUNKEL, THOMAS A.; GASCHE, CHRISTOPH

2007-01-01

Background & Aims Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. PMID:17241873

Clustered localization of STAT3 during the cell cycle detected by super-resolution fluorescence microscopy

NASA Astrophysics Data System (ADS)

Gao, Jing; Chen, Junling; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tong, Ti; Wang, Hongda

2017-06-01

Signal transducer and activator of transcription 3 (STAT3) plays a key role in various cellular processes such as cell proliferation, differentiation, apoptosis and immune responses. In particular, STAT3 has emerged as a potential molecular target for cancer therapy. The functional role and standard activation mechanism of STAT3 have been well studied, however, the spatial distribution of STAT3 during the cell cycle is poorly known. Therefore, it is indispensable to study STAT3 spatial arrangement and nuclear-cytoplasimic localization at the different phase of cell cycle in cancer cells. By direct stochastic optical reconstruction microscopy imaging, we find that STAT3 forms various number and size of clusters at the different cell-cycle stage, which could not be clearly observed by conventional fluorescent microscopy. STAT3 clusters get more and larger gradually from G1 to G2 phase, during which time transcription and other related activities goes on consistently. The results suggest that there is an intimate relationship between the clustered characteristic of STAT3 and the cell-cycle behavior. Meanwhile, clustering would facilitate STAT3 rapid response to activating signals due to short distances between molecules. Our data might open a new door to develop an antitumor drug for inhibiting STAT3 signaling pathway by destroying its clusters.

Cell-cycle control in the face of damage--a matter of life or death.

PubMed

Clarke, Paul R; Allan, Lindsey A

2009-03-01

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

Antitumor potential of crown ethers: structure-activity relationships, cell cycle disturbances, and cell death studies of a series of ionophores.

PubMed

Marjanović, Marko; Kralj, Marijeta; Supek, Fran; Frkanec, Leo; Piantanida, Ivo; Smuc, Tomislav; Tusek-Bozić, Ljerka

2007-03-08

The present paper demonstrates the antiproliferative ability and structure-activity relationships (SAR) of 14 crown and aza-crown ether analogues on five tumor-cell types. The most active compounds were di-tert-butyldicyclohexano-18-crown-6 (3), which exhibited cytotoxicity in the submicromolar range, and di-tert-butyldibenzo-18-crown-6 (5) (IC50 values of approximately 2 microM). Also, 3 and 5 induced marked influence on the cell cycle phase distribution--strong G1 arrest, followed by the induction of apoptosis. A computational SAR modeling effort offers insight into possible mechanisms of crown ether biological activity, presumably involving penetration into cell membranes, and points out structural features of molecules important for this activity. The results reveal that crown ethers possess marked tumor-cell growth inhibitory activity, the extent of which depends on the characteristics of the hydrophilic macrocylic cavity and the surrounding hydrophobic ring. Our work supports the hypothesis that crown ether compounds inhibit tumor-cell growth by disrupting potassium ion homeostasis, which in turn leads to cell cycle perturbations and apoptosis.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

PubMed Central

Levasseur, Mark; Dumollard, Remi; Chambon, Jean-Philippe; Hebras, Celine; Sinclair, Maureen; Whitaker, Michael; McDougall, Alex

2013-01-01

The fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/Ccdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates. PMID:24194472

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

PubMed Central

Schraufstätter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

1985-01-01

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells. PMID:3840176

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

LPS-induced inflammatory response triggers cell cycle reactivation in murine neuronal cells through retinoblastoma proteins induction.

PubMed

D'Angelo, Barbara; Astarita, Carlo; Boffo, Silvia; Massaro-Giordano, Mina; Antonella Ianuzzi, Carmelina; Caporaso, Antonella; Macaluso, Marcella; Giordano, Antonio

2017-01-01

Cell cycle reactivation in adult neurons is an early hallmark of neurodegeneration. The lipopolysaccharide (LPS) is a well-known pro-inflammatory factor that provokes neuronal cell death via glial cells activation. The retinoblastoma (RB) family includes RB1/p105, retinoblastoma-like 1 (RBL1/p107), and retinoblastoma-like 2 (Rb2/p130). Several studies have indicated that RB proteins exhibit tumor suppressor activities, and play a central role in cell cycle regulation. In this study, we assessed LPS-mediated inflammatory effect on cell cycle reactivation and apoptosis of neuronally differentiated cells. Also, we investigated whether the LPS-mediated inflammatory response can influence the function and expression of RB proteins. Our results showed that LPS challenges triggered cell cycle reactivation of differentiated neuronal cells, indicated by an accumulation of cells in S and G2/M phase. Furthermore, we found that LPS treatment also induced apoptotic death of neurons. Interestingly, we observed that LPS-mediated inflammatory effect on cell cycle re-entry and apoptosis was concomitant with the aberrant expression of RBL1/p107 and RB1/p105. To the best of our knowledge, our study is the first to indicate a role of LPS in inducing cell cycle re-entry and/or apoptosis of differentiated neuronal cells, perhaps through mechanisms altering the expression of specific members of RB family proteins. This study provides novel information on the biology of post-mitotic neurons and could help in identifying novel therapeutic targets to prevent de novo cell cycle reactivation and/or apoptosis of neurons undergoing neurodegenerative processes.

Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression.

PubMed

Lee, Suk Jun; Bae, Joonbeom; Kim, Sunhee; Jeong, Seonah; Choi, Chang-Yong; Choi, Sang-Pil; Kim, Hyun-Sook; Jung, Woon-Won; Imm, Jee-Young; Kim, Sae Hun; Chun, Taehoon

2013-02-01

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A(b)) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G(1) to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27(KIP1). Saponins also increased stability of p27(KIP1) in Th cells after antigenic stimulation.

Delayed Cell Cycle Progression and Apoptosis Induced by Hemicellulase-Treated Agaricus blazei

PubMed Central

Kasai, Hirotake

2007-01-01

We examined the effects of hemicellulase-treated Agaricus blazei (AB fraction H, ABH) on growth of several tumor cell lines. ABH inhibited the proliferation of some cell lines without cytotoxic effects. It markedly prolonged the S phase of the cell cycle. ABH also induced mitochondria-mediated apoptosis in different cell lines. However, it had no impact on the growth of other cell lines. ABH induced strong activation of p38 mitogen-activated protein kinase (MAPK) in the cells in which it evoked apoptosis. On the other hand, ABH showed only a weak p38 activation effect in those cell lines in which it delayed cell cycle progression with little induction of apoptosis. However, p38 MAPK-specific inhibitor inhibited both ABH-induced effects, and ABH also caused apoptosis in the latter cells under conditions of high p38 MAPK activity induced by combined treatment with TNF-α. These results indicate that the responsiveness of p38 MAPK to ABH, which differs between cell lines, determines subsequent cellular responses on cell growth. PMID:17342245

IL-7 splicing variant IL-7{delta}5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

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

Pan, Deshun; Department of Pharmaceutical science, Guangdong Pharmaceutical University, Guangzhou, Guangdong; Liu, Bing

2012-06-15

Highlights: Black-Right-Pointing-Pointer This study confirms the role of IL-7{delta}5 in breast cancer cell proliferation. Black-Right-Pointing-Pointer IL-7{delta}5 promotes breast cancer cell proliferation and cell cycle progression. Black-Right-Pointing-Pointer IL-7{delta}5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7{delta}5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The resultsmore » showed that IL-7{delta}5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27{sup kip1} expression. Mechanistically, we found that IL-7{delta}5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7{delta}5. In conclusion, our findings demonstrate that IL-7{delta}5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7{delta}5 may be a potential target for human breast cancer therapeutics intervention.« less

Cdk2 Phosphorylation on Threonine39 by AKT and Its Implication on Cyclin Binding, Cellular Localization, and Cell Cycle Progression

DTIC Science & Technology

2008-10-01

cell cycle progression in most cell types. Mouse embryos develop normally until mid gestation without all interphase Cdks 28. Pertinent to the...Ciemerych and P. Sicinski, "Cell cycle in mouse development ," 24(17), 2877 (2005). Ref Type: Journal 5 K. Coulonval, et al., "Phosphorylations of...34 Development 135(20), 3389 (2008). Ref Type: Journal 30 J. P. Tassan, et al., "Cell cycle analysis of the activity, subcellular localization, and subunit

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

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

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

Juglans mandshurica Maxim extracts exhibit antitumor activity on HeLa cells in vitro.

PubMed

Xin, Nian; Hasan, Murtaza; Li, Wei; Li, Yan

2014-04-01

The present study examined the potential application of Juglans mandshurica Maxim extracts (HT) for cancer therapy by assessing their anti‑proliferative activity, reduction of telomerase activity, induction of apoptosis and cell cycle arrest in S phase in HeLa cells. From the perspective of using HT as a herbal medicine, photomicroscopy and florescent microscopy techniques were utilized to characterize the effect of the extracts on telomerase activity and cell morphology. Flow cytometry was employed to study apoptosis and cell cycle of HeLa cells, and DNA laddering was performed. The results showed that HT inhibited cell proliferation and telomerase activity, induced apoptosis and caused S phase arrest of HeLa cells in vitro. HT inhibited HeLa cell proliferation significantly, and the highest inhibition rate was 83.7%. A trap‑silver staining assay showed that HT was capable of markedly decreasing telomerase activity of HeLa cells and this inhibition was enhanced in a time‑ and dose‑dependent manner. Results of a Hoechst 33258 staining assay showed that HeLa cells treated by HT induced cell death. Through DNA agarose gel electrophoresis, DNA ladders of HeLa cells treated with HT were observed, indicating apoptosis. In conclusion, the present study demonstrated that HT exhibited anti‑tumor effects comprising the inhibition of growth and telomerase activity as well as apoptosis and cell cycle arrest in HeLa cells.

Self-Organizing and Stochastic Behaviors During the Regeneration of Hair Stem Cells

PubMed Central

Plikus, Maksim V.; Baker, Ruth E.; Chen, Chih-Chiang; Fare, Clyde; de la Cruz, Damon; Andl, Thomas; Maini, Philip K.; Millar, Sarah E.; Widelitz, Randall; Chuong, Cheng-Ming

2012-01-01

Stem cells cycle through active and quiescent states. Large populations of stem cells in an organ may cycle randomly or in a coordinated manner. Although stem cell cycling within single hair follicles has been studied, less is known about regenerative behavior in a hair follicle population. By combining predictive mathematical modeling with in vivo studies in mice and rabbits, we show that a follicle progresses through cycling stages by continuous integration of inputs from intrinsic follicular and extrinsic environmental signals based on universal patterning principles. Signaling from the WNT/bone morphogenetic protein activator/inhibitor pair is coopted to mediate interactions among follicles in the population. This regenerative strategy is robust and versatile because relative activator/inhibitor strengths can be modulated easily, adapting the organism to different physiological and evolutionary needs. PMID:21527712

Effectiveness and student perceptions of an active learning activity using a headline news story to enhance in-class learning of cell cycle regulation.

PubMed

Dirks-Naylor, Amie J

2016-06-01

An active learning activity was used to engage students and enhance in-class learning of cell cycle regulation in a PharmD level integrated biological sciences course. The aim of the present study was to determine the effectiveness and perception of the in-class activity. After completion of a lecture on the topic of cell cycle regulation, students completed a 10-question multiple-choice quiz before and after engaging in the activity. The activity involved reading of a headline news article published by ScienceDaily.com entitled "One Gene Lost Equals One limb Regained." The name of the gene was deleted from the article and, thus, the end goal of the activity was to determine the gene of interest by the description in the story. The activity included compiling a list of all potential gene candidates before sufficient information was given to identify the gene of interest (p21). A survey was completed to determine student perceptions of the activity. Quiz scores improved by an average of 20% after the activity (40.1 ± 1.95 vs. 59.9 ± 2.14,P< 0.0001,n= 96). Students enjoyed the activity, found the news article interesting, and believed that the activity improved their understanding of cell cycle regulation. The majority of students agreed that the in-class activity piqued their interest for learning the subject matter and also agreed that if they understand a concept during class, they are more likely to want to study that concept outside of class. In conclusion, the activity improved in-class understanding and enhanced interest in cell cycle regulation. Copyright © 2016 The American Physiological Society.

Distinct mechanisms act in concert to mediate cell cycle arrest.

PubMed

Toettcher, Jared E; Loewer, Alexander; Ostheimer, Gerard J; Yaffe, Michael B; Tidor, Bruce; Lahav, Galit

2009-01-20

In response to DNA damage, cells arrest at specific stages in the cell cycle. This arrest must fulfill at least 3 requirements: it must be activated promptly; it must be sustained as long as damage is present to prevent loss of genomic information; and after the arrest, cells must re-enter into the appropriate cell cycle phase to ensure proper ploidy. Multiple molecular mechanisms capable of arresting the cell cycle have been identified in mammalian cells; however, it is unknown whether each mechanism meets all 3 requirements or whether they act together to confer specific functions to the arrest. To address this question, we integrated mathematical models describing the cell cycle and the DNA damage signaling networks and tested the contributions of each mechanism to cell cycle arrest and re-entry. Predictions from this model were then tested with quantitative experiments to identify the combined action of arrest mechanisms in irradiated cells. We find that different arrest mechanisms serve indispensable roles in the proper cellular response to DNA damage over time: p53-independent cyclin inactivation confers immediate arrest, whereas p53-dependent cyclin downregulation allows this arrest to be sustained. Additionally, p21-mediated inhibition of cyclin-dependent kinase activity is indispensable for preventing improper cell cycle re-entry and endoreduplication. This work shows that in a complex signaling network, seemingly redundant mechanisms, acting in a concerted fashion, can achieve a specific cellular outcome.

Loss of DLK expression in WI-38 human diploid fibroblasts induces a senescent-like proliferation arrest

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

Daviau, Alex; Couture, Jean-Philippe; Blouin, Richard, E-mail: Richard.Blouin@USherbrooke.ca

Highlights: {yields} Role of DLK in cell proliferation. {yields} Modulation of DLK expression during cell cycle progression. {yields} DLK knockdown induces proliferation arrest and senescence. {yields} DLK-depleted cells display loss of cyclin D1 and up-regulation of p21. {yields} DLK participates in cell proliferation by modulating cell cycle regulator expression. -- Abstract: DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, althoughmore » little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins.« less

Long Life Nickel Electrodes for a Nickel-hydrogen Cell: Cycle Life Tests

NASA Technical Reports Server (NTRS)

Lim, H. S.; Verzwyvelt, S. A.

1984-01-01

In order to develop a long life nickel electrode for a Ni/H2 cell, cycle life tests of nickel electrodes were carried out in Hi/H2 boiler plate cells. A 19 test cell matrix was made of various nickel electrode designs including three levels each of plaque mechanical strength, median pore size of the plaque, and active material loading. Test cells were cycled to the end of their life (0.5v) in a 45-minute low earth orbit cycle regime at 80% depth-of-discharge. The results show that the active material loading level affects the cycle life the most with the optimum loading at 1.6 g/cc void. Mechanical strength did not affect the cycle life noticeably in the bend strength range of 400 to 700 psi. The best plaque type appears to be one which is made of INCO nickel powder type 287 and has a median pore size of 13 micron.

xCT expression reduces the early cell cycle requirement for calcium signaling

PubMed Central

Lastro, Michele; Kourtidis, Antonis; Farley, Kate; Conklin, Douglas S.

2009-01-01

Calcium has long been recognized as an important regulator of cell cycle transitions although the mechanisms are largely unknown. A functional genomic screen has identified genes involved in the regulation of early cell cycle progression by calcium. These genes when overexpressed confer the ability to bypass the G1/S arrest induced by Ca2+- channel antagonists in mouse fibroblasts. Overexpression of the cystine-glutamate exchanger, xCT, had the greatest ability to evade calcium antagonist-induced cell cycle arrest. xCT carries out the rate limiting step of glutathione synthesis in many cell types and is responsible for the uptake of cystine in most human cancer cell lines. Functional analysis indicates that the cystine uptake activity of xCT overcomes the G1/S arrest induced by Ca2+- channel antagonists by bypassing the requirement for calcium signaling. Since cells overexpressing xCT were found to have increased levels and activity of the AP-1 transcription factor in G1, redox stimulation of AP-1 activity accounts for the observed growth of these cells in the presence of calcium channel antagonists. These results suggest that reduced calcium signaling impairs AP-1 activation and that xCT expression may directly affect cell proliferation. PMID:18054200

Structure related effects of flavonoid aglycones on cell cycle progression of HepG2 cells: Metabolic activation of fisetin and quercetin by catechol-O-methyltransferase (COMT).

PubMed

Poór, Miklós; Zrínyi, Zita; Kőszegi, Tamás

2016-10-01

Dietary flavonoids are abundant in the Plant Kingdom and they are extensively studied because of their manifold pharmacological activities. Recent studies highlighted that cell cycle arrest plays a key role in their antiproliferative effect in different tumor cells. However, structure-activity relationship of flavonoids is poorly characterized. In our study the influence of 18 flavonoid aglycones (as well as two metabolites) on cell cycle distribution was investigated. Since flavonoids are extensively metabolized by liver cells, HepG2 tumor cell line was applied, considering the potential metabolic activation/inactivation of flavonoids. Our major observations are the followings: (1) Among the tested compounds diosmetin, fisetin, apigenin, lutelin, and quercetin provoked spectacular extent of G2/M phase cell cycle arrest. (2) Inhibition of catechol-O-methyltransferase enzyme by entacapone decreased the antiproliferative effects of fisetin and quercetin. (3) Geraldol and isorhamnetin (3'-O-methylated metabolites of fisetin and quercetin, respectively) demonstrated significantly higher antiproliferative effect on HepG2 cells compared to the parent compounds. Based on these results, O-methylated flavonoid metabolites or their chemically modified derivatives may be suitable candidates of tumor therapy in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Effects of exogenous hormones on spermatogenesis in the male prairie dog (Cynomys ludovicianus).

PubMed

Foreman, D

1998-01-01

Male prairie dogs (Cynomys ludovicianus) breed anually and have complete testicular regression. Changes in the seminiferous tubules during the annual cycle have been described recently (Foreman, 1997). This is the first description of spermatogenesis in such a species. The definition of tubular stages during the cycle allows for evaluation of the effects of exogenous hormones, hemicastration, and hemicryptorchidism on spermatogenesis during the annual cycle. Hemicastration was performed during stages of the annual cycle to determine effects of exogenous hormones on remaining testes. Hemicryptorchidism was also done during stages of the annual cycle. FSH, LH, and testosterone were given in high and low doses for short- or long-term treatment periods during stages of the annual cycle. Testicular weights and counts of cell types in tubules of control and treated testes were made on testis tissues. Hemicastration during the out-of-season period does not cause compensatory hypertrophy of the remaining testis, but during recrudescence, hypertrophy of the remaining testis occurs. Hemicastration does not prevent loss of weight by the remaining testis during regression. The seminiferous epithelium of hemicryptorchid prairie dog testes shows damage during spermatogenic activity but not during testicular inactivity. Similarly, hemicryptorchid 15-day-old rat testes do not show damage from hemicryptorchidism. Long-term treatment with FSH preparations during testicular inactivity increased testis weights, spermatogonial proliferation, and spermatocyte differentiation in conjunction with Sertoli cell differentiation. Short-term treatments with low doses increased spermatogonial proliferation and abnormal meiotic activity. Both long- and short-term treatments with LH caused increased sloughing of germ cells and stimulated Leydig and Sertoli cells. Testosterone propionate injections stimulated Sertoli secretions but not Leydig cell activity. Hemicastration during inactivity does not stimulate gonadotropin secretion. Hemicryptorchidism does not affect tubular morphology during inactivity in either rats or prairie dogs. Prompt responses to FSH depend on scrotal location of the testis. FSH has its major effects on germ cell proliferation and differentiation, both directly and through activation of Sertoli cells, whereas LH affects Sertoli and Leydig cell activation but has no effect on germ cell activity. Testosterone activates Sertoli cells.

Jellyfish extract induces apoptotic cell death through the p38 pathway and cell cycle arrest in chronic myelogenous leukemia K562 cells

PubMed Central

Kwak, Choong-Hwan; Abekura, Fukushi; Park, Jun-Young; Park, Nam Gyu; Chang, Young-Chae; Lee, Young-Choon; Chung, Tae-Wook; Ha, Ki-Tae; Son, Jong-Keun

2017-01-01

Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells. PMID:28133573

Cell cycle proteins as promising targets in cancer therapy.

PubMed

Otto, Tobias; Sicinski, Piotr

2017-01-27

Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

The Slow Cycling Phenotype: A Growing Problem for Treatment Resistance in Melanoma.

PubMed

Ahn, Antonio; Chatterjee, Aniruddha; Eccles, Michael R

2017-06-01

Treatment resistance in metastatic melanoma is a longstanding issue. Current targeted therapy regimes in melanoma largely target the proliferating cancer population, leaving slow-cycling cancer cells undamaged. Consequently, slow-cycling cells are enriched upon drug therapy and can remain in the body for years until acquiring proliferative potential that triggers cancer relapse. Here we overview the molecular mechanisms of slow-cycling cells that underlie treatment resistance in melanoma. Three main areas of molecular reprogramming are discussed that mediate slow cycling and treatment resistance. First, a low microphthalmia-associated transcription factor (MITF) dedifferentiated state activates various signaling pathways. This includes WNT5A, EGFR, as well as other signaling activators, such as AXL and NF-κB. Second, the chromatin-remodeling factor Jumonji/ARID domain-containing protein 1B (JARID1B, KDM5B ) orchestrates and maintains slow cycling and treatment resistance in a small subpopulation of melanoma cells. Finally, a shift in metabolic state toward oxidative phosphorylation has been demonstrated to regulate treatment resistance in slow-cycling cells. Elucidation of the underlying processes of slow cycling and its utilization by melanoma cells may reveal new vulnerable characteristics as therapeutic targets. Moreover, combining current therapies with targeting slow-cycling subpopulations of melanoma cells may allow for more durable and greater treatment responses. Mol Cancer Ther; 16(6); 1002-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Regulation of TGF-β signaling, exit from the cell cycle, and cellular migration through cullin cross-regulation: SCF-FBXO11 turns off CRL4-Cdt2.

PubMed

Abbas, Tarek; Keaton, Mignon; Dutta, Anindya

2013-07-15

Deregulation of the cell cycle and genome instability are common features of cancer cells and various mechanisms exist to preserve the integrity of the genome and guard against cancer. The cullin 4-RING ubiquitin ligase (CRL4) with the substrate receptor Cdt2 (CRL4 (Cdt2)) promotes cell cycle progression and prevents genome instability through ubiquitylation and degradation of Cdt1, p21, and Set8 during S phase of the cell cycle and following DNA damage. Two recently published studies report the ubiquitin-dependent degradation of Cdt2 via the cullin 1-RING ubiquitin ligase (CRL1) in association with the substrate specificity factor and tumor suppressor FBXO11 (CRL1 (FBXO11)). The newly identified pathway restrains the activity of CRL4 (Cdt2) on p21 and Set8 and regulates cellular response to TGF-β, exit from the cell cycle and cellular migration. Here, we show that the CRL1 (FBXO11) also promotes the degradation of Cdt2 during an unperturbed cell cycle to promote efficient progression through S and G 2/M phases of the cell cycle. We discuss how this new method of regulating the abundance of Cdt2 participates in various cellular activities.

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

PubMed Central

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-01-01

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

PubMed

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-09-08

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Nitric oxide is involved in hydrogen gas-induced cell cycle activation during adventitious root formation in cucumber.

PubMed

Zhu, Yongchao; Liao, Weibiao; Niu, Lijuan; Wang, Meng; Ma, Zhanjun

2016-06-28

Adventitious root development is a complex process regulated through a variety of signaling molecules. Hydrogen gas (H2) and nitric oxide (NO), two new signaling molecules are both involved in plant development and stress tolerance. To investigate the mechanism of adventitious root development induced by hydrogen-rich water (HRW), a combination of fluorescence microscopy and molecular approaches was used to study cell cycle activation and cell cycle-related gene expression in cucumber (Cucumis sativus 'Xinchun 4') explants. The results revealed that the effect of HRW on adventitious root development was dose-dependent, with maximal biological responses at 50 % HRW. HRW treatment increased NO content in a time-dependent fashion. The results also indicated that HRW and NO promoted the G1-to-S transition and up-regulated cell cycle-related genes: CycA (A-type cyclin), CycB (B-type cyclin), CDKA (cyclin-dependent kinase A) and CDKB (cyclin-dependent kinase B) expression. Additionally, target genes related to adventitious rooting were up-regulated by HRW and NO in cucumber explants. While, the responses of HRW-induced adventitious root development and increase of NO content were partially blocked by a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt, NO synthase (NOS)-like enzyme inhibitor N(G) -nitro-L-arginine methylester hydrochloride, or nitrate reductase inhibitors tungstate and NaN3. These chemicals also partially reversed the effect of HRW on cell cycle activation and the transcripts of cell cycle regulatory genes and target genes related adventitious root formation. Together, NO may emerge as a downstream signaling molecule in H2-induced adventitious root organogenesis. Additionally, H2 mediated cell cycle activation via NO pathway during adventitious root formation.

The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

PubMed

Yin, Jiuheng; Sheng, Baifa; Han, Bin; Pu, Aimin; Yang, Kunqiu; Li, Ping; Wang, Qimeng; Xiao, Weidong; Yang, Hua

2016-05-01

Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation. © 2016 International Federation for Cell Biology.

Cell Cycle Status of CD34+ Hemopoietic Stem Cells Determines Lentiviral Integration in Actively Transcribed and Development-related Genes

PubMed Central

Papanikolaou, Eleni; Paruzynski, Anna; Kasampalidis, Ioannis; Deichmann, Annette; Stamateris, Evangelos; Schmidt, Manfred; von Kalle, Christof; Anagnou, Nicholas P

2015-01-01

Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis. PMID:25523760

The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms.

PubMed

Croft, Daniel R; Olson, Michael F

2006-06-01

The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.

C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling

PubMed Central

Falin, Rebecca A.; Miyazaki, Hiroaki

2011-01-01

Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl− cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes. PMID:21160027

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.

The yeast DNA ligase gene CDC9 is controlled by six orientation specific upstream activating sequences that respond to cellular proliferation but which alone cannot mediate cell cycle regulation.

PubMed Central

White, J H; Johnson, A L; Lowndes, N F; Johnston, L H

1991-01-01

By fusing the CDC9 structural gene to the PGK upstream sequences and the CDC9 upstream to lacZ, we showed that the cell cycle expression of CDC9 is largely due to transcriptional regulation. To investigate the role of six ATGATT upstream repeats in CDC9 regulation, synthetic copies of the sequence were attached to a heterologous gene. The repeats stimulated transcription strongly and additively, but, unlike conventional yeast UAS elements, only when present in one orientation. Transcription driven by the repeats declines in cells held at START of the cell cycle or in stationary phase, as occurs with CDC9. However, the repeats by themselves cannot impart cell cycle regulation to a heterologous gene. CDC9 may therefore be controlled by an activating system operating through the repeats that is sensitive to cellular proliferation and a separate mechanism that governs the periodic expression in the cell cycle. Images PMID:1901644

A nontranscriptional role for Oct4 in the regulation of mitotic entry

PubMed Central

Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

PubMed Central

Burkhart, Deborah L.; Wirt, Stacey E.; Zmoos, Anne-Flore; Kareta, Michael S.; Sage, Julien

2010-01-01

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells. PMID:20585628

In Vitro Effects of Bromoalkyl Phenytoin Derivatives on Regulated Death, Cell Cycle and Ultrastructure of Leukemia Cells.

PubMed

Śladowska, Katarzyna; Opydo-Chanek, Małgorzata; Król, Teodora; Trybus, Wojciech; Trybus, Ewa; Kopacz-Bednarska, Anna; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna; Mazur, Lidia

2017-11-01

To search for new antileukemic agents, the chemical structure of phenytoin was modified. A possible cytotoxic activity of three bromoalkyl phenytoin analogs, methyl 2-(1-(3-bromopropyl)-2,4-dioxo-5,5-diphenylimidazolidin-3-yl) propanoate (PH2), 1-(3-bromopropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH3) and 1-(4-bromobutyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH4) on regulated cell death, the cell cycle and cell ultrastructure was assessed. The experiments were performed in vitro on HL-60 and U937 cells, using flow cytometry and electron microscopy methods. Application of PH2, PH3, and PH4 resulted in cell surface exposure of phosphatidylserine and plasma membrane impairment, caspase-8, -9, and -3/7 activation, dissipation of mitochondrial membrane potential, DNA breakage, cell-cycle disturbance and cell ultrastructural changes. In general, PH3 appeared to be the most active against the leukemia cells, and all bromoalkyl hydantoins, PH2-PH4, were more active in HL-60 cells than in U937 cells. The antileukemic activity of the bromoalkyl phenytoin analogs depended on the combination of N-hydantoin substituents and the human cell line used. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Interplay between cancer cell cycle and metabolism: Challenges, targets and therapeutic opportunities.

PubMed

Roy, Debmalya; Sheng, Gao Ying; Herve, Semukunzi; Carvalho, Evandro; Mahanty, Arpan; Yuan, Shengtao; Sun, Li

2017-05-01

A growing interest has emerged in the field of studying the cross-talk between cancer cell cycle and metabolism. In this review, we aimed to present how metabolism and cell cycle are correlated and how cancer cells get energy to drive cell cycle. Cell proliferation and cell death largely depend on the metabolic activity of the cell. Cell cycle proteins, e.g. cyclin D, cyclin dependent kinase (CDK), some pro-apoptotic and anti-apoptotic proteins, and P53 have been shown to be regulated by metabolic crosstalk. Dysregulation of this cross-talk between metabolism and cell cycle leads to degenerative disorder(s) and cancer. It is not fully understood the actual reason of aberration between metabolism and cell cycle, but it is a hallmark of cancer research. Herein, we discussed the role of some regulatory molecules relative of cell cycle and metabolism and highlight how they control the function of each other. We also pointed out, current therapeutic opportunities and some additional crucial therapeutic targets on these fields that could be a breakthrough in cancer research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Intrinsic and extrinsic mechanisms regulating satellite cell function

PubMed Central

Dumont, Nicolas A.; Wang, Yu Xin; Rudnicki, Michael A.

2015-01-01

Muscle stem cells, termed satellite cells, are crucial for skeletal muscle growth and regeneration. In healthy adult muscle, satellite cells are quiescent but poised for activation. During muscle regeneration, activated satellite cells transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent studies have demonstrated that satellite cells are heterogeneous and that subpopulations of satellite stem cells are able to perform asymmetric divisions to generate myogenic progenitors or symmetric divisions to expand the satellite cell pool. Thus, a complex balance between extrinsic cues and intrinsic regulatory mechanisms is needed to tightly control satellite cell cycle progression and cell fate determination. Defects in satellite cell regulation or in their niche, as observed in degenerative conditions such as aging, can impair muscle regeneration. Here, we review recent discoveries of the intrinsic and extrinsic factors that regulate satellite cell behaviour in regenerating and degenerating muscles. PMID:25922523

Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

PubMed

Ma, Zhaowu; Yu, Guanghui

2010-02-15

The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

Recovery from the DNA Replication Checkpoint

PubMed Central

Chaudhury, Indrajit; Koepp, Deanna M.

2016-01-01

Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β.

PubMed

Jung, Hye-Won; Park, Inae; Ghil, Sungho

2014-09-01

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

Curcumin induces apoptosis and cell cycle arrest via the activation of reactive oxygen species-independent mitochondrial apoptotic pathway in Smad4 and p53 mutated colon adenocarcinoma HT29 cells.

PubMed

Agarwal, Ayushi; Kasinathan, Akiladdevi; Ganesan, Ramamoorthi; Balasubramanian, Akhila; Bhaskaran, Jahnavi; Suresh, Samyuktha; Srinivasan, Revanth; Aravind, K B; Sivalingam, Nageswaran

2018-03-01

Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53. Copyright © 2018. Published by Elsevier Inc.

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

PubMed

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

2011-01-01

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

Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.

PubMed

Cedeño, Cesyen; La Monaca, Esther; Esposito, Mara; Gutierrez, Gustavo J

2016-01-01

The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.

Chronic restraint stress inhibits hair growth via substance P mediated by reactive oxygen species in mice.

PubMed

Liu, Nan; Wang, Lin-Hui; Guo, Ling-Ling; Wang, Guo-Qing; Zhou, Xi-Ping; Jiang, Yan; Shang, Jing; Murao, Koji; Chen, Jing-Wei; Fu, Wen-Qing; Zhang, Guo-Xing

2013-01-01

Solid evidence has demonstrated that psychoemotional stress induced alteration of hair cycle through neuropeptide substance P (SP) mediated immune response, the role of reactive oxygen species (ROS) in brain-skin-axis regulation system remains unknown. The present study aims to investigate possible mechanisms of ROS in regulation of SP-mast cell signal pathway in chronic restraint stress (CRS, a model of chronic psychoemotional stress) which induced abnormal of hair cycle. Our results have demonstrated that CRS actually altered hair cycle by inhibiting hair follicle growth in vivo, prolonging the telogen stage and delaying subsequent anagen and catagen stage. Up-regulation of SP protein expression in cutaneous peripheral nerve fibers and activation of mast cell were observed accompanied with increase of lipid peroxidation levels and reduction of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in CRS mice skin. In addition, SP receptor antagonist (RP67580) reduced mast cell activations and lipid peroxidation levels as well as increased GSH-Px activity and normalized hair cycle. Furthermore, antioxidant Tempol (a free radical scavenger) also restored hair cycle, reduced SP protein expression and mast cell activation. Our study provides the first solid evidence for how ROS play a role in regulation of psychoemotional stress induced SP-Mast cell pathway which may provide a convincing rationale for antioxidant application in clinical treatment with psychological stress induced hair loss.

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.

Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly

PubMed Central

Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.

2012-01-01

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853

Physical and chemical analysis of a Ni/H2 cell

NASA Technical Reports Server (NTRS)

Vaidyanathan, H.; Earl, M. W.; Kirkendall, T. D.

1991-01-01

A cycled aerospace nickel hydrogen (Ni/H2) cell was subjected to destructive physical analysis to determine the reason for a capacity loss after 5,967 cycles at 60 percent depth of discharge. The positive plates in the cell were analyzed in terms of chemical composition, active material utilization, charge efficiency, and thickness increase. The microstructure of a cross section of the positive plate was determined by backscattered electron image analysis. The results suggest that the capacity loss in the cell is caused by low charge acceptance and low active material utilization at the positive plate. The oxidized nickel species content of the positive plate increased due to corrosion of the nickel sintered skeleton. This appears to circumvent the orderly reaction of the active material. Microstructural analysis has indicated that a new phase of active material is formed with cycling.

Berberine Induces Cell Cycle Arrest in Cholangiocarcinoma Cell Lines via Inhibition of NF-κB and STAT3 Pathways.

PubMed

Puthdee, Nattapong; Seubwai, Wunchana; Vaeteewoottacharn, Kulthida; Boonmars, Thidarut; Cha'on, Ubon; Phoomak, Chatchai; Wongkham, Sopit

2017-01-01

Berberine is a natural compound found in several herbs. Anticancer activity of berberine was reported in several cancers, however, little is known regarding the effects of berberine against cholangiocarcinoma (CCA). In this study, the growth inhibitory effects of berberine on CCA cell lines and its molecular mechanisms were explored. Cell growth and cell cycle distribution were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The expression levels of cell cycle regulatory proteins were determined by Western blot analysis. Berberine significantly inhibited growth of CCA cell lines in a dose and time dependent fashion. The inhibition was largely attributed to cell cycle arrest at the G1 phase through the reduction of cyclin D1, and cyclin E. Moreover, berberine could reduce the expression and activation of signal transducers and activator of transcription 3 (STAT3) and probably nuclear factor-kappaB (NF-κB) via suppression of extracellular signal-regulated kinase (ERK) 1/2 action. These results highlight the potential of berberine to be a multi-target agent for CCA treatment.

Non-stochastic reprogramming from a privileged somatic cell state

PubMed Central

Guo, Shangqin; Zi, Xiaoyuan; Schulz, Vincent P.; Cheng, Jijun; Zhong, Mei; Koochaki, Sebastian H.J.; Megyola, Cynthia M.; Pan, Xinghua; Heydari, Kartoosh; Weissman, Sherman M.; Gallagher, Patrick G.; Krause, Diane S.; Fan, Rong; Lu, Jun

2014-01-01

SUMMARY Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PMID:24486105

Aging behavior of lithium iron phosphate based 18650-type cells studied by in situ neutron diffraction

NASA Astrophysics Data System (ADS)

Paul, Neelima; Wandt, Johannes; Seidlmayer, Stefan; Schebesta, Sebastian; Mühlbauer, Martin J.; Dolotko, Oleksandr; Gasteiger, Hubert A.; Gilles, Ralph

2017-03-01

The aging behavior of commercially produced 18650-type Li-ion cells consisting of a lithium iron phosphate (LFP) based cathode and a graphite anode based on either mesocarbon microbeads (MCMB) or needle coke (NC) is studied by in situ neutron diffraction and standard electrochemical techniques. While the MCMB cells showed an excellent cycle life with only 8% relative capacity loss (i.e., referenced to the capacity after formation) after 4750 cycles and showed no capacity loss on storage for two years, the needle coke cells suffered a 23% relative capacity loss after cycling and a 11% loss after storage. Based on a combination of neutron diffraction and electrochemical characterization, it is shown that the entire capacity loss for both cell types is dominated by the loss of active lithium; no other aging mechanisms like structural degradation of anode or cathode active materials or deactivation of active material could be found, highlighting the high structural stability of the active material and the excellent quality of the investigated cells.

Two inhibitory systems and CKIs regulate cell cycle exit of mammalian cardiomyocytes after birth

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

Tane, Shoji; Okayama, Hitomi; Ikenishi, Aiko

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which they exit their cell cycle after birth, and the exit is maintained. Previously, we showed that two inhibitory systems (the G1-phase inhibitory system: repression of cyclin D1 expression; the M-phase inhibitory system: inhibition of CDK1 activation) maintain the cell cycle exit of mouse adult cardiomyocytes. We also showed that two CDK inhibitors (CKIs), p21{sup Cip1} and p27{sup Kip1}, regulate the cell cycle exit in a portion of postnatal cardiomyocytes. It remains unknown whether the two inhibitory systems are involved in the cell cycle exit of postnatal cardiomyocytes and whether p21{sup Cip1}more » and p27{sup Kip1} also inhibit entry to M-phase. Here, we showed that more than 40% of cardiomyocytes entered an additional cell cycle by induction of cyclin D1 expression at postnatal stages, but M-phase entry was inhibited in the majority of cardiomyocytes. Marked cell cycle progression and endoreplication were observed in cardiomyocytes of p21{sup Cip1} knockout mice at 4 weeks of age. In addition, tri- and tetranucleated cardiomyocytes increased significantly in p21{sup Cip1} knockout mice. These data showed that the G1-phase inhibitory system and two CKIs (p21{sup Cip1} and p27{sup Kip1}) inhibit entry to an additional cell cycle in postnatal cardiomyocytes, and that the M-phase inhibitory system and p21{sup Cip1} inhibit M-phase entry of cardiomyocytes which have entered the additional cell cycle. - Highlights: • Many postnatal cardiomyocytes entered an additional cell cycle by cyclin D1 induction. • The majority of cardiomyocytes could not enter M-phase after cyclin D1 induction. • Cell cycle progressed markedly in p21{sup Cip1} knockout mice after postnatal day 14. • Tri- and tetranucleated cardiomyocytes increased in p21{sup Cip1} knockout mice.« less

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc- cells

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.

1994-01-01

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the -49 lymphoma variant (cyc-) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A1 (dmPGA1) inhibits DNA synthesis and cell growth in cyc- cells. DNA synthesis is inhibited 42% by dmPGA1 (50 microM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the alpha, beta unsaturated ketone ring. Dimethyl PGA1 is most effective in inhibiting DNA synthesis in cyc- cells, with prostaglandins PGE1 and PGB1 being less potent inhibitors of DNA synthesis. DmPGE2 caused a significant stimulation of DNA synthesis. S-49 cyc- variant cells exposed to (30-50 microns) dmPGA1, arrested in the G1 phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc- cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G1, S, G2, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G1 cell cycle block.(ABSTRACT TRUNCATED AT 250 WORDS).

Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

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

Ma, Yong-Cheng; Su, Nan; Shi, Xiao-Jing

2015-01-15

Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore,more » Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.« less

Regulation of Cell Cycle and Stress Responses to Hydrostatic Pressure in Fission Yeast

PubMed Central

George, Vinoj T.; Brooks, Gavin

2007-01-01

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress. PMID:17699598

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

PubMed

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

2008-04-01

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

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

PubMed Central

Buckle, A. M.; Mottram, R.; Pierce, A.; Lucas, G. S.; Russell, N.; Miyan, J. A.; Whetton, A. D.

2000-01-01

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation. PMID:11126203

Levels of Ycg1 Limit Condensin Function during the Cell Cycle

PubMed Central

Arsenault, Heather E.; Benanti, Jennifer A.

2016-01-01

During mitosis chromosomes are condensed to facilitate their segregation, through a process mediated by the condensin complex. Although several factors that promote maximal condensin activity during mitosis have been identified, the mechanisms that downregulate condensin activity during interphase are largely unknown. Here, we demonstrate that Ycg1, the Cap-G subunit of budding yeast condensin, is cell cycle-regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. This cyclical expression pattern is established by a combination of cell cycle-regulated transcription and constitutive degradation. Interestingly, overexpression of YCG1 and mutations that stabilize Ycg1 each result in delayed cell-cycle entry and an overall proliferation defect. Overexpression of no other condensin subunit impacts the cell cycle, suggesting that Ycg1 is limiting for condensin complex formation. Consistent with this possibility, we find that levels of intact condensin complex are reduced in G1 phase compared to mitosis, and that increased Ycg1 expression leads to increases in both levels of condensin complex and binding to chromatin in G1. Together, these results demonstrate that Ycg1 levels limit condensin function in interphase cells, and suggest that the association of condensin with chromosomes must be reduced following mitosis to enable efficient progression through the cell cycle. PMID:27463097

Radiotherapy induces cell cycle arrest and cell apoptosis in nasopharyngeal carcinoma via the ATM and Smad pathways.

PubMed

Li, Ming-Yi; Liu, Jin-Quan; Chen, Dong-Ping; Li, Zhou-Yu; Qi, Bin; He, Lu; Yu, Yi; Yin, Wen-Jin; Wang, Meng-Yao; Lin, Ling

2017-09-02

Nasopharyngeal carcinoma (NPC) is a common malignant neoplasm of the head and neck which is harmful to human's health. Radiotherapy is commonly used in the treatment of NPC and it induces immediate cell cycle arrest and cell apoptosis. However, the mechanism remains unknown. Evidences suggested the activation of Ataxia telangiectasia mutated (ATM) pathway and Smad pathway are 2 of the important crucial mediators in the function of radiotherapy. In this study, we performed in vitro assays with human nasopharyngeal carcinoma CNE-2 cells and in vivo assays with nude mice to investigate the role of the ATM and Smad pathways in the treatment of nasopharyngeal carcinoma with radiotherapy. The results suggested that radiation induced activation of ATM pathway by inducing expression of p-ATM, p-CHK1, p-CHK2, p15 and inhibiting expression of p-Smad3. In addition, Caspase3 expression was increased while CDC25A was decreased, leading to cell cycle arrest and cell apoptosis. On the other hand, activation of Smad3 can inhibited the ATM pathway and attenuated the efficacy of radiation. In summary, we suggest that both ATM and Smad pathways contribute to the cell cycle arrest and cell apoptosis during nasopharyngeal carcinoma cells treated with radiation.

Characterisation of cell cycle arrest and terminal differentiation in a maximally proliferative human epithelial tissue: Lessons from the human hair follicle matrix.

PubMed

Purba, Talveen S; Brunken, Lars; Peake, Michael; Shahmalak, Asim; Chaves, Asuncion; Poblet, Enrique; Ceballos, Laura; Gandarillas, Alberto; Paus, Ralf

2017-09-01

Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21 CIP1 , p27 KIP1 and p57 KIP2 ) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21 CIP1 , p27 KIP1 and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57 KIP2 protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that were >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds of DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ-biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically relevant model system for cell cycle physiology research of human epithelial cells within their natural tissue habitat. Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.

Co-production of tannase and pectinase by free and immobilized cells of the yeast Rhodotorula glutinis MP-10 isolated from tannin-rich persimmon (Diospyros kaki L.) fruits.

PubMed

Taskin, Mesut

2013-02-01

Hyper tannase and pectinase-producing yeast Rhodotorula glutinis MP-10 was isolated from persimmon (Diospyros kaki L.) fruits. The main pectinase activity of yeast was exo-polygalacturonase. No pectin methyl esterase and too low pectin lyase activities were detected for this yeast. The maximum exo-activities of tannase and polygalacturonase were determined as 15.2 and 26.9 U/mL for free cells and 19.8 and 28.6 U/mL for immobilized cells, respectively. Immobilized cells could be reused in 13 successive reaction cycles without any loss in the maximum tannase and polygalacturonase activities. Besides, too little decreases in activities of these enzymes were recorded between 14 and 18 cycles. At the end of 18 successive reaction cycles, total 503.1 U/mL of polygalacturonase and 349.6 U/mL of tannase could be produced using the same immobilized cells. This is the first report on the use of free and/or immobilized cells of a microorganism for the co-production of tannase and pectinase.

Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation

PubMed Central

Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J.; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L.; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D.; Weninger, Wolfgang

2015-01-01

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8+ T cells. During influenza virus infection in vivo, naive T cells enter a CD62Lintermediate state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62Lhi central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62Lhi memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways. PMID:25709008

Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation.

PubMed

Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D; Weninger, Wolfgang

2015-02-24

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L(hi) central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L(hi) memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.

The Nuclear and Adherent Junction Complex Component Protein Ubinuclein Negatively Regulates the Productive Cycle of Epstein-Barr Virus in Epithelial Cells▿

PubMed Central

Gruffat, Henri; Lupo, Julien; Morand, Patrice; Boyer, Véronique; Manet, Evelyne

2011-01-01

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus. PMID:21084479

A mitosis block links active cell cycle with human epidermal differentiation and results in endoreplication.

PubMed

Zanet, Jennifer; Freije, Ana; Ruiz, María; Coulon, Vincent; Sanz, J Ramón; Chiesa, Jean; Gandarillas, Alberto

2010-12-20

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation.

A Mitosis Block Links Active Cell Cycle with Human Epidermal Differentiation and Results in Endoreplication

PubMed Central

Zanet, Jennifer; Freije, Ana; Ruiz, María; Coulon, Vincent; Sanz, J. Ramón; Chiesa, Jean; Gandarillas, Alberto

2010-01-01

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation. PMID:21187932

Lactate dehydrogenase activity drives hair follicle stem cell activation

PubMed Central

Aimee, Flores; John, Schell; Abby, Krall; David, Jelinek; Matilde, Miranda; Melina, Grigorian; Daniel, Braas; White Andrew, C; Jessica, Zhou; Nick, Graham; Thomas, Graeber; Pankaj, Seth; Denis, Evseenko; Hilary, Coller; Jared, Rutter; Heather, Christofk; Lowry William, E

2017-01-01

Summary While normally dormant, Hair Follicle Stem Cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allow them to remain dormant and yet quickly respond to appropriate proliferative stimuli. PMID:28812580

Coordination of Satellite Cell Activation and Self-Renewal by Par-Complex-Dependent Asymmetric Activation of p38α/β MAPK

PubMed Central

Troy, Andrew; Cadwallader, Adam B.; Fedorov, Yuri; Tyner, Kristina; Tanaka, Kathleen Kelly; Olwin, Bradley B.

2014-01-01

SUMMARY In response to muscle injury, satellite cells activate the p38α/β MAPK pathway to exit quiescence, then proliferate, repair skeletal muscle, and self-renew, replenishing the quiescent satellite cell pool. Although satellite cells are capable of asymmetric division, the mechanisms regulating satellite cell self-renewal are not understood. We found that satellite cells, once activated, enter the cell cycle and a subset undergoes asymmetric division, renewing the satellite cell pool. Asymmetric localization of the Par complex activates p38α/β MAPK in only one daughter cell, inducing MyoD, which permits cell cycle entry and generates a proliferating myoblast. The absence of p38α/β MAPK signaling in the other daughter cell prevents MyoD induction, renewing the quiescent satellite cell. Thus, satellite cells employ a mechanism to generate distinct daughter cells, coupling the Par complex and p38α/β MAPK signaling to link the response to muscle injury with satellite cell self-renewal. PMID:23040480

Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

PubMed

Yap, W H; Khoo, K S; Lim, S H; Yeo, C C; Lim, Y M

2012-01-15

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Critical Role of AMPK/FoxO3A Axis in Globular Adiponectin-Induced Cell Cycle Arrest and Apoptosis in Cancer Cells.

PubMed

Shrestha, Anup; Nepal, Saroj; Kim, Mi Jin; Chang, Jae Hoon; Kim, Sang-Hyun; Jeong, Gil-Saeng; Jeong, Chul-Ho; Park, Gyu Hwan; Jung, Sunghee; Lim, Jaecheong; Cho, Eunha; Lee, Soyoung; Park, Pil-Hoon

2016-02-01

Adiponectin predominantly secreted from adipose tissue has exhibited potent anti-proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti-cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF-7) cancer cells. Gene ablation of FoxO3A prevented gAcrp-induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp-induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase-3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF-7 cells. Transfection with FoxO3A siRNA inhibited gAcrp-induced caspase-3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp-induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp-induced nuclear translocation of FoxO3A in HepG2 and MCF-7 cells. In addition, suppression of AMPK also blocked gAcrp-induced cell cycle arrest and further attenuated gAcrp-induced caspase-3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp-induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti-proliferative effect of adiponectin in cancer cells. © 2015 Wiley Periodicals, Inc.

A stochastic spatiotemporal model of a response-regulator network in the Caulobacter crescentus cell cycle

NASA Astrophysics Data System (ADS)

Li, Fei; Subramanian, Kartik; Chen, Minghan; Tyson, John J.; Cao, Yang

2016-06-01

The asymmetric cell division cycle in Caulobacter crescentus is controlled by an elaborate molecular mechanism governing the production, activation and spatial localization of a host of interacting proteins. In previous work, we proposed a deterministic mathematical model for the spatiotemporal dynamics of six major regulatory proteins. In this paper, we study a stochastic version of the model, which takes into account molecular fluctuations of these regulatory proteins in space and time during early stages of the cell cycle of wild-type Caulobacter cells. We test the stochastic model with regard to experimental observations of increased variability of cycle time in cells depleted of the divJ gene product. The deterministic model predicts that overexpression of the divK gene blocks cell cycle progression in the stalked stage; however, stochastic simulations suggest that a small fraction of the mutants cells do complete the cell cycle normally.

Cell Cycle Deregulation in the Neurons of Alzheimer’s Disease

PubMed Central

Moh, Calvin; Kubiak, Jacek Z.; Bajic, Vladan P.; Zhu, Xiongwei; Smith, Mark A.

2018-01-01

The cell cycle consists of four main phases: G1, S, G2, and M. Most cells undergo these cycles up to 40–60 times in their life. However, neurons remain in a nondividing, nonreplicating phase, G0. Neurons initiate but do not complete cell division, eventually entering apoptosis. Research has suggested that like cancer, Alzheimer’s disease (AD) involves dysfunction in neuronal cell cycle reentry, leading to the development of the two-hit hypothesis of AD. The first hit is abnormal cell cycle reentry, which typically results in neuronal apoptosis and prevention of AD. However, with the second hit of chronic oxidative damage preventing apoptosis, neurons gain “immortality” analogous to tumor cells. Once both of these hits are activated, AD can develop and produce senile plaques and neurofibrillary tangles throughout brain tissue. In this review, we propose a mechanism for neuronal cell cycle reentry and the development of AD. PMID:21630160

Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

PubMed

Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan

2016-10-01

Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

700 F hybrid capacitors cells composed of activated carbon and Li4Ti5O12 microspheres with ultra-long cycle life

NASA Astrophysics Data System (ADS)

Ruan, Dianbo; Kim, Myeong-Seong; Yang, Bin; Qin, Jun; Kim, Kwang-Bum; Lee, Sang-Hyun; Liu, Qiuxiang; Tan, Lei; Qiao, Zhijun

2017-10-01

To address the large-scale application demands of high energy density, high power density, and long cycle lifetime, 700-F hybrid capacitor pouch cells have been prepared, comprising ∼240-μm-thick activated carbon cathodes, and ∼60-μm-thick Li4Ti5O12 anodes. Microspherical Li4Ti5O12 (M-LTO) synthesized by spray-drying features 200-400 nm primary particles and interconnected nanopore structures. M-LTO half-cells exhibits high specific capacities (175 mAhh g-1), good rate capabilities (148 mAhh g-1 at 20 C), and ultra-long cycling stabilities (90% specific capacity retention after 10,000 cycles). In addition, the obtained hybrid capacitors comprising activated carbon (AC) and M-LTO shows excellent cell performances, achieving a maximum energy density of 51.65 Wh kg-1, a maximum power density of 2466 W kg-1, and ∼92% capacitance retention after 10,000 cycles, thus meeting the demands for large-scale applications such as trolleybuses.

Single-cell analysis of transcription kinetics across the cell cycle

PubMed Central

Skinner, Samuel O; Xu, Heng; Nagarkar-Jaiswal, Sonal; Freire, Pablo R; Zwaka, Thomas P; Golding, Ido

2016-01-01

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. DOI: http://dx.doi.org/10.7554/eLife.12175.001 PMID:26824388

The synthetic purine reversine selectively induces cell death of cancer cells.

PubMed

Piccoli, Marco; Palazzolo, Giacomo; Conforti, Erika; Lamorte, Giuseppe; Papini, Nadia; Creo, Pasquale; Fania, Chiara; Scaringi, Raffaella; Bergante, Sonia; Tringali, Cristina; Roncoroni, Leda; Mazzoleni, Stefania; Doneda, Luisa; Galli, Rossella; Venerando, Bruno; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2012-10-01

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. Copyright © 2012 Wiley Periodicals, Inc.

Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

PubMed Central

Ruijtenberg, Suzan; van den Heuvel, Sander

2016-01-01

ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227

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

Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

PubMed

Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

2017-04-01

Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Benzene-induced myelotoxicity: application of flow cytofluorometry for the evaluation of early proliferative change in bone marrow.

PubMed Central

Irons, R D

1981-01-01

A detailed description of flow cytofluorometric DNA cell cycle analysis is presented. A number of studies by the author and other investigators are reviewed in which a method is developed for the analysis of cell cycle phase in bone marrow of experimental animals. Bone marrow cell cycle analysis is a sensitive indicator of changes in bone marrow proliferative activity occurring early in chemically-induced myelotoxicity. Cell cycle analysis, used together with other hematologic methods, has revealed benzene-induced toxicity in proliferating bone marrow cells to be cycle specific, appearing to affect a population in late S phase which then accumulate in G2/M. PMID:7016521

LPA Induces Colon Cancer Cell Proliferation through a Cooperation between the ROCK and STAT-3 Pathways.

PubMed

Leve, Fernanda; Peres-Moreira, Rubem J; Binato, Renata; Abdelhay, Eliana; Morgado-Díaz, José A

2015-01-01

Lysophosphatidic acid (LPA) plays a critical role in the proliferation and migration of colon cancer cells; however, the downstream signaling events underlying these processes remain poorly characterized. The aim of this study was to investigate the signaling pathways triggered by LPA to regulate the mechanisms involved in the progression of colorectal cancer (CRC). We have used three cell line models of CRC, and initially analyzed the expression profile of LPA receptors (LPAR). Then, we treated the cells with LPA and events related to their tumorigenic potential, such as migration, invasion, anchorage-independent growth, proliferation as well as apoptosis and cell cycle were evaluated. We used the Chip array technique to analyze the global gene expression profiling that occurs after LPA treatment, and we identified cell signaling pathways related to the cell cycle. The inhibition of these pathways verified the conclusions of the transcriptomic analysis. We found that the cell lines expressed LPAR1, -2 and -3 in a differential manner and that 10 μM LPA did not affect cell migration, invasion and anchorage-independent growth, but it did induce proliferation and cell cycle progression in HCT-116 cells. Although LPA in this concentration did not induce transcriptional activity of β-catenin, it promoted the activation of Rho and STAT-3. Moreover, ROCK and STAT-3 inhibitors prevented LPA-induced proliferation, but ROCK inhibition did not prevent STAT-3 activation. Finally, we observed that LPA regulates the expression of genes related to the cell cycle and that the combined inhibition of ROCK and STAT-3 prevented cell cycle progression and increased the LPA-induced expression of cyclins E1, A2 and B1 to a greater degree than either inhibitor alone. Overall, these results demonstrate that LPA increases the proliferative potential of colon adenocarcinoma HCT-116 cells through a mechanism involving cooperation between the Rho-ROCK and STAT3 pathways involved in cell cycle control.

Nox2-derived ROS in PPARγ signaling and cell-cycle progression of lung alveolar epithelial cells

PubMed Central

Tickner, Jennifer; Fan, Lampson M.; Du, Junjie; Meijles, Daniel; Li, Jian-Mei

2011-01-01

Reactive oxygen species (ROS) play important roles in peroxisome proliferator-activated receptor γ (PPARγ) signaling and cell-cycle regulation. However, the PPARγ redox-signaling pathways in lung alveolar epithelial cells remain unclear. In this study, we investigated the in vivo and in vitro effects of PPARγ activation on the levels of lung ROS production and cell-cycle progression using C57BL/6J wild-type and Nox2 knockout mice (n = 10) after intraperitoneal injection of a selective PPARγ agonist (GW1929, 5 mg/kg body wt, daily) for 14 days. Compared to vehicle-treated mice, GW1929 increased significantly the levels of ROS production in wild-type lungs, and this was accompanied by significant up-regulation of PPARγ, Nox2, PCNA, and cyclin D1 and phosphorylation of ERK1/2 and p38MAPK. These effects were absent in Nox2 knockout mice. In cultured alveolar epithelial cells, GW1929 (5 μM for 24 h) increased ROS production and promoted cell-cycle progression from G0/G1 into S and G2/M phases, and these effects were abolished by (1) adding a PPARγ antagonist (BADGE, 1 μM), (2) knockdown of PPARγ using siRNA, or (3) knockout of Nox2. In conclusion, PPARγ activation through Nox2-derived ROS promotes cell-cycle progression in normal mouse lungs and in cultured normal alveolar epithelial cells. PMID:21664456

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

SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1

PubMed Central

Leal, Paulo C.; Bhasin, Manoj K.; Zenatti, Priscila Pini; Nunes, Ricardo J.; Yunes, Rosendo A.; Nowill, Alexandre E.; Libermann, Towia A.; Zerbini, Luiz Fernando; Yunes, José Andrés

2015-01-01

Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002. PMID:26302043

Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

PubMed

Solomon, Lauren A; Podder, Shreya; He, Jessica; Jackson-Chornenki, Nicholas L; Gibson, Kristen; Ziliotto, Rachel G; Rhee, Jess; DeKoter, Rodney P

2017-05-15

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1 , an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. Copyright © 2017 American Society for Microbiology.

Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.

PubMed

Liu, Yongji; Shi, Ling; Liu, Yuan; Li, Peng; Jiang, Guoping; Gao, Xiaoning; Zhang, Yongbin; Jiang, Chuanwu; Zhu, Weiping; Han, Hongxing; Ju, Fang

2018-04-01

Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A Unifying Theory of Prostate Cancer

DTIC Science & Technology

2001-09-01

of cell cycle arrest or apoptosis, may lead to genomic instability and the development of cancer. 10 * One of our goals was to determine whether p53...and cell cycle arrest. Therefore, mechanisms responsible for upregulation and activation of p53 are intact in prostatic epithelial cells. We also...such as y-irradiation (Girinsky et al., 1995). The p53 protein is known to be a key regulator of cell cycle arrest and/or apoptosis (Wiman, 1997

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

PubMed

Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

2012-10-01

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity.

PubMed

Duncan, Robin E; Lau, Dominic; El-Sohemy, Ahmed; Archer, Michael C

2004-11-01

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.

Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models.

PubMed

Lehmann, Christian; Friess, Thomas; Birzele, Fabian; Kiialainen, Anna; Dangl, Markus

2016-06-28

Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies. Our findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.

Clinical trial tests drug for tumors associated with Krebs-cycle dysfunction | Center for Cancer Research

Cancer.gov

The Krebs cycle is part of the complex process where cells turn food into energy. One of the elements of the Krebs cycle is succinate dehydrogenase (SDH). Loss of SDH activity in cells has been linked to tumor formation. This new trial is studying guadecitabine for tumors associated with Krebs cycle dysfunction. Learn more...

Spatial and temporal characterization of endometrial mesenchymal stem-like cells activity during the menstrual cycle

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

Shan, Xu; Chan, Rachel W.S., E-mail: rwschan@hku.hk; Centre of Reproduction, Development of Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR

The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal stem-like cells (eMSCs) located throughout the endometrium have shown to functionally contribute to endometrial regeneration. In this study we examine whether the menstrual cycle stage and the location in the endometrial bilayer (superficial and deep portions of the endometrium) has an effect on stem cell activities of eMSCs (CD140b{sup +}CD146{sup +} cells). Here we show the percentage and clonogenic ability of eMSCs were constant in the various stages of the menstrual cycle (menstrual, proliferative and secretory). However, eMSCs from themore » menstrual endometrium underwent significantly more rounds of self-renewal and enabled a greater total cell output than those from the secretory phase. Significantly more eMSCs were detected in the deeper portion of the endometrium compared to the superficial layer but their clonogenic and self-renewal activities remained similar. Our findings suggest that eMSCs are activated in the menstrual phase for the cyclical regeneration of the endometrium. - Highlights: • The percentages of endometrial mesenchymal-like stem cells (eMSCs) were constant across the menstrual cycle. • Menstruation eMSCs display superior self-renewal and long-term proliferative activities. • More eMSCs reside in the deeper portion of the endometrium than the superficial layer.« less

p21 induction plays a dual role in anti-cancer activity of ursolic acid

PubMed Central

Zhang, Xudong; Song, Xinhua; Yin, Shutao; Zhao, Chong; Fan, Lihong

2015-01-01

Previous studies have shown that induction of G1 arrest and apoptosis by ursolic acid is associated with up-regulation of cyclin-dependent kinase inhibitor (CDKI) protein p21 in multiple types of cancer cells. However, the functional role of p21 induction in G1 cell cycle arrest and apoptosis, and the mechanisms of p21 induction by ursolic acid have not been critically addressed. In the current study, we demonstrated that p21 played a mediator role in G1 cell cycle arrest by ursolic acid, whereas p21-mediated up-regulation of Mcl-1 compromised apoptotic effect of ursolic acid. These results suggest that p21 induction plays a dual role in the anti-cancer activity of ursolic acid in terms of cell cycle and apoptosis regulation. p21 induction by ursolic acid was attributed to p53 transcriptional activation. Moreover, we found that ursolic acid was able to inhibit murine double minute-2 protein (MDM2) and T-LAK cell-originated protein kinase (TOPK), the two negative regulator of p53, which in turn contributed to ursolic acid-induced p53 activation. Our findings provided novel insights into understanding of the mechanisms involved in cell cycle arrest and apoptosis induction in response to ursolic acid exposure. PMID:26582056

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

PubMed Central

de Bruin, R. A. M.; Kalashnikova, T. I.; Aslanian, A.; Wohlschlegel, J.; Chahwan, C.; Yates, J. R.; Russell, P.; Wittenberg, C.

2008-01-01

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication. PMID:18682565

Synthesis, Biological Evaluation, and Structure–Activity Relationships of Novel Substituted N-Phenyl Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression in S-Phase and Inducing DNA Double-Strand Breaks

PubMed Central

2012-01-01

Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents. PMID:22694057

Synthesis, biological evaluation, and structure-activity relationships of novel substituted N-phenyl ureidobenzenesulfonate derivatives blocking cell cycle progression in S-phase and inducing DNA double-strand breaks.

PubMed

Turcotte, Vanessa; Fortin, Sébastien; Vevey, Florence; Coulombe, Yan; Lacroix, Jacques; Côté, Marie-France; Masson, Jean-Yves; C-Gaudreault, René

2012-07-12

Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents.

Cell cycle effect on the activity of deoxynucleoside analogue metabolising enzymes

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

Fyrberg, Anna; Albertioni, Freidoun; Lotfi, Kourosh

Deoxynucleoside analogues (dNAs) are cytotoxic towards both replicating and indolent malignancies. The impact of fluctuations in the metabolism of dNAs in relation to cell cycle could have strong implications regarding the activity of dNAs. Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are important enzymes for phosphorylation/activation of dNAs. These drugs can be dephosphorylated/deactivated by 5'-nucleotidases (5'-NTs) and elevated activities of 5'-NTs and decreased dCK and/or dGK activities represent resistance mechanisms towards dNAs. The activities of dCK, dGK, and three 5'-NTs were investigated in four human leukemic cell lines in relationship to cell cycle progression and cytotoxicity of dNAs. Synchronization ofmore » cell cultures to arrest in G0/G1 by serum-deprivation was performed followed by serum-supplementation for cell cycle progression. The activities of dCK and dGK increased up to 3-fold in CEM, HL60, and MOLT-4 cells as they started to proliferate, while the activity of cytosolic nucleotidase I was reduced in proliferating cells. CEM, HL60, and MOLT-4 cells were also more sensitive to cladribine, cytarabine, 9-{beta}-D-arabinofuranosylguanine and clofarabine than K562 cells which demonstrated lower levels and less alteration of these enzymes and were least susceptible to the cytotoxic effects of most dNAs. The results suggest that, in the cell lines studied, the proliferation process is associated with a general shift in the direction of activation of dNAs by inducing activities of dCK/dGK and reducing the activity of cN-I which is favourable for the cytotoxic effects of cladribine, cytarabine and, 9-{beta}-D-arabinofuranosylguanine. These results emphasize the importance of cellular proliferation and dNA metabolism by both phosphorylation and dephosphorylation for susceptibility to dNAs. It underscores the need to understand the mechanisms of action and resistance to dNAs in order to increase efficacy of dNAs treatment by new rational.« less

3,3′-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung Injury through Alterations in the Expression of MicroRNA that Target Apoptosis and Cell-Cycle Arrest in Activated T Cells

PubMed Central

Elliott, David M.; Nagarkatti, Mitzi

2016-01-01

3,3′-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables, has significant anti-cancer and anti-inflammatory properties. In this current study, we investigated the effects of DIM on acute lung injury (ALI) induced by exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of mice with DIM led to attenuation of SEB-induced inflammation in the lungs, vascular leak, and IFN-γ secretion. Additionally, DIM could induce cell-cycle arrest and cell death in SEB-activated T cells in a concentration-dependent manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and regulation networks indicated that DIM alters miRNA in the cell death and cell-cycle progression pathways. Specifically, DIM treatment significantly downregulated several miRNA and a correlative increase associated gene targets. Furthermore, overexpression and inhibition studies demonstrated that DIM-induced cell death, at least in part, used miR-222. Collectively, these studies demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and may do so through the induction of microRNAs that promote apoptosis and cell-cycle arrest in SEB-activated T cells. PMID:26818958

Evodiamine Induces Cell Growth Arrest, Apoptosis and Suppresses Tumorigenesis in Human Urothelial Cell Carcinoma Cells.

PubMed

Shi, Chung-Sheng; Li, Jhy-Ming; Chin, Chih-Chien; Kuo, Yi-Hung; Lee, Ying-Ray; Huang, Yun-Ching

2017-03-01

Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities including vasodilatation, analgesia, anti-cardiovascular disease, anti-Alzheimer's disease, anti-inflammation, and anti-tumor activity. This study analyzes the anti-tumor effects of evodiamine on cellular growth, tumorigenesis, cell cycle and apoptosis induction of human urothelial cell carcinoma (UCC) cells. The present study showed that evodiamine significantly inhibited the proliferation of UCC cells in a dose- and time-dependent manner. Also, evodiamine suppressed the tumorigenesis of UCC cells in vitro. Moreover, evodiamine caused G 2 /M cell-cycle arrest and induced caspase-dependent apoptosis in UCC cells. Finally, we demonstrated that evodiamine exhibits better cytotoxic than 5-fluorouracil, a clinical chemotherapeutic drug, for UCC cells. Evodiamine induces growth inhibition, tumorigenesis suppression, cell-cycle arrest, and apoptosis induction in human UCC cells. Therefore, this agent displays a therapeutic potential for treating human UCC cells and is worthy for further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Cycling to Maintain and Improve Fitness: Line-1 Modes of Nuclear Entrance and Retrotransposition.

PubMed

Mita, Paolo; Boeke, Jef D

2018-04-01

The LINE-1/L1 retrotransposon is a transposable element still active in the human genome. Most retrotransposons in the genome are inactive or repressed by several host mechanisms. In specific contexts, active L1 retrotransposons may evade repression and copy themselves into new genomic loci. Despite a general knowledge of the L1 life cycle, little was known about the dynamics of L1 proteins and function during the different stages of the host cell cycle. Our work highlighted a well-orchestrated localization of L1 proteins and mRNA that take advantage of mitotic nuclear membrane breakdown. Once in the nucleus, L1 ribonucleoproteins (RNPs) are able to retrotranspose during the S phase when L1 retrotransposition peaks. Our conclusions highlight previously unappreciated features of the L1 life cycle, such as the differences between cytoplasmic and nuclear RNPs and the cycling of L1 ORF1 protein and L1 activity during progression through the cell cycle. These new observations are discussed here in light of the evolutionary arms race between L1 retrotransposons and the host cell.

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.

Synchronization ability of coupled cell-cycle oscillators in changing environments

PubMed Central

2012-01-01

Background The biochemical oscillator that controls periodic events during the Xenopus embryonic cell cycle is centered on the activity of CDKs, and the cell cycle is driven by a protein circuit that is centered on the cyclin-dependent protein kinase CDK1 and the anaphase-promoting complex (APC). Many studies have been conducted to confirm that the interactions in the cell cycle can produce oscillations and predict behaviors such as synchronization, but much less is known about how the various elaborations and collective behavior of the basic oscillators can affect the robustness of the system. Therefore, in this study, we investigate and model a multi-cell system of the Xenopus embryonic cell cycle oscillators that are coupled through a common complex protein, and then analyze their synchronization ability under four different external stimuli, including a constant input signal, a square-wave periodic signal, a sinusoidal signal and a noise signal. Results Through bifurcation analysis and numerical simulations, we obtain synchronization intervals of the sensitive parameters in the individual oscillator and the coupling parameters in the coupled oscillators. Then, we analyze the effects of these parameters on the synchronization period and amplitude, and find interesting phenomena, e.g., there are two synchronization intervals with activation coefficient in the Hill function of the activated CDK1 that activates the Plk1, and different synchronization intervals have distinct influences on the synchronization period and amplitude. To quantify the speediness and robustness of the synchronization, we use two quantities, the synchronization time and the robustness index, to evaluate the synchronization ability. More interestingly, we find that the coupled system has an optimal signal strength that maximizes the synchronization index under different external stimuli. Simulation results also show that the ability and robustness of the synchronization for the square-wave periodic signal of cyclin synthesis is strongest in comparison to the other three different signals. Conclusions These results suggest that the reaction process in which the activated cyclin-CDK1 activates the Plk1 has a very important influence on the synchronization ability of the coupled system, and the square-wave periodic signal of cyclin synthesis is more conducive to the synchronization and robustness of the coupled cell-cycle oscillators. Our study provides insight into the internal mechanisms of the cell cycle system and helps to generate hypotheses for further research. PMID:23046815

Cell wall mannoprotein of Candida albicans induces cell cycle alternation and inhibits apoptosis of HaCaT cells via NF-κB signal pathway.

PubMed

Han, Yang; Jiang, Hang-Hang; Zhang, Yu-Jing; Hao, Xing-Jia; Sun, Yu-Zhe; Qi, Rui-Qun; Chen, Hong-Duo; Gao, Xing-Hua

2017-10-01

Candida albicans (C. albicans) is a commensal organism in human and a well-known dimorphic opportunistic pathogenic fungus. Though plenty of researches on the pathogenesis of C. albicans have been performed, the mechanism is not fully understood. The cell wall components of C. albicans have been documented to play important roles in its pathogenic processes. To further study the infectious mechanism of C. albicans, we investigated the potential functional role of its cell wall mannoprotein in cell cycle and apoptosis of HaCaT cells. We found that mannoprotein could promote the transition of cell cycle from G1/G0 to S phase, in which Cyclin D1, CDK4 and p-Rb, the major regulators of the cell cycle progression, showed significant upregulation, and CDKN1A (cyclin dependent kinase inhibitor 1A (p21)) showed significant downregulation. Mannoprotein also could inhibit apoptosis of HaCaT cells, which was well associated with increased expression of BCL2 (Bcl-2). Moreover, mannoprotein could increase the phosphorylation levels of RELA (p65) and NFKBIA (IκBα), as the key factors of NF-κB signal pathway in HaCaT cells, suggesting the activation of NF-κB signal pathway. Additionally, a NF-κB specific inhibitor, PDTC, could rescue the effect of mannoprotein on cell cycle and apoptosis of HaCaT cells, which suggested that mannoprotein could activate NF-κB signal pathway to mediate cell cycle alternation and inhibit apoptosis. Copyright © 2017. Published by Elsevier Ltd.

Identification of Novel Saccharomyces cerevisiae Proteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

PubMed Central

Jensen, Torben Heick; Neville, Megan; Rain, Jean Christophe; McCarthy, Terri; Legrain, Pierre; Rosbash, Michael

2000-01-01

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner. PMID:11027275

Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.

PubMed

Mohammadi, Saeed; Seyedhosseini, Fakhri Sadat; Behnampour, Nasser; Yazdani, Yaghoub

2017-10-01

The role of aryl hydrocarbon receptor (AhR) in carcinogenesis has been studied recently. Indole-3-carbinol (I3C) is an AhR agonist and a potential anticancer agent. Here, we investigated the effects of I3C on cell cycle progression and apoptosis through activation of AhR on THP-1 acute myeloid leukemia (AML) cell line. MTT viability assay was used to measure the cytotoxic effects of I3C on THP-1 cells. Apoptosis and cell cycle assays were investigated using flow cytometry. Real time RT-PCR was conducted to measure the alterations in the expression of AhR gene, key genes associated with AhR activation (IL1β and CYP1A1) and major genes involved in cell cycle regulation and apoptosis including P27, P21, CDK2, P53, BCL2 and FasR. Our findings revealed that I3C inhibits the proliferation of THP-1 cells in a dose- and time-dependent manner with minimal toxicity over normal monocytes. The AhR target genes (CYP1A1, IL1β) were overexpressed upon I3C treatment (p < .05 to p < .001). The antiproliferative effects of I3C were in association with programed cell death. I3C downregulated BCL2 and upregulated FasR in THP-1 cells (p < .05 to p < .001). G1 cell cycle arrest was also observed using flow cytometry. G1-acting cell cycle genes (P21, P27 and P53) were overexpressed (p < .05 to p < .001), while CDK2 was downregulated upon I3C treatment (p < .01 to p < .001). I3C could exert its antileukemic effects through AhR activation which is associated with programed cell death and G1 cell cycle arrest in a dose- and time-dependent manner. Therefore, AhR could be targeted as a novel treatment possibility in AML.

Cytostatic response of HepG2 to 0.57 MHz electric currents mediated by changes in cell cycle control proteins.

PubMed

Hernández-Bule, María Luisa; Cid, María Antonia; Trillo, María Angeles; Leal, Jocelyne; Ubeda, Alejandro

2010-12-01

The capacitive-resistive electric transfer (CRet) therapy is a non-invasive technique that applies electrical currents of 0.4-0.6 MHz to the treatment of musculoskeletal injuries. Although this therapy has proved effective in clinical studies, its interaction mechanisms at the cellular level still are insufficiently investigated. Results from previous studies have shown that the application of CRet currents at subthermal doses causes alterations in cell cycle progression and decreased proliferation in hepatocarcinoma (HepG2) and neuroblastoma (NB69) human cell lines. The aim of the present study was to investigate the antiproliferative response of HepG2 to CRet currents. The results showed that 24-h intermittent treatment with 50 µA/mm(2) current density induced in HepG2 statistically significant changes in expression and activation of cell cycle control proteins p27Kip1 and cyclins D1, A and B1. The chronology of these changes is coherent with that of the alterations reported in the cell cycle of HepG2 when exposed to the same electric treatment. We propose that the antiproliferative effect exerted by the electric stimulus would be primarily mediated by changes in the expression and activation of proteins intervening in cell cycle regulation, which are among the targets of emerging chemical therapies. The capability to arrest the cell cycle through electrically-induced changes in cell cycle control proteins might open new possibilities in the field of oncology.

Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana

NASA Technical Reports Server (NTRS)

Velasco, P. J.; Tischner, R.; Huffaker, R. C.; Whitaker, J. R.

1989-01-01

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

Versatile function of the circadian protein CIPC as a regulator of Erk activation

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

Matsunaga, Ryota; Nishino, Tasuku; Yokoyama, Atsushi

2016-01-15

The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase,more » and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.« less

Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override

PubMed Central

Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

2014-01-01

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25. PMID:24666325

Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

PubMed Central

Arango, Daniel; Parihar, Arti; Villamena, Frederick A.; Wang, Liwen; Freitas, Michael A.; Grotewold, Erich; Doseff, Andrea I.

2014-01-01

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid. PMID:22985621

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.

Chemopreventive activity of ellagitannins and their derivatives from black raspberry seeds on HT-29 colon cancer cells.

PubMed

Cho, Hyunnho; Jung, Hana; Lee, Heejae; Yi, Hae Chang; Kwak, Ho-kyung; Hwang, Keum Taek

2015-05-01

Black raspberry (BRB) seeds are a major waste product after fruit processing. The seeds are abundant in ellagitannins (ET), a class of hydrolysable tannins, which are hydrolyzed to ellagic acid (EA) and further metabolized to urolithin A (UA) and urolithin B (UB), known to be bioavailable in the colon and the prostate. In this study, the anti-cancer activities of these compounds were evaluated on HT-29 colon cancer cells. ET, EA, UA and UB inhibited the proliferation of the cancer cells. EA caused a slight, but significant cell cycle arrest at the G1 phase, and urolithins caused cell cycle arrest at the G2/M phase and upregulated p21 expression. Apoptotic cells were detected by Annexin V-FITC/PI assay when treated with the compounds. Disruption in mitochondrial membrane potential and activation of caspases 8 and 9 suggest that both extrinsic and intrinsic apoptotic pathways may be involved. Activation of caspase 3 and cleavage of PARP further confirmed the induction of the apoptosis. ET, EA, UA and UB showed anti-cancer activity by arresting the cell cycle and inducing apoptosis on HT-29 human colon cancer cells. This study suggests that the BRB seeds could be a potential source of anti-cancer ET.

Chromatin Structure and the Cell Cycle

PubMed Central

Pederson, Thoru

1972-01-01

Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G1, S, and G2 phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing, subconfluent cultures. These cell cycle-dependent chromatin changes were observed consistently at all enzyme concentrations (5000-fold range) and incubation times (15 min-2 hr) tested. The results indicate that the degree of complexing between DNA and chromosomal proteins changes during interphase, and they suggest that the chromosome coiling cycle of visible mitosis may extend in more subtle form over the entire cell cycle. PMID:4626402

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

PubMed

Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

2006-05-01

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

TGFβ lengthens the G1 phase of stem cells in aged mouse brain.

PubMed

Daynac, Mathieu; Pineda, Jose R; Chicheportiche, Alexandra; Gauthier, Laurent R; Morizur, Lise; Boussin, François D; Mouthon, Marc-André

2014-12-01

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique, we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition, we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells, but not in transit-amplifying cells, and directly impacts on neurogenesis. Finally, we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. © 2014 AlphaMed Press.

Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures.

PubMed Central

Stoop, JMH.; Pharr, D. M.

1993-01-01

Little information exists concerning the biochemical route of mannitol catabolism in higher plant cells. In this study, the role of a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery (Apium graveolens L. var dulce [Mill.] Pers.) were successfully grown on nutrient media with either mannitol, mannose, or sucrose as the sole carbon source. Cell cultures grown on any of the three carbon sources did not differ in relative growth rate, as measured by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had high concentrations of sucrose early in the growth cycle and contained a substantial hexose pool. Mannose-grown cells had a high mannose concentration early in the cycle, which decreased during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle. Celery suspension cultures on all three carbon substrates contained an NAD-dependent MDH. Throughout the growth cycle, MDH activity was 2- to 4-fold higher in mannitol-grown cells compared with sucrose- or mannose-grown cells, which did not contain detectable levels of mannitol, indicating that MDH functions pre-dominantly in an oxidative capacity in situ. The MDH activity observed in celery cells was 3-fold higher than the minimum amount required to account for the observed rate of mannitol utilization from the media. Cultures transferred from mannitol to mannose underwent a decrease in MDH activity over a period of days, and transfer from mannose to mannitol resulted in an increase in MDH activity. These data provide strong evidence that MDH plays an important role in mannitol utilization in celery suspension cultures. PMID:12231996

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

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

PubMed

Lange, S; Viergutz, T; Simkó, M

2004-10-01

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining.

PubMed

Montijn, M B; ten Hoopen, R; Fransz, P F; Oud, J L; Nanninga, N

1998-05-01

The cell cycle-dependent spatial position, morphology and activity of the four nucleolar organising regions (NORs) of the Petunia hybrida cultivar Mitchell and the inbred line V26 have been analysed. Application of the silver staining technique and fluorescence in situ hybridisation on fixed root-tip material revealed that these interspecific hybrids possess four NORs of which only those of chromosome 2 are active during interphase, which implies that the NOR activity is not of parental origin. However, at the end of mitosis, activity of all NOR regions could be detected, suggesting that the high demand for ribosomes at this stage of the cell cycle requires temporal activity of all NORs. Using actin DNA probes as markers in fluorescence in situ hybridisation experiments enabled the identification of the individual petunia chromosomes.

The mechanistic effects of the dioxonaphthoimidazolium analog YM155 in renal cell carcinoma cell cycling and apoptosis.

PubMed

Sim, Mei Yi; Go, Mei Lin; Yuen, John Shyi Peng

2018-06-15

To investigate the effect of dioxonaphthoimidazolium analog YM155 on cell cycle progression of the clear-cell variant of renal cell carcinoma (ccRCC). Cell cycle analysis was performed using bromodeoxyuridine (BrdU) and PI, apoptosis initiation was monitored using Annexin V and proteins expression was determined using western immunoblotting. Here, we showed that YM155 activated stress-related molecules (histone H2AX, checkpoint kinases Chk1 and Chk2, p53) that mediate DNA damage checkpoint responses. The coordinated activation of these effector molecules disrupts progression of the cell cycle at the S phase as deduced from BrdU pulsing experiments and the ensuing changes in the levels of proteins (cyclins, CDKs, CDK inhibitors, phosphatases) that control cell cycle progression. Notably, we found increases in cyclin E and Cdc2 which regulate transition of cells from G1 to S, even as losses were observed for other CDKs and their cyclin partners. Furthermore, by inducing a loss in total pRb possibly by promoting its degradation, YM155 promoted the E2F transcription of genes that regulate entry into the S phase. After 24 h, cell cycle arrest to repair YM155-inflicted DNA damage was overtaken by p53-mediated apoptosis. YM155 induced increases in pro-apoptotic proteins (Bax and Bad), diminished anti-apoptotic proteins (Mcl-1, Bcl-xl, XIAP, survivin) and initiated cleavage of apoptotic marker proteins caspase 3 and PARP. Taken together, the added insight provided on the cell cycle perturbative effects of YM155 may assist clinicians in framing rational choices for combining YM155 with other anti-cancer drugs or treatment modalities in ccRCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

PubMed Central

Wang, Lintao; Peng, Yanyan; Shi, Kaikai; Wang, Haixiao; Lu, Jianlei; Li, Yanli; Ma, Changyan

2015-01-01

Abstract Recent studies have revealed that osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, a traditional Chinese medicine, possesses anticancer activity. However, its effect on breast cancer cells so far has not been elucidated clearly. In the present study, we evaluated the effects of osthole on the proliferation, cell cycle and apoptosis of human breast cancer cells MDA-MB 435. We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells, The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole, as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation. The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression. Were observed taken together, these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer. PMID:25859268

Cell cycle progression is regulated by intertwined redox oscillators.

PubMed

da Veiga Moreira, Jorgelindo; Peres, Sabine; Steyaert, Jean-Marc; Bigan, Erwan; Paulevé, Loïc; Nogueira, Marcel Levy; Schwartz, Laurent

2015-05-29

The different phases of the eukaryotic cell cycle are exceptionally well-preserved phenomena. DNA decompaction, RNA and protein synthesis (in late G1 phase) followed by DNA replication (in S phase) and lipid synthesis (in G2 phase) occur after resting cells (in G0) are committed to proliferate. The G1 phase of the cell cycle is characterized by an increase in the glycolytic metabolism, sustained by high NAD+/NADH ratio. A transient cytosolic acidification occurs, probably due to lactic acid synthesis or ATP hydrolysis, followed by cytosolic alkalinization. A hyperpolarized transmembrane potential is also observed, as result of sodium/potassium pump (NaK-ATPase) activity. During progression of the cell cycle, the Pentose Phosphate Pathway (PPP) is activated by increased NADP+/NADPH ratio, converting glucose 6-phosphate to nucleotide precursors. Then, nucleic acid synthesis and DNA replication occur in S phase. Along with S phase, unpublished results show a cytosolic acidification, probably the result of glutaminolysis occurring during this phase. In G2 phase there is a decrease in NADPH concentration (used for membrane lipid synthesis) and a cytoplasmic alkalinization occurs. Mitochondria hyperfusion matches the cytosolic acidification at late G1/S transition and then triggers ATP synthesis by oxidative phosphorylation. We hypothesize here that the cytosolic pH may coordinate mitochondrial activity and thus the different redox cycles, which in turn control the cell metabolism.

Repeated B cell depletion in treatment of refractory systemic lupus erythematosus

PubMed Central

Ng, K P; Leandro, M J; Edwards, J C; Ehrenstein, M R; Cambridge, G; Isenberg, D A

2006-01-01

Objectives To report the clinical outcome and safety profile of repeated B cell depletion in seven patients with refractory systemic lupus erythematosus (SLE). Methods Since June 2000, seven patients with refractory SLE had repeated cycles of B cell depletion (18 cycles in total, up to three cycles per patient) because of disease relapse. The clinical response (assessed by the British Isles Lupus Activity Guide (BILAG) activity index), duration of B cell depletion, and adverse events in these patients was reviewed. Results Four patients (Nos 1, 2, 3, 6) had three cycles of treatment and three (Nos 4, 5, 7) had two cycles. Four of the seven patients (Nos 1, 3, 5, 6) improved. The mean global BILAG scores dropped from 15 to 6 at 5–7 months. The median duration of clinical response and B cell depletion was 13 months and 6 months, respectively. After the third cycle, 2/4 patients (Nos 1 and 2) improved. The median duration of clinical benefit was 12 months. Most patients tolerate re‐treatment very well. Conclusion Re‐treatment with B cell depletion of patients with severe SLE is safe and may be effective for 6–12 months on average. PMID:16269424

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

PubMed Central

Matusali, Giulia; Arena, Giuseppe; De Leo, Alessandra; Di Renzo, Livia; Mattia, Elena

2009-01-01

Background EBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ), sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitt's lymphoma (BL) cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents. Results Our data show that, following EBV activation, the latent membrane protein 1 (LMP1) and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU)2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression. Conclusion These findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells. PMID:19272151

Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells.

PubMed

Kito, Hiroaki; Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto; Ohya, Susumu; Asai, Kiyofumi; Imaizumi, Yuji

2015-04-10

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Foxi3 deficiency compromises hair follicle stem cell specification and activation

PubMed Central

Shirokova, Vera; Biggs, Leah C.; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K.; Mikkola, Marja L.

2017-01-01

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ. Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary hair germ marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary hair germ activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. PMID:26992132

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

PubMed Central

Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; Whitfield, Michael L.

2012-01-01

We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. PMID:22740631

Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

PubMed

Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

Temperature oscillations drive cycles in the activity of MMP-2,9 secreted by a human trabecular meshwork cell line.

PubMed

Li, Stanley Ka-Lok; Banerjee, Juni; Jang, Christopher; Sehgal, Amita; Stone, Richard A; Civan, Mortimer M

2015-02-05

Aqueous humor inflow falls 50% during sleeping hours without proportional fall in IOP, partly reflecting reduced outflow facility. The mechanisms underlying outflow facility cycling are unknown. One outflow facility regulator is matrix metalloproteinase (MMP) release from trabecular meshwork (TM) cells. Because anterior segment temperature must oscillate due to core temperature cycling and eyelid closure during sleep, we tested whether physiologically relevant temperature oscillations drive cycles in the activity of secreted MMP. Temperature of transformed normal human TM cells (hTM5 line) was fixed or alternated 12 hours/12 hours between 33°C and 37°C. Activity of secreted MMP-2 and MMP-9 was measured by zymography, and gene expression by RT-PCR and quantitative PCR. Raising temperature to 37°C increased, and lowering to 33°C reduced, activity of secreted MMP. Switching between 37°C and 33°C altered MMP-9 by 40% ± 3% and MMP-2 by 22% ± 2%. Peripheral circadian clocks did not mediate temperature-driven cycling of MMP secretion because MMP-release oscillations did not persist at constant temperature after 3 to 6 days of alternating temperatures, and temperature cycles did not entrain clock-gene expression in these cells. Furthermore, inhibiting heat shock transcription factor 1, which links temperature and peripheral clock-gene oscillations, inhibited MMP-9 but not MMP-2 temperature-driven MMP cycling. Inhibition of heat-sensitive TRPV1 channels altered total MMP secretion but not temperature-induced modulations. Inhibiting cold-sensitive TRPM-8 channels had no effect. Physiologically relevant temperature oscillations drive fluctuations of secreted MMP-2 and MMP-9 activity in hTM5 cells independent of peripheral clock genes and temperature-sensitive TRP channels. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

Aqueous lithium air batteries

DOEpatents

Visco, Steven J.; Nimon, Yevgeniy S.; De Jonghe, Lutgard C.; Petrov, Alexei; Goncharenko, Nikolay

2017-05-23

Aqueous Li/Air secondary battery cells are configurable to achieve high energy density and prolonged cycle life. The cells include a protected a lithium metal or alloy anode and an aqueous catholyte in a cathode compartment. The aqueous catholyte comprises an evaporative-loss resistant and/or polyprotic active compound or active agent that partakes in the discharge reaction and effectuates cathode capacity for discharge in the acidic region. This leads to improved performance including one or more of increased specific energy, improved stability on open circuit, and prolonged cycle life, as well as various methods, including a method of operating an aqueous Li/Air cell to simultaneously achieve improved energy density and prolonged cycle life.

Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

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

Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O., E-mail: pustylnyak@ngs.ru

Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p′-DDT (85%) and o,p′-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CARmore » and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. - Highlights: • DDT activated both CAR and ERα and their cell cycle and apoptosis target genes. • DDT produced increases in cell cycle and anti-apoptosis proteins and decrease in p53. • DDT mixture was unable to stimulate the β-catenin signalling pathway in mouse livers.« less

DREAMs make plant cells to cycle or to become quiescent.

PubMed

Magyar, Zoltán; Bögre, László; Ito, Masaki

2016-12-01

Cell cycle phase specific oscillation of gene transcription has long been recognized as an underlying principle for ordered processes during cell proliferation. The G1/S-specific and G2/M-specific cohorts of genes in plants are regulated by the E2F and the MYB3R transcription factors. Mutant analysis suggests that activator E2F functions might not be fully required for cell cycle entry. In contrast, the two activator-type MYB3Rs are part of positive feedback loops to drive the burst of mitotic gene expression, which is necessary at least to accomplish cytokinesis. Repressor MYB3Rs act outside the mitotic time window during cell cycle progression, and are important for the shutdown of mitotic genes to impose quiescence in mature organs. The two distinct classes of E2Fs and MYB3Rs together with the RETINOBLATOMA RELATED are part of multiprotein complexes that may be evolutionary related to what is known as DREAM complex in animals. In plants, there are multiple such complexes with distinct compositions and functions that may be involved in the coordinated cell cycle and developmental regulation of E2F targets and mitotic genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

PubMed Central

Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

2012-01-01

SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

Towards Predicting the Response of a Solid Tumour to Chemotherapy and Radiotherapy Treatments: Clinical Insights from a Computational Model

PubMed Central

Powathil, Gibin G.; Adamson, Douglas J. A.; Chaplain, Mark A. J.

2013-01-01

In this paper we use a hybrid multiscale mathematical model that incorporates both individual cell behaviour through the cell-cycle and the effects of the changing microenvironment through oxygen dynamics to study the multiple effects of radiation therapy. The oxygenation status of the cells is considered as one of the important prognostic markers for determining radiation therapy, as hypoxic cells are less radiosensitive. Another factor that critically affects radiation sensitivity is cell-cycle regulation. The effects of radiation therapy are included in the model using a modified linear quadratic model for the radiation damage, incorporating the effects of hypoxia and cell-cycle in determining the cell-cycle phase-specific radiosensitivity. Furthermore, after irradiation, an individual cell's cell-cycle dynamics are intrinsically modified through the activation of pathways responsible for repair mechanisms, often resulting in a delay/arrest in the cell-cycle. The model is then used to study various combinations of multiple doses of cell-cycle dependent chemotherapies and radiation therapy, as radiation may work better by the partial synchronisation of cells in the most radiosensitive phase of the cell-cycle. Moreover, using this multi-scale model, we investigate the optimum sequencing and scheduling of these multi-modality treatments, and the impact of internal and external heterogeneity on the spatio-temporal patterning of the distribution of tumour cells and their response to different treatment schedules. PMID:23874170

A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells.

PubMed

Schorpp, Kenji; Rothenaigner, Ina; Maier, Julia; Traenkle, Bjoern; Rothbauer, Ulrich; Hadian, Kamyar

2016-10-01

Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. © 2016 Society for Laboratory Automation and Screening.

TORC1 and class I HDAC inhibitors synergize to suppress mature B cell neoplasms.

PubMed

Simmons, John K; Patel, Jyoti; Michalowski, Aleksandra; Zhang, Shuling; Wei, Bih-Rong; Sullivan, Patrick; Gamache, Ben; Felsenstein, Kenneth; Kuehl, W Michael; Simpson, R Mark; Zingone, Adriana; Landgren, Ola; Mock, Beverly A

2014-03-01

Enhanced proliferative signaling and loss of cell cycle regulation are essential for cancer progression. Increased mitogenic signaling through activation of the mTOR pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of lymphoid malignancies, including plasmacytoma (PCT), multiple myeloma (MM), Burkitt's lymphoma (BL), and mantle cell lymphoma (MCL). Here we evaluate the synergy of pharmacologically affecting both of these critical pathways using the mTOR inhibitor sirolimus and the histone deacetylase inhibitor entinostat. A dose-matrix screening approach found this combination to be highly active and synergistic in a panel of genetically diverse human MM cell lines. Synergy and activity was observed in mouse PCT and human BL and MCL cell lines tested in vitro, as well as in freshly isolated primary MM patient samples tested ex vivo. This combination had minimal effects on healthy donor cells and retained activity when tested in a co-culture system simulating the protective interaction of cancer cells with the tumor microenvironment. Combining sirolimus with entinostat enhanced cell cycle arrest and apoptosis. At the molecular level, entinostat increased the expression of cell cycle negative regulators including CDKN1A (p21) and CDKN2A (p16), while the combination decreased critical growth and survival effectors including Cyclin D, BCL-XL, BIRC5, and activated MAPK. Published by Elsevier B.V.

Ubiquitin specific protease 2 acts as a key modulator for the regulation of cell cycle by adiponectin and leptin in cancer cells.

PubMed

Nepal, Saroj; Shrestha, Anup; Park, Pil-Hoon

2015-09-05

Adiponectin and leptin, both produced from adipose tissue, cause cell cycle arrest and progression, respectively in cancer cells. Ubiquitin specific protease-2 (USP-2), a deubiquitinating enzyme, is known to impair proteasome-induced degradation of cyclin D1, a critical cell cycle regulator. Herein, we investigated the effects of these adipokines on USP-2 expression and its potential role in the modulation of cell cycle. Treatment with globular adiponectin (gAcrp) decreased, whereas leptin increased USP-2 expression both in human hepatoma and breast cancer cells. In addition, overexpression or gene silencing of USP-2 affected cyclin D1 expression and cell cycle progression/arrest by adipokines. Adiponectin and leptin also modulated in vitro proteasomal activity, which was partially dependent on USP-2 expression. Taken together, our results reveal that modulation of USP-2 expression plays a crucial role in cell cycle regulation by adipokines. Thus, USP-2 would be a promising therapeutic target for the modulation of cancer cell growth by adipokines. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms.

PubMed

Wu, Jung-Ju; Omar, Hany A; Lee, Ying-Ray; Teng, Yen-Ni; Chen, Pin-Shern; Chen, Yu-Chung; Huang, Hsiao-Shan; Lee, Kuan-Han; Hung, Jui-Hsiang

2015-09-05

Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5' AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaol's clinical promise as a potential candidate for HCC therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

PubMed Central

Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

2015-01-01

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

Nucleosome architecture throughout the cell cycle

PubMed Central

Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

2016-01-01

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620

Watch Out for the "Living Dead": Cell-Free Enzymes and Their Fate.

PubMed

Baltar, Federico

2017-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the "gatekeepers" of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell's fate. In contrast, cell-free enzymes belong to a kind of "living dead" realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go "beyond the living things," studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

PubMed Central

Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2014-01-01

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

PubMed

Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

2008-03-01

Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

Beat-to-Beat Variation in Periodicity of Local Calcium Releases Contributes to Intrinsic Variations of Spontaneous Cycle Length in Isolated Single Sinoatrial Node Cells

PubMed Central

Monfredi, Oliver; Maltseva, Larissa A.; Spurgeon, Harold A.; Boyett, Mark R.; Lakatta, Edward G.; Maltsev, Victor A.

2013-01-01

Spontaneous, submembrane local Ca2+ releases (LCRs) generated by the sarcoplasmic reticulum in sinoatrial nodal cells, the cells of the primary cardiac pacemaker, activate inward Na+/Ca2+-exchange current to accelerate the diastolic depolarization rate, and therefore to impact on cycle length. Since LCRs are generated by Ca2+ release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence. Aim The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells. Methods We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp. Results LCRs begin to occur on the descending part of the action potential-induced whole-cell Ca2+ transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential (mean 54±7.7 ms, n = 14), the ensemble of waxing LCR activity converts the decay of the global Ca2+ transient into a rise, resulting in a late, whole-cell diastolic Ca2+ elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca2+ transient peak to an LCR’s subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length. Conclusion Intrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle. PMID:23826247

Geraniol and geranyl acetate induce potent anticancer effects in colon cancer Colo-205 cells by inducing apoptosis, DNA damage and cell cycle arrest.

PubMed

Qi, Fei; Yan, Qiang; Zheng, Zhaozheng; Liu, Jian; Chen, Yan; Zhang, Guiyang

2018-01-01

Colon cancer ranks second in mortality among all human malignancies, creating thus a need for exploration of novel molecules that would prove effective, cost-effective and with lower toxicity. In the recent past monoterpenes have gained tremendous attention for their anticancer activity. In the present study we evaluated the anticancer effects of two important monoterpenes, geraniol and geranyl acetate against colo-205 cancer cells. The antiproliferative activity was determined by MTT assay. Apoptosis was assessed by DAPI staining and DNA damage was checked by comet assay. The cell cycle analysis was carried out by flow cytometry and protein expression was examined by western blotting. The results showed that both geraniol and geranyl acetate exhibited significant anticancer activity against colo-205 cancer cell line with IC50 values of 20 and 30 μM respectively. To find out the underlying mechanism, DAPI staining was carried out and it was observed that both the monoterpenes, geraniol and geranyl acetate, induced apoptosis in colo-205 cells. The apoptosis was also associated with upregulation of Bax and downregulation of Bcl-2 expressions, indicative of mitochondrial apoptosis. Moreover, these two monoterpenes could trigger DNA damage and G2/M cell cycle arrest in colo-205 cells. Taken together, we propose that geraniol and geranyl acetate may prove to be important lead molecular candidates for the treatment of colon cancer. Their anticancer activity can be attributed to the ability to trigger apoptosis, DNA damage and cell cycle arrest.

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

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

Ji, Shuai

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

Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

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

Kito, Hiroaki; Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto; Yamamura, Hisao

2015-04-10

Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cyclemore » progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.« less

The prohibitin-repressive interaction with E2F1 is rapidly inhibited by androgen signalling in prostate cancer cells

PubMed Central

Koushyar, S; Economides, G; Zaat, S; Jiang, W; Bevan, C L; Dart, D A

2017-01-01

Prohibitin (PHB) is a tumour suppressor molecule with pleiotropic activities across several cellular compartments including mitochondria, cell membrane and the nucleus. PHB and the steroid-activated androgen receptor (AR) have an interplay where AR downregulates PHB, and PHB represses AR. Additionally, their cellular locations and chromatin interactions are in dynamic opposition. We investigated the mechanisms of cell cycle inhibition by PHB and how this is modulated by AR in prostate cancer. Using a prostate cancer cell line overexpressing PHB, we analysed the gene expression changes associated with PHB-mediated cell cycle arrest. Over 1000 gene expression changes were found to be significant and gene ontology analysis confirmed PHB-mediated repression of genes essential for DNA replication and synthesis, for example, MCMs and TK1, via an E2F1 regulated pathway—agreeing with its G1/S cell cycle arrest activity. PHB is known to inhibit E2F1-mediated transcription, and the PHB:E2F1 interaction was seen in LNCaP nuclear extracts, which was then reduced by androgen treatment. Upon two-dimensional western blot analysis, the PHB protein itself showed androgen-mediated charge differentiation (only in AR-positive cells), indicating a potential dephosphorylation event. Kinexus phosphoprotein array analysis indicated that Src kinase was the main interacting intracellular signalling hub in androgen-treated LNCaP cells, and that Src inhibition could reduce this AR-mediated charge differentiation. PHB charge change may be associated with rapid dissociation from chromatin and E2F1, allowing the cell cycle to proceed. The AR and androgens may deactivate the repressive functions of PHB upon E2F1 leading to cell cycle progression, and indicates a role for AR in DNA replication licensing. PMID:28504694

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

PubMed

Chandler-Brown, Devon; Schmoller, Kurt M; Winetraub, Yonatan; Skotheim, Jan M

2017-09-25

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth. Copyright © 2017 Elsevier Ltd. All rights reserved.

Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway.

PubMed

Shen, Ziying; Ma, Yunqing; Ji, Zhonghao; Hao, Yang; Yan, Xuan; Zhong, Yuan; Tang, Xiaochun; Ren, Wenzhi

2018-02-09

Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. AA exposure reduced the viability and number of macrophages in a dose- and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose- and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.

New evidence for antioxidant properties of vitamin C.

PubMed

Vojdani, A; Bazargan, M; Vojdani, E; Wright, J

2000-01-01

This study was designed to examine the effect of 500 to 5,000 mg of ascorbic acid on DNA adducts, natural killer (NK) cell activity, programmed cell death, and cell cycle analysis of human peripheral blood leukocytes. According to our hypothesis, if ascorbic acid is a pro-oxidant, doses between 500 and 5,000 mg should enhance DNA adduct formation, decrease immune function, change the cell cycle progression, and increase the rate of apoptosis. Twenty healthy volunteers were divided into four groups and given either placebo or daily doses of 500, 1,000 or 5,000 mg of ascorbic acid for a period of 2 weeks. On days 0, 1, 7, 15, and 21, blood was drawn from them, and the leukocytes were separated and examined for intracellular levels of ascorbic acid, the level of 8-hydroxyguanosine, NK cell activity, cell cycle progression, and apoptosis. Depending on the subjects, between a 0% and a 40% increase in cellular absorption of ascorbic acid was observed when daily doses of 500 mg were used. At doses greater than 500 mg, this cellular absorption was not increased further, and all doses produced equivalent increases in ascorbic acid on days 1 to 15. This increase in cellular concentration of ascorbic acid resulted in no statistically meaningful changes in the level of 8-hydroxyguanosine, increased NK cytotoxic activity, a reduced percentage of cells undergoing apoptosis, and switched cell cycle phases from S and G2/M to G0/G1. After a period of 1 week, with no placebo or vitamin washout, ascorbic acid levels along with functional assays returned to the baseline and became equivalent to placebos. In comparison with baseline values, no change (not more than daily assays variation) was seen in ascorbate concentrations or other assays during oral placebo treatment. We concluded that ascorbic acid is an antioxidant and that doses up to 5,000 mg neither induce mutagenic lesions nor have negative effects on NK cell activity, apoptosis, or cell cycle.

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

Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes.

PubMed

Lacroix, Isabelle; Lipcey, Carol; Imbert, Jean; Kahn-Perlès, Brigitte

2002-03-15

We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

CARMA3 is overexpressed in colon cancer and regulates NF-{kappa}B activity and cyclin D1 expression

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

Miao, Zhifeng; Zhao, Tingting; Wang, Zhenning

2012-09-07

Highlights: Black-Right-Pointing-Pointer CARMA3 expression is elevated in colon cancers. Black-Right-Pointing-Pointer CARMA3 promotes proliferation and cell cycle progression in colon cancer cells. Black-Right-Pointing-Pointer CARMA3 upregulates cyclinD1 through NF-{kappa}B activation. -- Abstract: CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression andmore » TNM stage (p = 0.0383), lymph node metastasis (p = 0.0091) and Ki67 proliferation index (p = 0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-I{kappa}B levels and NF-{kappa}B activity and its overexpression increased p-I{kappa}B expression and NF-{kappa}B activity. NF-{kappa}B inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-{kappa}B mediated upregulation of cyclin D1.« less

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

Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells.

PubMed

Resnick, M S; Kang, B S; Luu, D; Wickham, J T; Sando, J J; Hahn, C S

1998-10-16

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.

Cell cycle in egg cell and its progression during zygotic development in rice.

PubMed

Sukawa, Yumiko; Okamoto, Takashi

2018-03-01

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.

Capacity loss on storage and possible capacity recovery for HST nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Lowery, John E.

1992-01-01

Negatively precharged nickel hydrogen cells will experience a useable capacity loss during extended open circuit storage periods. Some of the lost capacity can be recovered through cycling. Capacity recovery through cycling can be enhanced by cycling at high depths of discharge (DOD). The most timely procedure for recovering the faded capacity is to charge the cell fully and allow the cell to sit open-circuit at room temperature. This procedure seems to be effective in part because of the enlarged structure of the active materials. The compounds that formed during storage at the low electrode potentials can more easily dissolve and redistribute. All of the original capacity cannot be recovered because the lattice structure of the active material is irreversibly altered during storage. The recommendation is to use positively precharged cells activated with 26 percent KOH if possible. In aerospace applications, the benefits of negative precharge are offset by the possibility of delays and storage periods.

Annexin V-induced rat Leydig cell proliferation involves Ect2 via RhoA/ROCK signaling pathway.

PubMed

Jing, Jun; Chen, Li; Fu, Hai-Yan; Fan, Kai; Yao, Qi; Ge, Yi-Feng; Lu, Jin-Chun; Yao, Bing

2015-03-24

This study investigated the effect of annexin V on the proliferation of primary rat Leydig cells and the potential mechanism. Our results showed that annexin V promoted rat Leydig cell proliferation and cell cycle progression in a dose- and time-dependent manner. Increased level of annexin V also enhanced Ect2 protein expression. However, siRNA knockdown of Ect2 attenuated annexin V-induced proliferation of rat Leydig cells. Taken together, these data suggest that increased level of annexin V induced rat Leydig cell proliferation and cell cycle progression via Ect2. Since RhoA activity was increased following Ect2 activation, we further investigated whether Ect2 was involved in annexin V-induced proliferation via the RhoA/ROCK pathway, and the results showed that annexin V increased RhoA activity too, and this effect was abolished by the knockdown of Ect2. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated annexin V-induced proliferation and cell cycle progression. We thus conclude that Ect2 is involved in annexin V-induced rat Leydig cell proliferation through the RhoA/ROCK pathway.

The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle

PubMed Central

Samuel, Temesgen; Fadlalla, Khalda; Turner, Timothy; Yehualaeshet, Teshome E.

2010-01-01

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose dependent manner, 12.5-50μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 hours. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that while long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M. PMID:21058190

The cell-cycle interactome: a source of growth regulators?

PubMed

Blomme, Jonas; Inzé, Dirk; Gonzalez, Nathalie

2014-06-01

When plants develop, cell proliferation and cell expansion are tightly controlled in order to generate organs with a determinate final size such as leaves. Several studies have demonstrated the importance of the cell proliferation phase for leaf growth, illustrating that cell-cycle regulation is crucial for correct leaf development. A large and complex set of interacting proteins that constitute the cell-cycle interactome controls the transition from one cell-cycle phase to another. Here, we review the current knowledge on cell-cycle regulators from this interactome affecting final leaf size when their expression is altered, mainly in Arabidopsis. In addition to the description of mutants of CYCLIN-DEPENDENT KINASES (CDKs), CYCLINS (CYCs), and their transcriptional and post-translational regulators, a phenotypic analysis of gain- and loss-of-function mutants for 27 genes encoding proteins that interact with cell-cycle proteins is presented. This compilation of information shows that when cell-cycle-related genes are mis-expressed, leaf growth is often altered and that, seemingly, three main trends appear to be crucial in the regulation of final organ size by cell-cycle-related genes: (i) cellular compensation; (ii) gene dosage; and (iii) correct transition through the G2/M phase by ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) activation. In conclusion, this meta-analysis shows that the cell-cycle interactome is enriched in leaf growth regulators, and illustrates the potential to identify new leaf growth regulators among putative new cell-cycle regulators. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

PubMed Central

Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

2007-01-01

Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

Enhanced performance of Zn(II)-doped lead-acid batteries with electrochemical active carbon in negative mass

NASA Astrophysics Data System (ADS)

Xiang, Jiayuan; Hu, Chen; Chen, Liying; Zhang, Dong; Ding, Ping; Chen, Dong; Liu, Hao; Chen, Jian; Wu, Xianzhang; Lai, Xiaokang

2016-10-01

The effect and mechanism of Zn(II) on improving the performances of lead-acid cell with electrochemical active carbon (EAC) in negative mass is investigated. The hydrogen evolution of the cell is significantly reduced due to the deposition of Zn on carbon surface and the increased porosity of negative mass. Zn(II) additives can also improve the low-temperature and high-rate capacities of the cell with EAC in negative mass, which ascribes to the formation of Zn on lead and carbon surface that constructs a conductive bridge among the active mass. Under the co-contribution of EAC and Zn(II), the partial-state-of-charge cycle life is greatly prolonged. EAC optimizes the NAM structure and porosity to enhance the charge acceptance and retard the lead sulfate accumulation. Zn(II) additive reduces the hydrogen evolution during charge process and improves the electric conductivity of the negative electrode. The cell with 0.6 wt% EAC and 0.006 wt% ZnO in negative mass exhibits 90% reversible capacity of the initial capacity after 2100 cycles. In contrast, the cell with 0.6 wt% EAC exhibits 84% reversible capacity after 2100 cycles and the control cell with no EAC and Zn(II) exhibits less than 80% reversible capacity after 1350 cycles.

Activation of BRCA1/BRCA2-Associated Helicase BACH1 Is Required for Timely Progression through S Phase▿

PubMed Central

Kumaraswamy, Easwari; Shiekhattar, Ramin

2007-01-01

BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G1 phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability. PMID:17664283

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

Exogenous hydrogen sulfide exerts proliferation, anti-apoptosis, migration effects and accelerates cell cycle progression in multiple myeloma cells via activating the Akt pathway.

PubMed

Zheng, Dong; Chen, Ziang; Chen, Jingfu; Zhuang, Xiaomin; Feng, Jianqiang; Li, Juan

2016-10-01

Hydrogen sulfide (H2S), regarded as the third gaseous transmitter, mediates and induces various biological effects. The present study investigated the effects of H2S on multiple myeloma cell progression via amplifying the activation of Akt pathway in multiple myeloma cells. The level of H2S produced in multiple myeloma (MM) patients and healthy subjects was measured using enzyme-linked immunosorbent assay (ELISA). MM cells were treated with 500 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated-Akt (p-Akt), Bcl-2 and caspase-3 were measured by western blot assay. Cell viability was detected by Cell Counting Kit 8 (CCK-8). The cell cycle was analyzed by flow cytometry. Our results show that the concentration of H2S was higher in MM patients and that it increased in parallel with disease progression. Treating MM cells with 500 µmol/l NaHS for 24 h markedly increased the expression level of Bcl-2 and the activation of p-Akt, however, the expression level of caspase-3 was decreased, cell viability was increased, and cell cycle progression was accelerated in MM cells. NaHS also induced migration in MM cells in transwell migration assay. Furthermore, co-treatment of MM cells with 500 µmol/l NaHS and 50 µmol/l LY294002 for 24 h significantly overset these effects. In conclusion, our findings demonstrate that the Akt pathway contributes to NaHS-induced cell proliferation, migration and acceleration of cell cycle progression in MM cells.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

PubMed

Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M

2004-11-01

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

A Transient Expression of Prospero Promotes Cell Cycle Exit of Drosophila Postembryonic Neurons through the Regulation of Dacapo

PubMed Central

Colonques, Jordi; Ceron, Julian; Reichert, Heinrich; Tejedor, Francisco J.

2011-01-01

Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor PROSPERO is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that PROSPERO is not expressed in postmitotic neuronal progeny. Thus, PROSPERO is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. Here we focus on postembryonic neurogenesis and show that the expression of PROSPERO is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, we provide evidence that this expression of PROSPERO in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) DACAPO. These findings imply that PROSPERO, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development. PMID:21552484

Vorinostat, a histone deacetylase (HDAC) inhibitor, promotes cell cycle arrest and re-sensitizes rituximab- and chemo-resistant lymphoma cells to chemotherapy agents.

PubMed

Xue, Kai; Gu, Juan J; Zhang, Qunling; Mavis, Cory; Hernandez-Ilizaliturri, Francisco J; Czuczman, Myron S; Guo, Ye

2016-02-01

Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab-chemotherapy-resistant preclinical models. A panel of rituximab-chemotherapy-sensitive (RSCL) and rituximab-chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were evaluated by Western blotting. Finally, cell lines were pre-exposed to vorinostat for 48 h and subsequently exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine); changes in cell viability were determined by CellTiter-Glo(®) luminescence assay (Promega, Fitchburg, WI), and synergistic activity was evaluated using the CalcuSyn software. Vorinostat induced dose-dependent cell death in RRCL and in primary tumor cells. In addition, in vitro exposure of RRCL to vorinostat resulted in an increase in p21 and acetylation of histone H3 leading to G1 cell cycle arrest. Vorinostat exposure resulted in apoptosis in RSCL cell lines but not in RRCL. This finding suggests that in RRCL, vorinostat induces cell death by alternative pathways (i.e., irreversible cell cycle arrest). Of interest, vorinostat was found to reverse acquired chemotherapy resistance in RRCL. Our data suggest that vorinostat is active in RRCL with a known defective apoptotic machinery, it can active alternative cell death pathways. Given the multiple pathways affected by HDAC inhibition, vorinostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B cell lymphoma.

Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

PubMed

Princz, Lissa N; Wild, Philipp; Bittmann, Julia; Aguado, F Javier; Blanco, Miguel G; Matos, Joao; Pfander, Boris

2017-03-01

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution. © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle.

PubMed

Pramila, Tata; Wu, Wei; Miles, Shawna; Noble, William Stafford; Breeden, Linda L

2006-08-15

Transcription patterns shift dramatically as cells transit from one phase of the cell cycle to another. To better define this transcriptional circuitry, we collected new microarray data across the cell cycle of budding yeast. The combined analysis of these data with three other cell cycle data sets identifies hundreds of new highly periodic transcripts and provides a weighted average peak time for each transcript. Using these data and phylogenetic comparisons of promoter sequences, we have identified a late S-phase-specific promoter element. This element is the binding site for the forkhead protein Hcm1, which is required for its cell cycle-specific activity. Among the cell cycle-regulated genes that contain conserved Hcm1-binding sites, there is a significant enrichment of genes involved in chromosome segregation, spindle dynamics, and budding. This may explain why Hcm1 mutants show 10-fold elevated rates of chromosome loss and require the spindle checkpoint for viability. Hcm1 also induces the M-phase-specific transcription factors FKH1, FKH2, and NDD1, and two cell cycle-specific transcriptional repressors, WHI5 and YHP1. As such, Hcm1 fills a significant gap in our understanding of the transcriptional circuitry that underlies the cell cycle.

Smad4 sensitizes colorectal cancer to 5-fluorouracil through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade.

PubMed

Zhang, Binhao; Leng, Chao; Wu, Chao; Zhang, Zhanguo; Dou, Lei; Luo, Xin; Zhang, Bixiang; Chen, Xiaoping

2016-03-01

5-Fluorouracil (5-FU), a cell cycle-specific antimetabolite, is one of the most commonly used chemotherapeutic agents for colorectal cancer (CRC). Yet, resistance to 5-FU-based chemotherapy is still an obstacle to the treatment of this malignancy. Mutation or loss of Smad4 in CRC is pivotal for chemoresistance. However, the mechanism by which Smad4 regulates the chemosensitivity of CRC remains unclear. In the present study, we investigated the role of Smad4 in the chemosensitivity of CRC to 5-FU, and whether Smad4-regulated cell cycle arrest is involved in 5-FU chemoresistance. We used Smad4-expressing CT26 and Smad4-null SW620 cell lines as experimental models, by knockdown or transgenic overexpression. Cells or tumors were treated with 5-FU to determine chemosensitivity by cell growth, tumorigenicity assay and a mouse model. Cell cycle distribution was examined with flow cytometric analysis, and cell cycle-related proteins were examined by western blotting. Smad4 deficiency in CT26 and SW620 cells induced chemoresistance to 5-FU both in vitro and in vivo. Smad4 deficiency attenuated G1 or G2 cell cycle arrest by activating the PI3K/Akt/CDC2/survivin pathway. The PI3K inhibitor, LY294002, reversed the activation of the Akt/CDC2/survivin cascade in the Smad4-deficient cells, while it had little effect on cells with high Smad4 expression. In conclusion, we discovered a novel mechanism mediated by Smad4 to trigger 5-FU chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade. The present study also implies that LY294002 has potential therapeutic value to reverse the chemosensitivity of CRC with low Smad4 expression.

Cryptotanshinone suppresses the proliferation and induces the apoptosis of pancreatic cancer cells via the STAT3 signaling pathway.

PubMed

Ge, Yuqing; Yang, Bo; Chen, Zhe; Cheng, Rubin

2015-11-01

Pancreatic cancer remains a challenging disease worldwide. Cryptotanshinone (CPT) is one of the active constituents of Salvia miltiorrhiza Bunge and exhibits significant antitumor activities in several human cancer cells. However, the efficacy and molecular mechanism of CPT in pancreatic cancer remains to be elucidated. In the present study, the effect of CPT on the proliferation, apoptosis and cell cycle of human pancreatic cancer cell BxPC‑3 cells was evaluated. The results demonstrated that CPT inhibited proliferation of the BxPC‑3 cells in a concentration‑dependent manner, and significantly induced cell apoptosis and cell cycle arrest. The protein levels of cleaved caspase‑3, caspase‑9 and poly ADP ribose polymerase were upregulated, while the levels of c‑myc, survivin and cyclin D1 were downregulated following treatment with CPT. In addition, CPT decreased the activities of signal transducer and activator of transcription 3 (STAT3) and several upstream regulatory signaling pathways after 24 h. However, CPT only inhibited the phosphorylation of STAT3 Tyr705 within 30 min, without marked effects on the phosphorylation of the other proteins. These results suggested that the inhibition of STAT3 activity by CPT was directly and independent of the upstream regulators in human pancreatic cancer. The present study demonstrated that CPT exerts anticancer effects by inducing apoptosis and cell cycle arrest via inhibition of the STAT3 signaling pathway in human BxPC-3 cells.

Dihydroartemisinin inhibits indoxyl sulfate (IS)-promoted cell cycle progression in mesangial cells by targeting COX-2/mPGES-1/PGE2 cascade.

PubMed

Mungun, Harr-Keshauve; Li, Shuzhen; Zhang, Yue; Huang, Songming; Jia, Zhanjun; Ding, Guixia; Zhang, Aihua

2018-01-01

Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and has been used as an antimalarial drug. Recently, roles of artemisinin and its derivatives in treating diseases besides antimalarial effect were documented. Thus, this study was undertaken to investigate the role of DHA in indoxyl sulfate (IS)-promoted cell cycle progression in glomerular mesangial cells, as well as the potential mechanisms. Under the basal condition, DHA significantly retarded the cell cycle progression as shown by decreased cell percentage in S phase and increased cell percentage in G1/G0 phases in line with reduced cell cycle proteins cyclin A2 and cyclin D1. Interestingly, DHA also inactivated the COX-2/mPGES-1/PGE 2 cascade which has been shown to play a critical role in promoting the mesangial cell cycle progression by our previous studies. Next, we investigated the role of DHA in IS-triggered cell cycle progression in this mesangial cell line. As expected, DHA treatment significantly retarded IS-induced cell cycle progression and inhibited the activation of COX-2/mPGES-1/PGE 2 cascade induced by IS. In summary, these data indicated that DHA inhibited the cell cycle progression in glomerular mesangial cells under normal condition or IS challenge possibly through the inhibition of COX-2/mPGES-1/PGE 2 cascade, suggesting a potential of DHA in treating glomerular diseases with mesangial cell proliferation.

Verteporfin inhibits papillary thyroid cancer cells proliferation and cell cycle through ERK1/2 signaling pathway

PubMed Central

Liao, Tian; Wei, Wen-Jun; Wen, Duo; Hu, Jia-Qian; Wang, Yu; Ma, Ben; Cao, Yi-Min; Xiang, Jun; Guan, Qing; Chen, Jia-Ying; Sun, Guo-Hua; Zhu, Yong-Xue; Li, Duan-Shu; Ji, Qing-Hai

2018-01-01

Verteporfin, a FDA approved second-generation photosensitizer, has been demonstrated to have anticancer activity in various tumors, but not including papillary thyroid cancer (PTC). In current pre-clinical pilot study, we investigate the effect of verteporfin on proliferation, apoptosis, cell cycle and tumor growth of PTC. Our results indicate verteporfin attenuates cell proliferation, arrests cell cycle in G2/S phase and induces apoptosis of PTC cells. Moreover, treatment of verteporfin dramatically suppresses tumor growth from PTC cells in xenograft mouse model. We further illustrate that exposure to MEK inhibitor U0126 inactivates phosphorylation of ERK1/2 and MEK in verteporfin-treated PTC cells. These data suggest verteporfin exhibits inhibitory effect on PTC cells proliferation and cell cycle partially via ERK1/2 signalling pathway, which strongly encourages the further application of verteporfin in the treatment against PTC. PMID:29721041

Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells

PubMed Central

Zhang, Yu; Xue, Ying-bo; Li, Hang; Qiu, Dong; Wang, Zhi-wei; Tan, Shi-sheng

2017-01-01

Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients. PMID:28165402

Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells.

PubMed

Zhang, Yu; Xue, Ying-Bo; Li, Hang; Qiu, Dong; Wang, Zhi-Wei; Tan, Shi-Sheng

2017-02-04

Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients.

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos.

PubMed

Zivkovic, Danica; Créton, Robbert; Dohmen, René

1991-08-01

During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca 2+ -stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm 2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca 2+ /Mg 2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca 2+ -stimulated ATPase. The ionic currents and Ca 2+ -stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca 2+ -stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca 2+ -stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.

CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression

PubMed Central

Pede, Valerie; Rombout, Ans; Vermeire, Jolien; Naessens, Evelien; Mestdagh, Pieter; Robberecht, Nore; Vanderstraeten, Hanne; Van Roy, Nadine; Vandesompele, Jo; Speleman, Frank; Philippé, Jan; Verhasselt, Bruno

2013-01-01

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation. PMID:23560086

Calotropin from Asclepias curasavica induces cell cycle arrest and apoptosis in cisplatin-resistant lung cancer cells.

PubMed

Mo, En-Pan; Zhang, Rong-Rong; Xu, Jun; Zhang, Huan; Wang, Xiao-Xiong; Tan, Qiu-Tong; Liu, Fang-Lan; Jiang, Ren-Wang; Cai, Shao-Hui

2016-09-16

Calotropin (M11), an active compound isolated from Asclepias curasavica L., was found to exert strong inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer (NSCLC) cells (A549/CDDP). Molecular mechanism study revealed that M11 induced cell cycle arrest at the G2/M phase through down-regulating cyclins, CDK1, CDK2 and up-regulating p53 and p21. Furthermore, M11 accelerated apoptosis through the mitochondrial apoptotic pathway which was accompanied by increase Bax/Bcl-2 ratio, decrease in mitochondrial membrane potential, increase in reactive oxygen species production, activations of caspases 3 and 9 as well as cleavage of poly ADP-ribose polymerase (PARP). The activation and phosphorylation of JNK was also found to be involved in M11-induced apoptosis, and SP610025 (specific JNK inhibitor) partially prevented apoptosis induced by M11. In contrast, all of the effects that M11 induce cell cycle arrest and apoptosis in A549/CDDP cells were not significant in A549 cells. Drugs with higher sensitivity against resistant tumor cells than the parent cells are rather rare. Results of this study supported the potential application of M11 on the non-small lung cancer (NSCLC) with cisplatin resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

PubMed

Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

Machineries regulating the activity of the small GTPase Arf6 in cancer cells are potential targets for developing innovative anti-cancer drugs.

PubMed

Yamauchi, Yohei; Miura, Yuki; Kanaho, Yasunori

2017-01-01

The Small GTPase ADP-ribosylation factor 6 (Arf6) functions as the molecular switch in cellular signaling pathways by cycling between GDP-bound inactive and GTP-bound active form, which is precisely regulated by two regulators, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Numerous studies have shown that these machineries play critical roles in tumor angiogenesis/growth and cancer cell invasion/metastasis through regulating the cycling of Arf6. Here, we summarize accumulating knowledge for involvement of Arf6 GEFs/GAPs and small molecule inhibitors of Arf6 signaling/cycling in cancer progression, and discuss possible strategies for developing innovative anti-cancer drugs targeting Arf6 signaling/cycling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

PubMed Central

Guo, Yongfeng; Flegel, Kerry; Kumar, Jayashree; McKay, Daniel J.

2016-01-01

ABSTRACT During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing. PMID:27737823

Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

PubMed

Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

2014-01-15

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers. © 2013 Elsevier Inc. All rights reserved.

In vitro growth inhibition of human cancer cells by novel honokiol analogs.

PubMed

Lin, Jyh Ming; Prakasha Gowda, A S; Sharma, Arun K; Amin, Shantu

2012-05-15

Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux

PubMed Central

Gangavarapu, Kalyan J.; Huss, Wendy J.

2011-01-01

This unit describes methods for the digestion of human prostate clinical specimens, dye cycle violet (DCV) staining procedure for the identification, isolation, and quantitation of radiolabeled dihydrotestosterone (DHT) retention of side population cells. The principle of the side population assay is based on differential efflux of DCV, a cell membrane permeable fluorescent dye, by cells with high ATP binding cassette (ABC) transporter activity. Cells with high ABC transporter activity efflux DCV and fall in the lower left quadrant of a flow cytograph are designated as “side population” cells. This unit emphasizes tissue digestion, DCV staining, flow settings for sorting side population cells and quantitation of radiolabeled DHT retention. PMID:21400686

Assessment of cell death mechanisms triggered by 177Lu-anti-CD20 in lymphoma cells.

PubMed

Azorín-Vega, E; Rojas-Calderón, E; Martínez-Ventura, B; Ramos-Bernal, J; Serrano-Espinoza, L; Jiménez-Mancilla, N; Ordaz-Rosado, D; Ferro-Flores, G

2018-08-01

The aim of this research was to evaluate the cell cycle redistribution and activation of early and late apoptotic pathways in lymphoma cells after treatment with 177 Lu-anti-CD20. Experimental and computer models were used to calculate the radiation absorbed dose to cancer cell nuclei. The computer model (Monte Carlo, PENELOPE) consisted of twenty spheres representing cells with an inner sphere (cell nucleus) embedded in culture media. Radiation emissions of the radiopharmaceutical located in cell membranes and in culture media were considered for nuclei dose calculations. Flow cytometric analyses demonstrated that doses as low as 4.8Gy are enough to induce cell cycle arrest and activate late apoptotic pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

PubMed

Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

2013-10-01

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed. © 2013 Blackwell Verlag GmbH.

Preventing the activation or cycling of the Rap1 GTPase alters adhesion and cytoskeletal dynamics and blocks metastatic melanoma cell extravasation into the lungs.

PubMed

Freeman, Spencer A; McLeod, Sarah J; Dukowski, Janet; Austin, Pamela; Lee, Crystal C Y; Millen-Martin, Brandie; Kubes, Paul; McCafferty, Donna-Marie; Gold, Michael R; Roskelley, Calvin D

2010-06-01

The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin-rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM. Copyright 2010 AACR.

Regulatory properties of polysaccharopeptide derived from Coriolus versicolor and its combined effect with ciclosporin on the homeostasis of human lymphocytes.

PubMed

Lee, Cheuk-Lun; Jiang, Pingping; Sit, Wai-Hung; Yang, Xiatong; Wan, Jennifer Man-Fan

2010-08-01

Lymphocyte homoeostasis is essential in inflammatory and autoimmune diseases. In search of natural fungal metabolites with effects on lymphocyte homoeostasis, we recently reported that polysaccharopeptide (PSP) from Coriolus versicolor exhibited ciclosporin-like activity in controlling aberrant lymphocyte activation. This object of this study was to investigate its effect on lymphocyte homoeostasis. This was done by investigating the mechanistic actions of PSP in relation to ciclosporin by performing cell cycle and cell death analysis of human lymphocytes in vitro. We investigated the effect of PSP in the presence and absence of ciclosporin on cell proliferation, cell cycle, cell death, immunophenotype and cell cycle regulatory proteins in human lymphocytes. The data showed that PSP exhibited homoeostatic activity by promoting and inhibiting the proliferation of resting and phytohaemagglutinin (PHA)-stimulated lymphocytes, respectively. PHA-stimulated lymphocytes exhibited G0/G1 cell cycle arrest that was accompanied by a reduction of cyclin E expression with PSP treatment. Both PSP and ciclosporin blocked the reduction of the CD4/CD8 ratio in stimulated lymphocytes. PSP did not induce cell death in human lymphocytes, but the suppression of the Fasreceptor suggested a protective role of PSP against extrinsic cell death signals. These homoeostatic effects were more potent with combined PSP and ciclosporin treatment than with either fungal metabolite alone. Collectively, the results reveal certain novel effects of PSP in lymphocyte homoeostasis and suggest potential as a specific immunomodulatory adjuvant for clinical applications in the treatment of autoimmune diseases.

Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

PubMed Central

Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

2016-01-01

Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

E2F activators signal and maintain centrosome amplification in breast cancer cells.

PubMed

Lee, Mi-Young; Moreno, Carlos S; Saavedra, Harold I

2014-07-01

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2(+) cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2(+) cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2(+) breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2.

Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1).

PubMed

Yamaguchi, Koushi; Honda, Mitsuo; Ikigai, Hajime; Hara, Yukihiko; Shimamura, Tadakatsu

2002-01-01

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-bacterial agent. In addition, anti-tumor promoting, anti-inflammatory, anti-oxidative and antiviral activities have been reported. In the present study, we investigated possible anti-human immunodeficiency virus type-1 (HIV-1) activity of EGCg and its mechanisms of action in the viral life cycle. EGCg impinges on each step of the HIV life cycle. Thus, destruction of the viral particles, viral attachment to cells, post-adsorption entry into cells, reverse transcription (RT), viral production from chronically-infected cells, and the level of expression of viral mRNA, were analyzed using T-lymphoid (H9) and monocytoid (THP-1) cell systems, and antiviral protease activity was measured using a cell-free assay. Inhibitory effects of EGCg on specific binding of the virions to the cellular surfaces and changes in the steady state viral regulation (mRNA expression) due to EGCg were not observed. However, EGCg had a destructive effect on the viral particles, and post-adsorption entry and RT in acutely infected monocytoid cells were significantly inhibited at concentrations of EGCg greater than 1 microM, and protease kinetics were suppressed at a concentration higher than 10 microM in the cell-free study. Viral production by THP-1 cells chronically-infected with HIV-1 was also inhibited in a dose-dependent manner and the inhibitory effect was enhanced by liposome modification of EGCg. As expected, increased viral mRNA production was observed in lipopolysaccharide (LPS)-activated chronically HIV-1-infected cells. This production was significantly inhibited by EGCg treatment of THP-1 cells. In contrast, production of HIV-1 viral mRNA in unstimulated or LPS-stimulated T-lymphoid cells (H9) was not inhibited by EGCg. Anti-HIV viral activity of EGCg may thus result from an interaction with several steps in the HIV-1 life cycle.

Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells

PubMed Central

Sumiyoshi, Tetsutaro; Sato, Kaoru; Yamamoto, Hitomi; Iwasaki, Yuka W.; Siomi, Haruhiko; Siomi, Mikiko C.

2016-01-01

In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Here, we show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila. PMID:27474440

Do lipids shape the eukaryotic cell cycle?

PubMed

Furse, Samuel; Shearman, Gemma C

2018-01-01

Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2012-01-01

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

Edge usage, motifs, and regulatory logic for cell cycling genetic networks

NASA Astrophysics Data System (ADS)

Zagorski, M.; Krzywicki, A.; Martin, O. C.

2013-01-01

The cell cycle is a tightly controlled process, yet it shows marked differences across species. Which of its structural features follow solely from the ability to control gene expression? We tackle this question in silico by examining the ensemble of all regulatory networks which satisfy the constraint of producing a given sequence of gene expressions. We focus on three cell cycle profiles coming from baker's yeast, fission yeast, and mammals. First, we show that the networks in each of the ensembles use just a few interactions that are repeatedly reused as building blocks. Second, we find an enrichment in network motifs that is similar in the two yeast cell cycle systems investigated. These motifs do not have autonomous functions, yet they reveal a regulatory logic for cell cycling based on a feed-forward cascade of activating interactions.

The Possible Crosstalk of MOB2 With NDR1/2 Kinases in Cell Cycle and DNA Damage Signaling.

PubMed

Gundogdu, Ramazan; Hergovich, Alexander

2016-09-06

This article is the authors' opinion of the roles of the signal transducer Mps one binder 2 (MOB2) in the control of cell cycle progression and the DNA Damage Response (DDR). We recently found that endogenous MOB2 is required to prevent the accumulation of endogenous DNA damage in order to prevent the undesired, and possibly detrimental, activation of cell cycle checkpoints. In this regard, it is noteworthy that MOB2 has been linked biochemically to the regulation of the NDR1/2 (aka STK38/STK38L) protein kinases, which themselves have functions at different steps of the cell cycle. Therefore, we are speculating in this article about the possible connections of MOB2 with NDR1/2 kinases in cell cycle and DDR Signaling.

Natural Compounds as Modulators of Cell Cycle Arrest: Application for Anticancer Chemotherapies

PubMed Central

Bailon-Moscoso, Natalia; Cevallos-Solorzano, Gabriela; Romero-Benavides, Juan Carlos; Orellana, Maria Isabel Ramirez

2017-01-01

Natural compounds from various plants, microorganisms and marine species play an important role in the discovery novel components that can be successfully used in numerous biomedical applications, including anticancer therapeutics. Since uncontrolled and rapid cell division is a hallmark of cancer, unraveling the molecular mechanisms underlying mitosis is key to understanding how various natural compounds might function as inhibitors of cell cycle progression. A number of natural compounds that inhibit the cell cycle arrest have proven effective for killing cancer cells in vitro, in vivo and in clinical settings. Significant advances that have been recently made in the understanding of molecular mechanisms underlying the cell cycle regulation using the chemotherapeutic agents is of great importance for improving the efficacy of targeted therapeutics and overcoming resistance to anticancer drugs, especially of natural origin, which inhibit the activities of cyclins and cyclin-dependent kinases, as well as other proteins and enzymes involved in proper regulation of cell cycle leading to controlled cell proliferation. PMID:28367072

Inhibition of NF-kappaB-mediated transcription and induction of apoptosis in human breast cancer cells by epoxypseudoisoeugenol-2-methyl butyrate.

PubMed

Ma, Guoyi; Tabanca, Nurhayat; Husnu Can Baser, K; Kirimer, Nese; Pasco, David S; Khan, Ikhlas A; Khan, Shabana I

2009-03-01

Breast cancer is one of the most prevalent woman cancers. Genomic instability, accumulative mutations, and subsequent changes in intracellular signaling cascades play key roles in the development of human breast cancers. Activation of nuclear factor-kappaB (NF-kappaB) has been implicated in oncogenesis of breast cancers and is known to be associated with resistance to anticancer agents and apoptosis. Blocking NF-kappaB signaling may represent a therapeutic strategy in breast cancer therapy. The objective of this study is to investigate the in vitro effects of epoxypseudoisoeugenol-2-methyl butyrate (EPB), a phenylpropranoid isolated from Pimpinella corymbosa, on the activation of NF-kappaB, cell growth, cell cycle progression and apoptosis in MCF-7 (estrogen-dependent) and BT-549 (estrogen-independent) breast cancer cells. Transcriptional activity of NF-kappaB was measured by cell based reporter gene assay. Cell proliferation was determined by MTT assay. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assy. EPB inhibited the NF-kappaB-mediated transcription activity induced by tumor necrosis factor-alpha (TNF-alpha) and phorbol myristate acetate (PMA) in MCF-7 cells. EPB also inhibited constitutive NF-kappaB transcriptional activity in BT-549 cells. EPB inhibited the proliferation of both MCF-7 and BT-549 cells in a concentration- and time-dependent manner. EPB induced cell cycle arrest in G(1)/G(0) phase and apoptosis in both MCF-7 and BT 549 cells. These in vitro results indicated that EPB has a potential for use against both hormone-dependent and hormone-independent breast cancers and its effects seem to be mediated by inhibiting the NF-kappaB activity.

Jungermannenone A and B induce ROS- and cell cycle-dependent apoptosis in prostate cancer cells in vitro

PubMed Central

Guo, Yan-xia; Lin, Zhao-min; Wang, Mei-juan; Dong, Yi-wen; Niu, Huan-min; Young, Charles YF; Lou, Hong-xiang; Yuan, Hui-qing

2016-01-01

Aim: Jungermannenone A and B (JA, JB) are new ent-kaurane diterpenoids isolated from Chinese liverwort Jungermannia fauriana, which show anti-proliferation activities in cancer cells. In this study we investigated the mechanisms underlying the anticancer action of JA and JB in PC3 human prostate cancer cells in vitro. Methods: A panel of 9 human cancer cell lines was tested. Cell proliferation was assessed with a real-time cell analyzer and MTT assay. Cell apoptosis, cell cycle distribution and ROS levels were measured using cytometry. Mitochondrial damage was examined by transmission electron microscopy. DNA damage was detected with comet assay. Apoptotic, DNA damage- and cell cycle-related proteins were analyzed using Western blotting. The expression of DNA repair genes was measured with qRT-PCR. Results: Both JA and JB exerted potent anti-proliferative action against the 9 cancer cell lines, and PC3 cells were more sensitive with IC50 values of 1.34±0.09 and 4.93±0.20 μmol/L, respectively. JA (1.5 μmol/L) and JB (5 μmol/L) induced PC3 cell apoptosis, which was attenuated by the caspase inhibitor Z-VAD. Furthermore, both JA and JB caused mitochondrial damage and ROS accumulation in PC3 cells, whereas vitamin C blocked the ROS accumulation and attenuated the cytotoxicity of JA and JB. Moreover, both JA and JB induced DNA damage, accompanied by downregulated DNA repair proteins Ku70/Ku80 and RDA51. JA induced marked cell cycle arrest at the G0/G1 phase, which was related to c-Myc suppression, whereas JB enforced the cell cycle blockade in the G2/M phase, which associated with activation of the JNK signaling. Conclusion: Both JA and JB induce prostate cancer apoptosis via ROS accumulation and induction of cell cycle arrest. PMID:27133304

Jungermannenone A and B induce ROS- and cell cycle-dependent apoptosis in prostate cancer cells in vitro.

PubMed

Guo, Yan-Xia; Lin, Zhao-Min; Wang, Mei-Juan; Dong, Yi-Wen; Niu, Huan-Min; Young, Charles Yf; Lou, Hong-Xiang; Yuan, Hui-Qing

2016-06-01

Jungermannenone A and B (JA, JB) are new ent-kaurane diterpenoids isolated from Chinese liverwort Jungermannia fauriana, which show anti-proliferation activities in cancer cells. In this study we investigated the mechanisms underlying the anticancer action of JA and JB in PC3 human prostate cancer cells in vitro. A panel of 9 human cancer cell lines was tested. Cell proliferation was assessed with a real-time cell analyzer and MTT assay. Cell apoptosis, cell cycle distribution and ROS levels were measured using cytometry. Mitochondrial damage was examined by transmission electron microscopy. DNA damage was detected with comet assay. Apoptotic, DNA damage- and cell cycle-related proteins were analyzed using Western blotting. The expression of DNA repair genes was measured with qRT-PCR. Both JA and JB exerted potent anti-proliferative action against the 9 cancer cell lines, and PC3 cells were more sensitive with IC50 values of 1.34±0.09 and 4.93±0.20 μmol/L, respectively. JA (1.5 μmol/L) and JB (5 μmol/L) induced PC3 cell apoptosis, which was attenuated by the caspase inhibitor Z-VAD. Furthermore, both JA and JB caused mitochondrial damage and ROS accumulation in PC3 cells, whereas vitamin C blocked the ROS accumulation and attenuated the cytotoxicity of JA and JB. Moreover, both JA and JB induced DNA damage, accompanied by downregulated DNA repair proteins Ku70/Ku80 and RDA51. JA induced marked cell cycle arrest at the G0/G1 phase, which was related to c-Myc suppression, whereas JB enforced the cell cycle blockade in the G2/M phase, which associated with activation of the JNK signaling. Both JA and JB induce prostate cancer apoptosis via ROS accumulation and induction of cell cycle arrest.

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors.

PubMed

Luniewski, Wojciech; Wietrzyk, Joanna; Godlewska, Joanna; Switalska, Marta; Piskozub, Malgorzata; Peczynska-Czoch, Wanda; Kaczmarek, Lukasz

2012-10-01

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells*

PubMed Central

Yang, Shaojun; Jan, Yi-Hua; Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2013-01-01

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury. PMID:23640889

The molecular basis of cytotoxicity of α-spinasterol from Ganoderma resinaceum: Induction of apoptosis and overexpression of p53 in breast and ovarian cancer cell lines.

PubMed

Sedky, Nada K; El Gammal, Zaynab H; Wahba, Amir E; Mosad, Eman; Waly, Zahraa Y; El-Fallal, Amira Ali; Arafa, Reem K; El-Badri, Nagwa

2018-05-01

Despite advances in therapy of breast and ovarian cancers, they still remain among the most imperative causes of cancer death in women. The first can be considered one of the most widespread diseases among females, while the latter is more lethal and needs prompt treatment. Thus, the research field can still benefit from discovery of new compounds that can be of potential use in management of these grave illnesses. We hereby aimed to assess the antitumor activity of the phytosterol α-spinasterol isolated from Ganoderma resinaceum mushroom on human breast cancer cell lines (MCF-7, MDA-MB-231), as well as, on human ovarian cancer cell line (SKOV-3). The anti-tumor activity of α-spinasterol, isolated from the mycelial extract of the Egyptian G. resinaceum, on human breast and ovarian cancer cell lines was evaluated by MTT cell viability assay and AnnexinV/propidium iodide apoptosis assay. The molecular mechanism underlying this effect was assessed by the relative expression of the following markers; tumor suppressor (p53, BRCA1, BRCA2), apoptotic marker (Bax) and cell cycle progression markers (cyclin dependent kinases cdk4/6) using real-time PCR. Cell cycle analysis was performed for the three investigated cancer cell lines to explore the effect on cell cycle progression. Our findings showed that α-spinasterol exhibited a higher antitumor activity on MCF-7 cells relative to SKOV-3 cells, while its lowest antitumor activity was against MDA-MB-231 cells. A significant increase in the expression of p53 and Bax was observed in cells treated with α-spinasterol, while cdk4/6 were significantly down-regulated upon exposure to α-spinasterol. Cell cycle analysis of α-spinasterol treated cells showed a G 0 -G 1 arrest. In conclusion, α-spinasterol isolated from G. resinaceum mushroom exerts a potent inhibitory activity on breast and ovarian cancer cell lines in a time- and dose-dependent manner. This can be reasonified in lights of the compound's ability to increase p53 and Bax expressions, and to lower the expression of cdk4/6. © 2017 Wiley Periodicals, Inc.

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

PubMed

Manokawinchoke, Jeeranan; Nattasit, Praphawi; Thongngam, Tanutchaporn; Pavasant, Prasit; Tompkins, Kevin A; Egusa, Hiroshi; Osathanon, Thanaphum

2017-08-31

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Pleiotrophin antagonizes Brd2 during neuronal differentiation

PubMed Central

Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J.; Garcia-Dominguez, Mario

2014-01-01

ABSTRACT Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system. PMID:24695857

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

Purified Brominated Indole Derivatives from Dicathais orbita Induce Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cell Lines

PubMed Central

Esmaeelian, Babak; Benkendorff, Kirsten; Johnston, Martin R.; Abbott, Catherine A.

2013-01-01

Dicathais orbita is a large Australian marine gastropod known to produce bioactive compounds with anticancer properties. In this research, we used bioassay guided fractionation from the egg mass extract of D. orbita using flash column chromatography and identified fractions containing tyrindoleninone and 6-bromoisatin as the most active against colon cancer cells HT29 and Caco-2. Liquid chromatography coupled with mass spectrometry (LCMS) and 1H NMR were used to characterize the purity and chemical composition of the isolated compounds. An MTT assay was used to determine effects on cell viability. Necrosis and apoptosis induction using caspase/LDH assay and flow cytometry (PI/Annexin-V) and cell cycle analysis were also investigated. Our results show that semi-purified 6-bromoisatin had the highest anti-cancer activity by inhibiting cell viability (IC50 = ~100 µM) and increasing caspase 3/7 activity in both of the cell lines at low concentration. The fraction containing 6-bromoisatin induced 77.6% apoptosis and arrested 25.7% of the cells in G2/M phase of cell cycle in HT29 cells. Tyrindoleninone was less potent but significantly decreased the viability of HT29 cells at IC50 = 390 µM and induced apoptosis at 195 µM by increasing caspase 3/7 activity in these cells. This research will facilitate the development of these molluscan natural products as novel complementary medicines for colorectal cancer. PMID:24152558

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

PubMed

Sales, Leilane; Pezuk, Julia Alejandra; Borges, Kleiton Silva; Brassesco, María Sol; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga; dos Santos, Marcelo Henrique; Ionta, Marisa; de Oliveira, Jaqueline Carvalho

2015-10-30

Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

Induction of the c-myc protooncogene following antigen binding to hapten-specific B cells

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

Snow, E.C.; Fetherston, J.; Zimmer, S.

1986-03-01

Considerable controversy has centered on the role that the surface immunoglobulin (sIg) receptor for antigen plays during the induction of B cell activation. Stimulation by anti-Ig reagents has been shown to activate G/sub 0/ B cells to enter the cell cycle. The binding of thymus-dependent antigens to hapten-specific B cell populations apparently does not result in the movement of the antigen-binding cells (ABC) into the G/sub 1/ stage of the cell cycle. However, the authors have recently demonstrated that antigen binding to such hapten-specific B cells does result in the initiation of the membrane phosphatidylinositol cycle. In the present experiments,more » hapten-specific B cells (80-90% ABC, 99% in G/sub 0/) were incubated with either the correct hapten-carrier conjugate, with the carrier protein, or only media for 2 hours at 37/sup 0/C. At that time, total cellular RNA was isolated and subsequently analyzed by either dot blots or Northern gel techniques. The blots were probed with a (/sup 32/P)-c-myc SstI-Xhol fragment. The results indicate that hapten carrier stimulation of the hapten-specific B cells induces enhanced transcription of the c-myc gene. These observations lend further support to the premise that antigen binding to the sIg receptor results in the transduction to the cell of important signals and implicates the active participation of sIg during the process of antigen-mediated B cell activation.« less

Porcine epidemic diarrhea virus through p53-dependent pathway causes cell cycle arrest in the G0/G1 phase.

PubMed

Sun, Pei; Wu, Haoyang; Huang, Jiali; Xu, Ying; Yang, Feng; Zhang, Qi; Xu, Xingang

2018-05-22

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus, has caused enormous economic losses in the swine industry. p53 protein exists in a wide variety of animal cells, which is involved in cell cycle regulation, apoptosis, cell differentiation and other biological functions. In this study, we investigated the effects of PEDV infection on the cell cycle of Vero cells and p53 activation. The results demonstrated that PEDV infection induces cell cycle arrest at G0/G1 phase in Vero cells, while UV-inactivated PEDV does not cause cell cycle arrest. PEDV infection up-regulates the levels of p21, cdc2, cdk2, cdk4, Cyclin A protein and down-regulates Cyclin E protein. Further research results showed that inhibition of p53 signaling pathway can reverse the cell cycle arrest in G0/G1 phase induced by PEDV infection and cancel out the up-regulation of p21 and corresponding Cyclin/cdk mentioned above. In addition, PEDV infection of the cells synchronized in various stages of cell cycle showed that viral subgenomic RNA and virus titer were higher in the cells released from G0/G1 phase synchronized cells than that in the cells released from the G1/S phase and G2/M phase synchronized or asynchronous cells after 18 h p.i.. This is the first report to demonstrate that the p53-dependent pathway plays an important role in PEDV induced cell cycle arrest and beneficially contributes to viral infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Helicobacter pylori Induced Phosphatidylinositol-3-OH Kinase/mTOR Activation Increases Hypoxia Inducible Factor-1α to Promote Loss of Cyclin D1 and G0/G1 Cell Cycle Arrest in Human Gastric Cells.

PubMed

Canales, Jimena; Valenzuela, Manuel; Bravo, Jimena; Cerda-Opazo, Paulina; Jorquera, Carla; Toledo, Héctor; Bravo, Denisse; Quest, Andrew F G

2017-01-01

Helicobacter pylori ( H. pylori ) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1α knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori -induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection.

Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin

PubMed Central

Hiratsuka, Toru; Fujita, Yoshihisa; Naoki, Honda; Aoki, Kazuhiro; Kamioka, Yuji; Matsuda, Michiyuki

2015-01-01

Extracellular signal-regulated kinase (ERK) is a key effector of many growth signalling pathways. In this study, we visualise epidermal ERK activity in living mice using an ERK FRET biosensor. Under steady-state conditions, the epidermis occasionally revealed bursts of ERK activation patterns where ERK activity radially propagated from cell to cell. The frequency of this spatial propagation of radial ERK activity distribution (SPREAD) correlated with the rate of epidermal cell division. SPREADs and proliferation were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in a manner dependent on EGF receptors and their cognate ligands. At the wounded skin, ERK activation propagated as trigger wave in parallel to the wound edge, suggesting that ERK activation propagation can be superimposed. Furthermore, by visualising the cell cycle, we found that SPREADs were associated with G2/M cell cycle progression. Our results provide new insights into how cell proliferation and transient ERK activity are synchronised in a living tissue. DOI: http://dx.doi.org/10.7554/eLife.05178.001 PMID:25668746

Molecular control of brain size: Regulators of neural stem cell life, death and beyond

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

Joseph, Bertrand; Hermanson, Ola, E-mail: ola.hermanson@ki.se

2010-05-01

The proper development of the brain and other organs depends on multiple parameters, including strictly controlled expansion of specific progenitor pools. The regulation of such expansion events includes enzymatic activities that govern the correct number of specific cells to be generated via an orchestrated control of cell proliferation, cell cycle exit, differentiation, cell death etc. Certain proteins in turn exert direct control of these enzymatic activities and thus progenitor pool expansion and organ size. The members of the Cip/Kip family (p21Cip1/p27Kip1/p57Kip2) are well-known regulators of cell cycle exit that interact with and inhibit the activity of cyclin-CDK complexes, whereas membersmore » of the p53/p63/p73 family are traditionally associated with regulation of cell death. It has however become clear that the roles for these proteins are not as clear-cut as initially thought. In this review, we discuss the roles for proteins of the Cip/Kip and p53/p63/p73 families in the regulation of cell cycle control, differentiation, and death of neural stem cells. We suggest that these proteins act as molecular interfaces, or 'pilots', to assure the correct assembly of protein complexes with enzymatic activities at the right place at the right time, thereby regulating essential decisions in multiple cellular events.« less

Molecular mechanism of action of oxazolinoanthracyclines in cells derived from human solid tumors. Part 2.

PubMed

Denel-Bobrowska, Marta; Łukawska, Małgorzata; Bukowska, Barbara; Gajek, Arkadiusz; Oszczapowicz, Irena; Marczak, Agnieszka

2018-02-01

Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are derivatives of anthracyclines (DOX and DAU) with a modified daunosamine moiety. We aimed to clarify their mechanisms of action by investigating intracellular accumulation and effects on the cell cycle, phosphatidylserine externalization, and proteasome 20S activity. Experimental model consisted of SKOV-3, A549 and HepG2 cells. Compounds were used at the concentration of 80nM. Intracellular accumulation, drug uptake, and proteasome 20S activity were evaluated by fluorimetric methods. The effects on the cell cycle and phosphatidylserine externalization were measured by flow cytometry. O-DOX was equivalent to DOX in terms of inducing G2/M arrest, but O-DAU was less potent in SKOV-3, HepG2, and A549 cells. O-DOX had the greatest effect on initiating apoptosis in all tested cells. Externalization of phosphatidylserine was significantly higher following O-DOX treatment compared with control cells and cells incubated with DOX. The intracellular accumulation and uptake of the derivatives were similar to those of the reference drugs. Tested compounds are able to activate proteasome 20S activity. Our results extended the understanding of the toxicity, mechanism of action, and biochemical properties of oxazoline derivatives of doxorubicin and daunorubicin, including their effects on cell cycle, apoptosis and DNA degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Krebs Cycle Component Limits Caspase Activation Rate through Mitochondrial Surface Restriction of CRL Activation.

PubMed

Aram, Lior; Braun, Tslil; Braverman, Carmel; Kaplan, Yosef; Ravid, Liat; Levin-Zaidman, Smadar; Arama, Eli

2016-04-04

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation. Copyright © 2016 Elsevier Inc. All rights reserved.

E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

PubMed

Carcagno, Abel L; Marazita, Mariela C; Ogara, María F; Ceruti, Julieta M; Sonzogni, Silvina V; Scassa, María E; Giono, Luciana E; Cánepa, Eduardo T

2011-01-01

A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

PubMed Central

Carcagno, Abel L.; Marazita, Mariela C.; Ogara, María F.; Ceruti, Julieta M.; Sonzogni, Silvina V.; Scassa, María E.; Giono, Luciana E.; Cánepa, Eduardo T.

2011-01-01

Background A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. Methodology/Principal Findings In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. Conclusions/Significance The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity. PMID:21765927

CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

PubMed Central

King, Jeffrey B.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Galanko, Joseph A.

2012-01-01

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies. PMID:22723265

Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

PubMed Central

Baltar, Federico

2018-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

PubMed Central

Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

2009-01-01

High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

PubMed

Ding, Xia; He, Zhuohao; Zhou, Kechun; Cheng, Ju; Yao, Hailan; Lu, Dongliang; Cai, Rong; Jin, Yening; Dong, Bin; Xu, Yinghui; Wang, Yizheng

2010-07-21

Patients with glioblastoma multiforme, the most aggressive form of glioma, have a median survival of approximately 12 months. Calcium (Ca(2+)) signaling plays an important role in cell proliferation, and some members of the Ca(2+)-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of many types of cancer cells. In this study, we investigated the role of TRPC6 in cell cycle progression and in the development of human glioma. TRPC6 protein and mRNA expression were assessed in glioma (n = 33) and normal (n = 17) brain tissues from patients and in human glioma cell lines U251, U87, and T98G. Activation of TRPC6 channels was tested by platelet-derived growth factor-induced Ca(2+) imaging. The effect of inhibiting TRPC6 activity or expression using the dominant-negative mutant TRPC6 (DNC6) or RNA interference, respectively, was tested on cell growth, cell cycle progression, radiosensitization of glioma cells, and development of xenografted human gliomas in a mouse model. The green fluorescent protein (GFP) and wild-type TRPC6 (WTC6) were used as controls. Survival of mice bearing xenografted tumors in the GFP, DNC6, and WTC6 groups (n = 13, 15, and 13, respectively) was compared using Kaplan-Meier analysis. All statistical tests were two-sided. Functional TRPC6 was overexpressed in human glioma cells. Inhibition of TRPC6 activity or expression attenuated the increase in intracellular Ca(2+) by platelet-derived growth factor, suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 activation and cell division cycle 25 homolog C expression regulated the cell cycle arrest. Inhibition of TRPC6 activity also reduced tumor volume in a subcutaneous mouse model of xenografted human tumors (P = .014 vs GFP; P < .001 vs WTC6) and increased mean survival in mice in an intracranial model (P < .001 vs GFP or WTC6). In this preclinical model, TRPC6 channels were essential for glioma development via regulation of G2/M phase transition. This study suggests that TRPC6 might be a new target for therapeutic intervention of human glioma.

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

PubMed

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

2010-11-01

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

TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

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

Wang Ling; Reinach, Peter; Lu, Luo

2005-11-15

Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases inmore » p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.« less

The architecture and conservation pattern of whole-cell control circuitry.

PubMed

McAdams, Harley H; Shapiro, Lucy

2011-05-27

The control circuitry that directs and paces Caulobacter cell cycle progression involves the entire cell operating as an integrated system. This control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates orderly activation of cell cycle subsystems and Caulobacter's asymmetric cell division. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus' functionality and peripheral connectivity to other cellular subsystems from species to species. This pattern is similar to that observed for the "kernels" of the regulatory networks that regulate development of metazoan body plans. The Caulobacter cell cycle control system has been exquisitely optimized as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty. When sufficient details accumulate, as for Caulobacter cell cycle regulation, the system design has been found to be eminently rational and indeed consistent with good design practices for human-designed asynchronous control systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

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

Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin

2014-03-28

Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined themore » impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.« less

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

Molecular identification of PpHDAC1, the first histone deacetylase fron the slime mold Physarum polycephalum.

PubMed

Brandtner, Eva-Maria; Lechner, Thomas; Loidl, Peter; Lusser, Alexandra

2002-01-01

The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression itself.

The gamma cycle.

PubMed

Fries, Pascal; Nikolić, Danko; Singer, Wolf

2007-07-01

Activated neuronal groups typically engage in rhythmic synchronization in the gamma-frequency range (30-100 Hz). Experimental and modeling studies demonstrate that each gamma cycle is framed by synchronized spiking of inhibitory interneurons. Here, we review evidence suggesting that the resulting rhythmic network inhibition interacts with excitatory input to pyramidal cells such that the more excited cells fire earlier in the gamma cycle. Thus, the amplitude of excitatory drive is recoded into phase values of discharges relative to the gamma cycle. This recoding enables transmission and read out of amplitude information within a single gamma cycle without requiring rate integration. Furthermore, variation of phase relations can be exploited to facilitate or inhibit exchange of information between oscillating cell assemblies. The gamma cycle could thus serve as a fundamental computational mechanism for the implementation of a temporal coding scheme that enables fast processing and flexible routing of activity, supporting fast selection and binding of distributed responses. This review is part of the INMED/TINS special issue Physiogenic and pathogenic oscillations: the beauty and the beast, based on presentations at the annual INMED/TINS symposium (http://inmednet.com).

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

PubMed

Marone, M; Scambia, G; Bonanno, G; Rutella, S; de Ritis, D; Guidi, F; Leone, G; Pierelli, L

2002-01-01

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.

PubMed Central

Minshull, J; Golsteyn, R; Hill, C S; Hunt, T

1990-01-01

Cyclins play a key role in the induction of mitosis. In this paper we report the isolation of a cyclin A cDNA clone from Xenopus eggs. Its cognate mRNA encodes a protein that shows characteristic accumulation and destruction during mitotic cell cycles. The cyclin A polypeptide is associated with a protein that cross-reacts with an antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the cyclin A-cdc2 complex exhibits protein kinase activity that oscillates with the cell cycle. This kinase activity rises more smoothly than that of the cyclin B-cdc2 complexes and reaches a peak earlier in the cell cycle; indeed, cyclin A is destroyed before nuclear envelope breakdown. None of the cyclin-cdc2 complexes show simple relationships between the concentration of the cyclin moiety and the kinase activity. All three cyclin associated kinases (A, B1 and B2) phosphorylate identical sites on histones with the consensus XSPXK/R, although they show significant differences in their substrate preferences. We discuss possible models for the different roles of the A- and B-type cyclins in the control of cell division. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2143983

Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels.

PubMed

Yang, Ke; Guo, Yang; Stacey, William C; Harwalkar, Jyoti; Fretthold, Jonathan; Hitomi, Masahiro; Stacey, Dennis W

2006-08-30

The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

PubMed Central

2010-01-01

Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models. PMID:20682067

ERK reinforces actin polymerization to power persistent edge protrusion during motility

PubMed Central

Mendoza, Michelle C.; Vilela, Marco; Juarez, Jesus E.; Blenis, John; Danuser, Gaudenz

2016-01-01

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. Here, we tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell-surface receptors and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy (qFSM) and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. Arp2/3 activity generates branched actin networks that can produce pushing force. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. PMID:25990957

Downregulation of gasdermin D promotes gastric cancer proliferation by regulating cell cycle-related proteins.

PubMed

Wang, Wei Jie; Chen, Di; Jiang, Ming Zuo; Xu, Bing; Li, Xiao Wei; Chu, Yi; Zhang, Yu Jie; Mao, Ren; Liang, Jie; Fan, Dai Ming

2018-02-01

To explore the relationship between gasdermin D (GSDMD) and gastric cancer (GC) cell proliferation, and to determine whether the downregulated expression of GSDMD contributed to the tumorigenesis and proliferation of GC cells. GSDMD expressions in GC tissues and matched adjacent non-cancerous tissues were assessed by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry. The effect of GSDMD on cell proliferation in vitro was assessed by the colony formation assay and cell viability assays. In vivo, xenografted tumors in nude mice were evaluated. The cell cycle was analyzed by flow cytometry. In addition, the alterations of several cell cycle-related and cell signaling pathway proteins were analyzed by Western blot. GSDMD expression was decreased in GC, and the decreased expression of GSDMD could markedly promote the proliferation of tumors in vivo and in vitro. The downregulation of GSDMD accelerated S/G 2 cell transition by activating extracellular signal regulated kinase, signal transducer and activator of transcription 3 and phosphatidylinositol 3 kinase/protein kinase B signaling pathways and regulating cell cycle-related proteins in GC. GSDMD may protect against cell proliferation of GC, and it may be used as a diagnostic and treatment strategy for GC. © 2018 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study.

PubMed

Ho, Si-Han Sherman; Sim, Mei-Yi; Yee, Wei-Loong Sherman; Yang, Tianming; Yuen, Shyi-Peng John; Go, Mei-Lin

2015-11-02

The anticancer agent YM155 is widely investigated as a specific survivin suppressant. More recently, YM155 was found to induce DNA damage and this has raised doubts as to whether survivin is its primary target. In an effort to assess the contribution of DNA damage to the anticancer activity of YM155, several analogs were prepared and evaluated for antiproliferative activity on malignant cells, participation in DNA intercalation and free radical generation by redox cycling. The intact positively charged scaffold was found to be essential for antiproliferative activity and intercalation but was less critical for redox cycling where the minimal requirement was a pared down bicyclic quinone. Side chain requirements at the N(1) and N(3) positions of the scaffold were more alike for redox cycling and intercalation than antiproliferative activity, underscoring yet again, the limited structural overlaps for these activities. Furthermore, antiproliferative activities were poorly correlated to DNA intercalation and redox cycling. Potent antiproliferative activity (IC50 9-23 nM), exceeding that of YM155, was found for a minimally substituted methyl analog AB7. Like YM155 and other dioxonaphthoimidazoliums, AB7 was a modest DNA intercalator but with weak redox cycling activity. Thus, the capacity of this scaffold to inflict direct DNA damage leading to cell death may not be significant and YM155 should not be routinely classified as a DNA damaging agent. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Inhibition of Krebs cycle and activation of glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae. Compensatory role of succinate oxidation.

PubMed

Samokhvalov, V; Ignatov, V; Kondrashova, M

2004-01-01

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), alpha-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.

Biochemical effects of veterinary antibiotics on proliferation and cell cycle arrest of human HEK293 cells.

PubMed

Kim, Hyeon Young; Kim, Ki-Tae; Kim, Sang Don

2012-08-01

The purpose of this study was to examine the effects of veterinary antibiotics, including amoxicillin (AMX), chlortetracycline (CTC) and tylosin (TYL), on the biochemical mechanism of human embryonic kidney cells (HEK293). CTC and TYL inhibited HEK293 cell proliferation, in both time- and dose-dependent manners, and changed the cell morphology; whereas, AMX showed no cytotoxic effects. The cell cycle analysis of CTC and TYL revealed G1-arrest in HEK293 cells. Western blot analysis also showed that CTC and TYL affected the activation of DNA damage responsive proteins, as well as cell cycle regulatory proteins, such as p53, p21(Waf1/Cip1) and Rb protein, which are crucial in the G1-S transition. The activation of p21(Waf1/Cip1) was significantly up-regulated over time, but there was no change in the level of CDK2 expression. The results of this study suggest that veterinary antibiotics, even at low level concentrations on continuous exposure, can potentially risk the development of human cells.

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

PubMed

Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

2018-02-01

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

The antiangiogenic activity of Kushecarpin D, a novel flavonoid isolated from Sophora flavescens Ait.

PubMed

Pu, Li-Ping; Chen, He-Ping; Cao, Mei-Ai; Zhang, Xiu-Li; Gao, Qing-Xiang; Yuan, Cheng-Shan; Wang, Chun-Ming

2013-11-13

Kushecarpin D (KD) is a novel flavonoid isolated from the traditional Chinese herbal medicine Kushen (the dried root of Sophora flavescens Ait). As part of our continuous effort to explore Chinese traditional medicinal herbs and to identify novel natural anticancer products, the antiangiogenic properties of KD were examined in vitro using a human umbilical vein endothelial cell line (ECV304). The SRB and Trypan Blue exclusion assays were used to evaluate the effect of KD on cell proliferation. The antiangiogenic activities of KD were evaluated through studies of cell migration, cell adhesion, and tube formation. DCFH-DA and DHE fluorescent assays were used to detect the reactive oxygen species (ROS) levels. Catalase activity was detected using the colorimetric ammonium molybdate method. Cell cycle and apoptosis were measured using flow cytometry and the Hoechst 33258 staining assay. The results indicated that KD showed antiangiogenic activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. ROS levels were down-regulated and catalase activity was up-regulated after treatment with KD. The cell cycle was arrested at the G2/M phase, while no apoptosis was observed using the Hoechst 33258 staining assay or following the flow cytometric analysis of the sub-G1 proportion. The antiangiogenic properties of KD, in combination with its anti-proliferative effect and ability to induce cell cycle arrest without inducing apoptosis, make it a good candidate for development as antitumor agent. However, further studies are essential to elucidate its mechanism of action. © 2013.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost.

PubMed

Ding, Xiang; Zhu, Hongqing; Hou, Yiling; Hou, Wanru; Zhang, Nan; Fu, Lei

2017-01-01

The mechanism of the immunoregulatory activities of polysaccharide is still not clear. Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages. These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups. An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways. Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide.

Criteria for robustness of heteroclinic cycles in neural microcircuits

PubMed Central

2011-01-01

We introduce a test for robustness of heteroclinic cycles that appear in neural microcircuits modeled as coupled dynamical cells. Robust heteroclinic cycles (RHCs) can appear as robust attractors in Lotka-Volterra-type winnerless competition (WLC) models as well as in more general coupled and/or symmetric systems. It has been previously suggested that RHCs may be relevant to a range of neural activities, from encoding and binding to spatio-temporal sequence generation. The robustness or otherwise of such cycles depends both on the coupling structure and the internal structure of the neurons. We verify that robust heteroclinic cycles can appear in systems of three identical cells, but only if we require perturbations to preserve some invariant subspaces for the individual cells. On the other hand, heteroclinic attractors can appear robustly in systems of four or more identical cells for some symmetric coupling patterns, without restriction on the internal dynamics of the cells. PMID:22656192

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

PubMed

Esteras, Noemí; Bartolomé, Fernando; Alquézar, Carolina; Antequera, Desireé; Muñoz, Úrsula; Carro, Eva; Martín-Requero, Ángeles

2012-09-01

Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

High content image based analysis identifies cell cycle inhibitors as regulators of Ebola virus infection.

PubMed

Kota, Krishna P; Benko, Jacqueline G; Mudhasani, Rajini; Retterer, Cary; Tran, Julie P; Bavari, Sina; Panchal, Rekha G

2012-09-25

Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI) assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential effects of cell cycle arrest on Ebola virus (EBOV) infection. Cells arrested in G1 phase by serum starvation or G1/S phase using aphidicolin or G2/M phase using nocodazole showed much reduced EBOV infection compared to the untreated control. Release of cells from serum starvation or aphidicolin block resulted in a time-dependent increase in the percentage of EBOV infected cells. The effect of EBOV infection on cell cycle progression was found to be cell-type dependent. Infection of asynchronous MCF-10A cells with EBOV resulted in a reduced number of cells in G2/M phase with concomitant increase of cells in G1 phase. However, these effects were not observed in HeLa or A549 cells. Together, our studies suggest that EBOV requires actively proliferating cells for efficient replication. Furthermore, multiplexing of HCI based assays to detect viral infection, cell cycle status and other phenotypic changes in a single cell population will provide useful information during screening campaigns using siRNA and small molecule therapeutics.

3,3′-Diindolylmethane Ameliorates Experimental Autoimmune Encephalomyelitis by Promoting Cell Cycle Arrest and Apoptosis in Activated T Cells through MicroRNA Signaling Pathways

PubMed Central

Rouse, Michael; Rao, Roshni; Nagarkatti, Mitzi

2014-01-01

3,3′-Diindolylmethane (DIM) is a naturally derived indole found in cruciferous vegetables that has great potential as a novel and effective therapeutic agent. In the current study, we investigated the effects of DIM post-treatment on the regulation of activated T cells during the development of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. We demonstrated that the administration of DIM 10 days after EAE induction was effective at ameliorating disease parameters, including inflammation and central nervous system cellular infiltration. MicroRNA (miRNA) microarray analysis revealed an altered miRNA profile in brain infiltrating CD4+ T cells following DIM post-treatment of EAE mice. Additionally, bioinformatics analysis suggested the involvement of DIM-induced miRNAs in pathways and processes that halt cell cycle progression and promote apoptosis. Additional studies confirmed that DIM impacted these cellular processes in activated T cells. Further evidence indicated that DIM treatment significantly upregulated several miRNAs (miR-200c, miR-146a, miR-16, miR-93, and miR-22) in brain CD4+ T cells during EAE while suppressing their associated target genes. Similarly, we found that overexpression of miR-16 in primary CD4+ T cells led to significant downregulation of both mRNA and protein levels of cyclin E1 and B-cell lymphoma-2, which play important roles in regulating cell cycle progression and apoptosis. Collectively, these studies demonstrate that DIM post-treatment leads to the amelioration of EAE development by suppressing T-cell responses through the induction of select miRNAs that control cell cycle progression and mediate apoptosis. PMID:24898268

Deoxyschizandrin, Isolated from Schisandra Berries, Induces Cell Cycle Arrest in Ovarian Cancer Cells and Inhibits the Protumoural Activation of Tumour-Associated Macrophages.

PubMed

Lee, Kijun; Ahn, Ji-Hye; Lee, Kyung-Tae; Jang, Dae Sik; Choi, Jung-Hye

2018-01-15

Deoxyschizandrin, a major lignan of Schisandra berries, has been demonstrated to have various biological activities such as antioxidant, hepatoprotective, and antidiabetic effects. However, the anti-cancer effects of deoxyschizandrin are poorly characterized. In the present study, we investigated the anti-cancer effect of deoxyschizandrin on human ovarian cancer cell lines and tumour-associated macrophages (TAMs). Deoxyschizandrin induced G₀/G₁ phase cell cycle arrest and inhibited cyclin E expression in human ovarian cancer cells. Overexpression of cyclin E significantly reversed the deoxyschizandrin-induced cell growth inhibition. Interestingly, increased production of reactive oxygen species and decreased activation of Akt were observed in A2780 cells treated with deoxyschizandrin, and the antioxidant compromised the deoxyschizandrin-induced cell growth inhibition and Akt inactivation. Moreover, deoxyschizandrin-induced cell growth inhibition was markedly suppressed by Akt overexpression. In addition, deoxyschizandrin was found to inhibit the expression of the M2 phenotype markers CD163 and CD209 in TAMs, macrophages stimulated by the ovarian cancer cells. Moreover, expression and production of the tumour-promoting factors MMP-9, RANTES, and VEGF, which are highly enhanced in TAMs, was significantly suppressed by deoxyschizandrin treatment. Taken together, these data suggest that deoxyschizandrin exerts anti-cancer effects by inducing G₀/G₁ cell cycle arrest in ovarian cancer cells and reducing the protumoural phenotype of TAMs.

Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

PubMed

Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

2004-06-18

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

PubMed Central

Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

2014-01-01

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overexpression of DYRK1A induced G0 cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

Cell cycle regulation and apoptosis mediated by p53 in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei.

PubMed

Nuñez-Hernandez, Dahlia M; Felix-Portillo, Monserrath; Peregrino-Uriarte, Alma B; Yepiz-Plascencia, Gloria

2018-01-01

Although hypoxic aquatic environments cause negative effects on shrimp, these animals can withstand somewhat hypoxia, but the cellular mechanisms underlying this capacity are still poorly understood. In humans, mild hypoxia causes the induction of many proteins to allow cell survival. In contrast, apoptosis is induced during severe hypoxia leading to cell death. p53 is a key transcription factor that determines cells fate towards cell cycle arrest or induction of apoptosis in humans. The aim of this work was to study the role of p53 in cell cycle regulation and apoptosis in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei. p53 was silenced by RNAi and afterwards the shrimp were exposed to hypoxia. Cdk-2 was used as indicator of cell cycle progression while caspase-3 expression and caspase activity were analyzed as indicators of apoptosis. p53 levels in hepatopancreas were significantly higher at 48 h after hypoxic treatment. Increased expression levels of Cdk-2 were found in p53-silenced shrimp after 24 and 48 h in the normoxic treatments as well as 48 h after hypoxia, indicating a possible role of p53 in cell cycle regulation. In response to hypoxia, unsilenced shrimp showed an increase in caspase-3 expression levels, however an increase was also observed in caspase activity at 24 h of normoxic conditions in p53-silenced shrimps. Taken together these results indicate the involvement of p53 in regulation of cell cycle and apoptosis in the white shrimp in response to hypoxia. Copyright © 2017 Elsevier Ltd. All rights reserved.

HTLV-I Tax and cell cycle progression.

PubMed

Neuveut, C; Jeang, K T

2000-01-01

Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T-cell leukemia (ATL) and various human myopathies/neuropathies. HTLV-I encodes a 40 kDa phosphoprotein, Tax, which has been implicated in cellular transformation. In similarity with several other oncoproteins such as Myc, Jun, and Fos, Tax is a transcriptional activator. How Tax mechanistically dysregulates the cell cycle remains unclear. Recent findings from us and others have shown that Tax targets key regulators of G1/S and M progression such as p16INK4a, cyclin D1, cyclin D3-cdk, and the mitotic spindle checkpoint apparatus. Thus, Tax influences the progression of cells in various phases of the cell cycle. In this regard, we will discuss three distinct mechanisms through which Tax affects cell-cycling: a) through direct association Tax can abrogate the inhibitory function of p16INK4a on the G1-cdks, b) Tax can also directly influence cyclin D-cdk activities by a protein-protein interaction, and c) Tax targets the HsMAD1 mitotic spindle-assembly checkpoint protein. Through these varied routes, the HTLV-I oncoprotein dysregulates cellular growth controls and engenders a proclivity of cells toward a loss of DNA-damage surveillance.

Sonic hedgehog initiates cochlear hair cell regeneration through downregulation of retinoblastoma protein

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

Lu, Na; Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114; Chen, Yan

Highlights: Black-Right-Pointing-Pointer Shh activation in neonatal cochleae enhances sensory cell proliferation. Black-Right-Pointing-Pointer Proliferating supporting cells can transdifferentiate into hair cells. Black-Right-Pointing-Pointer Shh promotes proliferation by transiently modulating pRb activity. Black-Right-Pointing-Pointer Shh inhibits pRb by inhibiting transcription and increasing phosphorylation of pRb. -- Abstract: Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We showmore » that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration.« less

The Concerted Action of Type 2 and Type 3 Deiodinases Regulates the Cell Cycle and Survival of Basal Cell Carcinoma Cells.

PubMed

Miro, Caterina; Ambrosio, Raffaele; De Stefano, Maria Angela; Di Girolamo, Daniela; Di Cicco, Emery; Cicatiello, Annunziata Gaetana; Mancino, Giuseppina; Porcelli, Tommaso; Raia, Maddalena; Del Vecchio, Luigi; Salvatore, Domenico; Dentice, Monica

2017-04-01

Thyroid hormones (THs) mediate pleiotropic cellular processes involved in metabolism, cellular proliferation, and differentiation. The intracellular hormonal environment can be tailored by the type 1 and 2 deiodinase enzymes D2 and D3, which catalyze TH activation and inactivation respectively. In many cellular systems, THs exert well-documented stimulatory or inhibitory effects on cell proliferation; however, the molecular mechanisms by which they control rates of cell cycle progression have not yet been entirely clarified. We previously showed that D3 depletion or TH treatment influences the proliferation and survival of basal cell carcinoma (BCC) cells. Surprisingly, we also found that BCC cells express not only sustained levels of D3 but also robust levels of D2. The aim of the present study was to dissect the contribution of D2 to TH metabolism in the BCC context, and to identify the molecular changes associated with cell proliferation and survival induced by TH and mediated by D2 and D3. We used the CRISPR/Cas9 technology to genetically deplete D2 and D3 in BCC cells and studied the consequences of depletion on cell cycle progression and on cell death. Cell cycle progression was analyzed by fluorescence activated cell sorting analysis of synchronized cells, and the apoptosis rate by annexin V incorporation. Mechanistic investigations revealed that D2 inactivation accelerates cell cycle progression thereby enhancing the proportion of S-phase cells and cyclin D1 expression. Conversely, D3 mutagenesis drastically suppressed cell proliferation and enhanced apoptosis of BCC cells. Furthermore, the basal apoptotic rate was oppositely regulated in D2- and D3-depleted cells. Our results indicate that BCC cells constitute an example in which the TH signal is finely tuned by the concerted expression of opposite-acting deiodinases. The dual regulation of D2 and D3 expression plays a critical role in cell cycle progression and cell death by influencing cyclin D1-mediated entry into the G1-S phase. These findings reinforce the concept that TH is a potential therapeutic target in human BCC.

Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

PubMed

Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

2014-05-15

The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.

Molecular mechanisms of cisplatin cytotoxicity in acute promyelocytic leukemia cells.

PubMed

Kumar, Sanjay; Tchounwou, Paul B

2015-12-01

Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anti-tumor drug for the treatment of a broad range of human malignancies with successful therapeutic outcomes for head and neck, ovarian, and testicular cancers. It has been found to inhibit cell cycle progression and to induce oxidative stress and apoptosis in acute promyelocytic leukemia (APL) cells. However, its molecular mechanisms of cytotoxic action are poorly understood. We hypothesized that cisplatin induces cytotoxicity through DNA adduct formation, oxidative stress, transcriptional factors (p53 and AP-1), cell cycle regulation, stress signaling and apoptosis in APL cells. We used the APL cell line as a model, and applied a variety of molecular tools to elucidate the cytotoxic mode of action of cisplatin. We found that cisplatin inhibited cell proliferation by a cytotoxicity, characterized by DNA damage and modulation of oxidative stress. Cisplatin also activated p53 and phosphorylated activator protein (AP-1) component, c-Jun at serine (63, 73) residue simultaneously leading to cell cycle arrest through stimulation of p21 and down regulation of cyclins and cyclin dependent kinases in APL cell lines. It strongly activated the intrinsic pathway of apoptosis through alteration of the mitochondrial membrane potential, release of cytochrome C, and up-regulation of caspase 3 activity. It also down regulated the p38MAPK pathway. Overall, this study highlights the molecular mechanisms that underline cisplatin toxicity to APL cells, and provides insights into selection of novel targets and/or design of therapeutic agents to treat APL.

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response

PubMed Central

Malzer, Elke; Daly, Marie-Louise; Moloney, Aileen; Sendall, Timothy J.; Thomas, Sally E.; Ryder, Edward; Ryoo, Hyung Don; Crowther, Damian C.; Lomas, David A.; Marciniak, Stefan J.

2010-01-01

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2α phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest. PMID:20682638

Cadmium promotes the proliferation of triple-negative breast cancer cells through EGFR-mediated cell cycle regulation

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

Wei, Zhengxi, E-mail: weizhengxi@gmail.com; Song, Xiulong; Shaikh, Zahir A., E-mail: zshaikh@uri.edu

Cadmium (Cd) is a carcinogenic metal which is implicated in breast cancer by epidemiological studies. It is reported to promote breast cancer cell growth in vitro through membrane receptors. The study described here examined Cd-mediated growth of non-metastatic human breast cancer derived cells that lack receptors for estrogen, progesterone, and HER2. Treatment of triple-negative HCC 1937 cells with 0.1–0.5 μM Cd increased cell growth by activation of AKT and ERK. Accelerated cell cycle progression was achieved by increasing the levels of cyclins A, B, and E, as well as those of CDKs 1 and 2. Although triple negative cells lackmore » estrogen receptor, they express high levels of EGFR. Therefore, further studies on HCC 1937 and another triple-negative cell line, HCC 38, were conducted using specific siRNA and an inhibitor of EGFR to determine whether EGFR was responsible for mediating the effect of Cd. The results revealed that in both cell types EGFR was not only activated upon Cd treatment, but was also essential for the downstream activation of AKT and ERK. Based on these observations, it is concluded that, in breast cancer cells lacking estrogen receptor, sub-micromolar concentration of Cd can promote cell proliferation. Furthermore, that EGFR plays a critical role in this process. - Highlights: • Sub-micromolar concentrations of Cd promote cell growth in breast cancer cells that lack ER, PR, and HER2. • The increase in cell number is not due to reduction in apoptosis. • Growth promotion involves AKT and ERK signaling and downstream stimulation of cell cycle progression. • Initiation of cell growth by Cd occurs at the cell membrane and requires the activation of EGFR.« less

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

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

Chiaro, Christopher, E-mail: cchiaro@tcmedc.org; Lazarova, Darina L., E-mail: dlazarova@tcmedc.org; Bordonaro, Michael, E-mail: mbordonaro@tcmedc.org

2012-11-09

Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116,more » does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.« less

E2F Activators Signal and Maintain Centrosome Amplification in Breast Cancer Cells

PubMed Central

Lee, Mi-Young; Moreno, Carlos S.

2014-01-01

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2+ cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2+ cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2+ breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2. PMID:24797070

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.

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

The Echinoid Mitotic Gradient: Effect of Cell Size on the Micromere Cleavage Cycle

PubMed Central

Langelan Duncan, Rosalie E.; Whiteley, Arthur H.

2012-01-01

SUMMARY Like other euechinoids, the fertilized eggs of the sand dollar Dendraster excentricus proceed through cleavages that produce a pattern of macromeres, mesomeres, and micromeres at the 4th division. The 8 cells of the macro-mesomere lineage proceed through 6 additional cleavages before hatching. At the fifth overall division, the 4 micromeres produce a lineage of large micromeres that will divide 3 additional times, and a lineage of small micromeres that will divide once more before hatching. Irrespective of lineage, the length of the cell cycles is closely related to the size of the blastomere; cells of the same size have the same cell cycle time. A consequence is that at the fourth cleavage, there is a gradient of mitotic activity from the fastest dividers at the animal pole and the slowest cleacing micromeres at the vegetal pole. By the time of hatching, which is the 10th division of meso-macromeres, all cells are the same small size, the metachronic pattern of division gives way to asynchrony, and the mitotic gradient along the polar axis is lost. Experimental pre-exposure to sodium dodecyl sulfate (SDS), however, blocks the appearance of the gradients in cell size, the mitotic gradient, and the differential in cell cycle times. It is proposed that the mitotic gradients, cell cycle times, and attainment of a state of asynchrony are functions of cell size. Developmental consequences of the transition are large, and include coordinated activation of transcriptions, synthesis of new patterns of proteins, alterations of metabolism, and onset of morphogenesis. PMID:22006441

Macroenvironmental regulation of hair cycling and collective regenerative behavior.

PubMed

Plikus, Maksim V; Chuong, Cheng-Ming

2014-01-01

The hair follicle (HF) regeneration paradigm provides a unique opportunity for studying the collective behavior of stem cells in living animals. Activation of HF stem cells depends on the core inhibitory BMP and activating WNT signals operating within the HF microenvironment. Additionally, HFs receive multilayered signaling inputs from the extrafollicular macroenvironment, which includes dermis, adipocytes, neighboring HFs, hormones, and external stimuli. These activators/inhibitors are integrated across multiple stem-cell niches to produce dynamic hair growth patterns. Because of their pigmentation, these patterns can be easily studied on live shaved animals. Comparing to autonomous regeneration of one HF, populations of HFs display coupled decision making, allowing for more robust and adaptable regenerative behavior to occur collectively. The generic cellular automata model used to simulate coordinated HF cycling here can be extended to study population-level behavior of other complex biological systems made of cycling elements.

Macroenvironmental Regulation of Hair Cycling and Collective Regenerative Behavior

PubMed Central

Plikus, Maksim V.; Chuong, Cheng-Ming

2014-01-01

The hair follicle (HF) regeneration paradigm provides a unique opportunity for studying the collective behavior of stem cells in living animals. Activation of HF stem cells depends on the core inhibitory BMP and activating WNT signals operating within the HF microenvironment. Additionally, HFs receive multilayered signaling inputs from the extrafollicular macroenvironment, which includes dermis, adipocytes, neighboring HFs, hormones, and external stimuli. These activators/inhibitors are integrated across multiple stem-cell niches to produce dynamic hair growth patterns. Because of their pigmentation, these patterns can be easily studied on live shaved animals. Comparing to autonomous regeneration of one HF, populations of HFs display coupled decision making, allowing for more robust and adaptable regenerative behavior to occur collectively. The generic cellular automata model used to simulate coordinated HF cycling here can be extended to study population-level behavior of other complex biological systems made of cycling elements. PMID:24384813

Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

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

Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

2010-09-01

Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results:more » IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.« less

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

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

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

2007-07-15

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

Overexpression of the growth arrest-specific homeobox gene Gax inhibits proliferation, migration, cell cycle progression, and apoptosis in serum-induced vascular smooth muscle cells.

PubMed

Zheng, H; Xue, S; Hu, Z L; Shan, J G; Yang, W G

2014-03-24

The Gax gene has been implicated in a variety of cell-developmental and biological processes, and aberrant Gax expression is linked to many diseases. In this study, to provide important insights for Gax-based gene therapy in vein graft restenosis and its anti-restenotic mechanism, we used rabbit vascular smooth muscle cells (VSMCs) to investigate the effects of Gax overexpression on proliferation, migration, cell cycle, and apoptosis in a serum-stimulated culture. Rabbit VSMC lines that stably overexpressed Gax were established by transfection with recombinant adenoviral vector Ad5-Gax. The effect of Gax overexpression on in vitro serum-induced VSMCs proliferation, migration, cell cycle, and apoptosis was assessed by MTT, wound healing, and flow cytometry assays, respectively. To investigate the effect of Gax overexpression on PCNA and MMP-2 in serum-induced VSMCs, immunocytochemistry, RT-PCR, and gelatin zymography were performed. The results clearly showed that Gax overexpression decreases PCNA expression in serum-induced VSMCs. Gax overexpression also significantly inhibited cell proliferation by blocking entry into the S-phase of the cell cycle, promoted cell apoptosis, and reduced cell migration activity by downregulating MMP-2 release and activity. These findings indicate that Gax would be an optimal target gene for gene therapy to treat vein graft restenosis.

A complex regulatory network coordinating cell cycles during C. elegans development is revealed by a genome-wide RNAi screen.

PubMed

Roy, Sarah H; Tobin, David V; Memar, Nadin; Beltz, Eleanor; Holmen, Jenna; Clayton, Joseph E; Chiu, Daniel J; Young, Laura D; Green, Travis H; Lubin, Isabella; Liu, Yuying; Conradt, Barbara; Saito, R Mako

2014-02-28

The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development. Copyright © 2014 Roy et al.

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

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis.

PubMed

Xu, Deyang; Huang, Weihua; Li, Yang; Wang, Hua; Huang, Hai; Cui, Xiaofeng

2012-03-01

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

DNA damage checkpoint recovery and cancer development

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

Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong, E-mail: lsteng@zju.edu.cn

2015-06-10

Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observedmore » to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint recovery pathway plays a role in overriding tumor barrier in tumorigenesis. • Recovery protein dysregulation and human cancer development is correlated.« less

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

PubMed

Chen, Wei-Qiang; Xu, Bin; Mao, Jian-Wen; Wei, Feng-Xiang; Li, Ming; Liu, Tao; Jin, Xiao-Bao; Zhang, Li-Rong

2014-01-01

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

Cellular STAT3 functions via PCBP2 to restrain Epstein-Barr Virus lytic activation in B lymphocytes.

PubMed

Koganti, Siva; Clark, Carissa; Zhi, Jizu; Li, Xiaofan; Chen, Emily I; Chakrabortty, Sharmistha; Hill, Erik R; Bhaduri-McIntosh, Sumita

2015-05-01

A major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV(+)) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV(+) cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers. Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV(+) cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control.

PubMed Central

Qian, D; Zhou, D; Ju, R; Cramer, C L; Yang, Z

1996-01-01

Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants. A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy. A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit. The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll. RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division. The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells. A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth. Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells. Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase. These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase. PMID:8989889

Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway.

PubMed

Yuan, Gangjun; Chen, Xin; Liu, Zhuowei; Wei, Wensu; Shu, Qinghai; Abou-Hamdan, Hussein; Jiang, Lijuan; Li, Xiangdong; Chen, Rixin; Désaubry, Laurent; Zhou, Fangjian; Xie, Dan

2018-02-07

Prohibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored. FL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB cell proliferation and to determine the underlying mechanisms. FL3 inhibited UCB cell proliferation and growth both in vitro and in vivo. By targeting the PHB protein, FL3 inhibited the interaction of Akt and PHB as well as Akt-mediated PHB phosphorylation, which consequently decreases the localization of PHB in the mitochondria. In addition, FL3 treatment resulted in cell cycle arrest in the G2/M phase, and this inhibitory effect of FL3 could be mimicked by knockdown of PHB. Through the microarray analysis of mRNA expression after FL3 treatment and knockdown of PHB, we found that the mRNA expression of the growth arrest and DNA damage-inducible alpha (GADD45α) gene were significantly upregulated. When knocked down the expression of GADD45α, the inhibitory effect of FL3 on cell cycle was rescued, suggesting that FL3-induced cell cycle inhibition is GADD45α dependent. Our data provide that FL3 inhibits the interaction of Akt and PHB, which in turn activates the GADD45α-dependent cell cycle inhibition in the G2/M phase.

DEC1 regulates breast cancer cell proliferation by stabilizing cyclin E protein and delays the progression of cell cycle S phase

PubMed Central

Bi, H; Li, S; Qu, X; Wang, M; Bai, X; Xu, Z; Ao, X; Jia, Z; Jiang, X; Yang, Y; Wu, H

2015-01-01

Breast cancer that is accompanied by a high level of cyclin E expression usually exhibits poor prognosis and clinical outcome. Several factors are known to regulate the level of cyclin E during the cell cycle progression. The transcription factor DEC1 (also known as STRA13 and SHARP2) plays an important role in cell proliferation and apoptosis. Nevertheless, the mechanism of its role in cell proliferation is poorly understood. In this study, using the breast cancer cell lines MCF-7 and T47D, we showed that DEC1 could inhibit the cell cycle progression of breast cancer cells independently of its transcriptional activity. The cell cycle-dependent timing of DEC1 overexpression could affect the progression of the cell cycle through regulating the level of cyclin E protein. DEC1 stabilized cyclin E at the protein level by interacting with cyclin E. Overexpression of DEC1 repressed the interaction between cyclin E and its E3 ligase Fbw7α, consequently reducing the level of polyunbiquitinated cyclin E and increased the accumulation of non-ubiquitinated cyclin E. Furthermore, DEC1 also promoted the nuclear accumulation of Cdk2 and the formation of cyclin E/Cdk2 complex, as well as upregulating the activity of the cyclin E/Cdk2 complex, which inhibited the subsequent association of cyclin A with Cdk2. This had the effect of prolonging the S phase and suppressing the growth of breast cancers in a mouse xenograft model. These events probably constitute the essential steps in DEC1-regulated cell proliferation, thus opening up the possibility of a protein-based molecular strategy for eliminating cancer cells that manifest a high-level expression of cyclin E. PMID:26402517

A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.

PubMed

Barik, Debashis; Ball, David A; Peccoud, Jean; Tyson, John J

2016-12-01

The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.

A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability

PubMed Central

Ball, David A.

2016-01-01

The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally. PMID:27935947

p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

PubMed

Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

2007-10-01

Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

Coordination of the cell cycle is an important determinant of the syndrome of acute renal failure.

PubMed

Megyesi, Judit; Andrade, Lucia; Vieira, Jose M; Safirstein, Robert L; Price, Peter M

2002-10-01

Recovery from injury is usually accompanied by cell replication, in which new cells replace those irreparably damaged. After acute renal failure, normally quiescent kidney cells enter the cell cycle, which in tubule segments is accompanied by the induction of cell cycle inhibitors. We found that after acute renal failure induced by either cisplatin injection or renal ischemia, induction of the p21 cyclin-dependent kinase (cdk) inhibitor is protective. Mice lacking this gene developed more widespread kidney cell death, more severe renal failure, and had reduced survival, compared with mice with a functional p21 gene. Here, we show induction of 14-3-3sigma, a regulator of G(2)-to-M transition, after acute renal failure. Our findings, using both in vivo and in vitro models of acute renal failure, show that this protein likely helps to coordinate cell cycle activity to maximize recovery of renal epithelial cells from injury and reduce the extent of the injury itself. Because in terminally differentiated cells, these proteins are highly expressed only after injury, we propose that cell cycle coordination by induction of these proteins could be a general model of tissue recovery from stress and injury.

The BMI1 inhibitor PTC-209 is a potential compound to halt cellular growth in biliary tract cancer cells

PubMed Central

Mayr, Christian; Wagner, Andrej; Loeffelberger, Magdalena; Bruckner, Daniela; Jakab, Martin; Berr, Frieder; Di Fazio, Pietro; Ocker, Matthias; Neureiter, Daniel; Pichler, Martin; Kiesslich, Tobias

2016-01-01

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and is up-regulated in biliary tract cancer (BTC), contributing to aggressive clinical features. In this study we investigated the cytotoxic effects of PTC-209, a recently developed inhibitor of BMI1, in BTC cells. PTC-209 reduced overall viability in BTC cell lines in a dose-dependent fashion (0.04 - 20 μM). Treatment with PTC-209 led to slightly enhanced caspase activity and stop of cell proliferation. Cell cycle analysis revealed that PTC-209 caused cell cycle arrest at the G1/S checkpoint. A comprehensive investigation of expression changes of cell cycle-related genes showed that PTC-209 caused significant down-regulation of cell cycle-promoting genes as well as of genes that contribute to DNA synthesis initiation and DNA repair, respectively. This was accompanied by significantly elevated mRNA levels of cell cycle inhibitors. In addition, PTC-209 reduced sphere formation and, in a cell line-dependent manner, aldehyde dehydrogease-1 positive cells. We conclude that PTC-209 might be a promising drug for future in vitro and in vivo studies in BTC. PMID:26623561

Linking morphology with activity through the lifetime of pretreated PtNi nanostructured thin film catalysts

DOE PAGES

Cullen, David A.; Lopez-Haro, Miguel; Bayle-Guillemaud, Pascale; ...

2015-04-10

In this study, the nanoscale morphology of highly active Pt 3Ni 7 nanostructured thin film fuel cell catalysts is linked with catalyst surface area and activity following catalyst pretreatments, conditioning and potential cycling. The significant role of fuel cell conditioning on the structure and composition of these extended surface catalysts is demonstrated by high resolution imaging, elemental mapping and tomography. The dissolution of Ni during fuel cell conditioning leads to highly complex, porous structures which were visualized in 3D by electron tomography. Quantification of the rendered surfaces following catalyst pretreatment, conditioning, and cycling shows the important role pore structure playsmore » in surface area, activity, and durability.« less

Cytotoxic effects of ultra-diluted remedies on breast cancer cells.

PubMed

Frenkel, Moshe; Mishra, Bal Mukund; Sen, Subrata; Yang, Peiying; Pawlus, Alison; Vence, Luis; Leblanc, Aimee; Cohen, Lorenzo; Banerji, Pratip; Banerji, Prasanta

2010-02-01

The use of ultra-diluted natural products in the management of disease and treatment of cancer has generated a lot of interest and controversy. We conducted an in vitro study to determine if products prescribed by a clinic in India have any effect on breast cancer cell lines. We studied four ultra-diluted remedies (Carcinosin, Phytolacca, Conium and Thuja) against two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and a cell line derived from immortalized normal human mammary epithelial cells (HMLE). The remedies exerted preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis. These effects were accompanied by altered expression of the cell cycle regulatory proteins, including downregulation of phosphorylated Rb and upregulation of the CDK inhibitor p27, which were likely responsible for the cell cycle delay/arrest as well as induction of the apoptotic cascade that manifested in the activation of caspase 7 and cleavage of PARP in the treated cells. The findings demonstrate biological activity of these natural products when presented at ultra-diluted doses. Further in-depth studies with additional cell lines and animal models are warranted to explore the clinical applicability of these agents.

Redistribution of cell cycle by arsenic trioxide is associated with demethylation and expression changes of cell cycle related genes in acute promyelocytic leukemia cell line (NB4).

PubMed

Hassani, Saeed; Khaleghian, Ali; Ahmadian, Shahin; Alizadeh, Shaban; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

2018-01-01

PML-RARα perturbs the normal epigenetic setting, which is essential to oncogenic transformation in acute promyelocytic leukemia (APL). Transcription induction and recruitment of DNA methyltransferases (DNMTs) by PML-RARα and subsequent hypermethylation are components of this perturbation. Arsenic trioxide (ATO), an important drug in APL therapy, concurrent with degradation of PML-RARα induces cell cycle change and apoptosis. How ATO causes cell cycle alteration has remained largely unexplained. Here, we investigated DNA methylation patterns of cell cycle regulatory genes promoters, the effects of ATO on the methylated genes and cell cycle distribution in an APL cell line, NB4. Analysis of promoter methylation status of 22 cell cycle related genes in NB4 revealed that CCND1, CCNE1, CCNF, CDKN1A, GADD45α, and RBL1 genes were methylated 60.7, 84.6, 58.6, 8.7, 33.4, and 73.7%, respectively, that after treatment with 2 μM ATO for 48 h, turn into 0.6, 13.8, 0.1, 6.6, 10.7, and 54.5% methylated. ATO significantly reduced the expression of DNMT1, 3A, and 3B. ATO induced the expression of CCND1, CCNE1, and GADD45α genes, suppressed the expression of CCNF and CDKN1A genes, which were consistent with decreased number of cells in G1 and S phases and increased number of cells in G2/M phase. In conclusion, demethylation and alteration in the expression level of the cell cycle related genes may be possible mechanisms in ATO-induced cell cycle arrest in APL cells. It may suggest that ATO by demethylation of CCND1 and CCNE1 and their transcriptional activation accelerates G1 and S transition into the G2/M cell cycle arrest.

Complexes of D-type cyclins with CDKs during maize germination

PubMed Central

Vázquez-Ramos, Jorge M.

2013-01-01

The importance of cell proliferation in plant growth and development has been well documented. The majority of studies on basic cell cycle mechanisms in plants have been at the level of gene expression and much less knowledge has accumulated in terms of protein interactions and activation. Two key proteins, cyclins and cyclin-dependent kinases (CDKs) are fundamental for cell cycle regulation and advancement. Our aim has been to understand the role of D-type cyclins and type A and B CDKs in the cell cycle taking place during a developmental process such as maize seed germination. Results indicate that three maize D-type cyclins—D2;2, D4;2, and D5;3—(G1-S cyclins by definition) bind and activate two different types of CDK—A and B1;1—in a differential way during germination. Whereas CDKA–D-type cyclin complexes are more active at early germination times than at later times, it was surprising to observe that CDKB1;1, a supposedly G2-M kinase, bound in a differential way to all D-type cyclins tested during germination. Binding to cyclin D2;2 was detectable at all germination times, forming a complex with kinase activity, whereas binding to D4;2 and D5;3 was more variable; in particular, D5;3 was only detected at late germination times. Results are discussed in terms of cell cycle advancement and its importance for seed germination. PMID:24127516

Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway

USDA-ARS?s Scientific Manuscript database

Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser33 in p53, which is associated with decreased p53...

In Vitro Antiproliferative Effect of Arthrocnemum indicum Extracts on Caco-2 Cancer Cells through Cell Cycle Control and Related Phenol LC-TOF-MS Identification

PubMed Central

Boulaaba, Mondher; Mkadmini, Khaoula; Tsolmon, Soninkhishig; Han, Junkyu; Smaoui, Abderrazak; Kawada, Kiyokazu; Ksouri, Riadh; Isoda, Hiroko; Abdelly, Chedly

2013-01-01

This study aimed to determinate phenolic contents and antioxidant activities of the halophyte Arthrocnemum indicum shoot extracts. Moreover, the anticancer effect of this plant on human colon cancer cells and the likely underlying mechanisms were also investigated, and the major phenols were identified by LC-ESI-TOF-MS. Results showed that shoot extracts had an antiproliferative effect of about 55% as compared to the control and were characterised by substantial total polyphenol content (19 mg GAE/g DW) and high antioxidant activity (IC50 = 40 μg/mL for DPPH test). DAPI staining revealed that these extracts decrease DNA synthesis and reduce the proliferation of Caco-2 cells which were stopped at the G2/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2) correlate with the changes in cell cycle distribution. Eight phenolic compounds were also identified. In conclusion, A. indicum showed interesting antioxidant capacities associated with a significant antiproliferative effect explained by a cell cycle blocking at the G2/M phase. Taken together, these data suggest that A. indicum could be a promising candidate species as a source of anticancer molecules. PMID:24348703

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFκB pathway

PubMed Central

Alvero, Ayesha B; Visintin, Irene

2011-01-01

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer. PMID:21623171

AMP-activated protein kinase is involved in neural stem cell growth suppression and cell cycle arrest by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and glucose deprivation by down-regulating phospho-retinoblastoma protein and cyclin D.

PubMed

Zang, Yi; Yu, Li-Fang; Nan, Fa-Jun; Feng, Lin-Yin; Li, Jia

2009-03-06

The fate of neural stem cells (NSCs), including their proliferation, differentiation, survival, and death, is regulated by multiple intrinsic signals and the extrinsic environment. We had previously reported that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) directly induces astroglial differentiation of NSCs by activation of the Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway independently of AMP-activated protein kinase (AMPK). Here, we reported the observation that AICAR inhibited NSC proliferation and its underlying mechanism. Analysis of caspase activity and cell cycle showed that AICAR induced G1/G0 cell cycle arrest in NSCs, associated with decreased levels of poly(ADP-ribose) polymerase, phospho-retinoblastoma protein (Rb), and cyclin D but did not cause apoptosis. Iodotubericidin and Compound C, inhibitors of adenosine kinase and AMPK, respectively, or overexpression of a dominant-negative mutant of AMPK, but not JAK inhibitor, were able to reverse the anti-proliferative effect of AICAR. Glucose deprivation also activated the AMPK pathway, induced G0/G1 arrest, and suppressed the proliferation of NSCs, an effect associated with decreased levels of phospho-Rb and cyclin D protein. Furthermore, Compound C and overexpression of dominant-negative AMPK in C17.2 NSCs could block the glucose deprivation-mediated down-regulation of cyclin D and partially reverse the suppression of proliferation. These results suggest that AICAR and glucose deprivation might induce G1/G0 cell cycle arrest and suppress proliferation of NSCs via phospho-Rb and cyclin D down-regulation. AMPK, but not JAK/STAT3, activation is key for this inhibitory effect and may play an important role in the responses of NSCs to metabolic stresses such as glucose deprivation.

Sodium valproate, a histone deacetylase inhibitor, enhances the efficacy of vinorelbine-cisplatin-based chemoradiation in non-small cell lung cancer cells.

PubMed

Gavrilov, Vladimir; Lavrenkov, Konstantin; Ariad, Samuel; Shany, Shraga

2014-11-01

To enhance the anticancer activity of vinorelbine, cisplatin and ionizing radiation (IR) combination against non-small cell lung cancer (NSCLC) cells by co-administration of sodium valproate (VPA), a histone deacetylase inhibitor, and to elucidate molecular events underpinning treatment efficacy. The NSCLC A549 cell line was treated with cisplatin (0.2 μg/ml), vinorelbine (2 nM), VPA (1 mM) and IR (2.5 Gy) alone, or in combination. Cell proliferation, cell-cycle distribution, apoptosis, and levels of DNA double-strand breaks, activated DNA damage checkpoint kinases pCHK1, pCHK2, cell-cycle inhibitors p21CIP1/WAF1 and p27KIP1 were assessed. VPA markedly enhanced the DNA-damaging effect of the cisplatin-vinorelbine-IR combination and induced increased DSBs, and expression of pCHK2, pCHK1, p21CIP1/WAF1 and p27KIP1. These molecular changes led to cell-cycle arrest and increased apoptosis and consequently markedly curtailed cancer cell growth. VPA markedly enhances the anticancer activity of cisplatin-vinorelbine-IR combination. This finding has translational implications for enhancing the efficacy of anticancer treatment and for reducing side-effects by reducing doses of radiation and drugs. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F.

PubMed

Oliveira, Amanda; Beyer, Georg; Chugh, Rohit; Skube, Steven J; Majumder, Kaustav; Banerjee, Sulagna; Sangwan, Veena; Li, Lihua; Dawra, Rajinder; Subramanian, Subbaya; Saluja, Ashok; Dudeja, Vikas

2015-06-01

Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

Chalepin: A Compound from Ruta angustifolia L. Pers Exhibits Cell Cycle Arrest at S phase, Suppresses Nuclear Factor-Kappa B (NF-κB) Pathway, Signal Transducer and Activation of Transcription 3 (STAT3) Phosphorylation and Extrinsic Apoptotic Pathway in Non-small Cell Lung Cancer Carcinoma (A549).

PubMed

Richardson, Jaime Stella Moses; Aminudin, Norhaniza; Abd Malek, Sri Nurestri

2017-10-01

Plants have been a major source of inspiration in developing novel drug compounds in the treatment of various diseases that afflict human beings worldwide. Ruta angustifolia L. Pers known locally as Garuda has been conventionally used for various medicinal purposes such as in the treatment of cancer. A dihydrofuranocoumarin named chalepin, which was isolated from the chloroform extract of the plant, was tested on its ability to inhibit molecular pathways of human lung carcinoma (A549) cells. Cell cycle analysis and caspase 8 activation were conducted using a flow cytometer, and protein expressions in molecular pathways were determined using Western blot technique. Cell cycle analysis showed that cell cycle was arrested at the S phase. Further studies using Western blotting technique showed that cell cycle-related proteins such as cyclins, cyclin-dependent kinases (CDKs), and inhibitors of CDKs correspond to a cell cycle arrest at the S phase. Chalepin also showed inhibition in the expression of inhibitors of apoptosis proteins. Nuclear factor-kappa B (NF-κB) pathway, signal transducer and activation of transcription 3 (STAT-3), cyclooxygenase-2, and c-myc were also downregulated upon treatment with chalepin. Chalepin was found to induce extrinsic apoptotic pathway. Death receptors 4 and 5 showed a dramatic upregulation at 24 h. Analysis of activation of caspase 8 with the flow cytometer showed an increase in activity in a dose- and time-dependent manner. Activation of caspase 8 induced cleavage of BH3-interacting domain death agonist, which initiated a mitochondrial-dependent or -independent apoptosis. Chalepin causes S phase cell cycle arrest, NF-κB pathway inhibition, and STAT-3 inhibition, induces extrinsic apoptotic pathway, and could be an excellent chemotherapeutic agent. This study reports the capacity of an isolated bioactive compound known as chalepin to suppress the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, signal transducer and activation of transcription 3, and extrinsic apoptotic pathway and also its ability to arrest cell cycle in S phase. This compound was from the leaves of Ruta angustifolia L. Pers. It provides new insight on the ability of this plant in suppressing certain cancers, especially the nonsmall cell lung carcinoma according to this study. Abbreviations used: °C: Degree Celsius, ANOVA: Analysis of variance, ATCC: American Type Culture Collection, BCL-2: B-Cell CLL/Lymphoma 2, Bcl-xL: B-cell lymphoma extra-large, BH3: Bcl-2 homology 3, BID: BH3-interacting domain death agonist, BIR: Baculovirus inhibitor of apoptosis protein repeat, Caspases: Cysteinyl aspartate-specific proteases, CDK: Cyclin-dependent kinase, CO 2 : Carbon dioxide, CST: Cell signaling technologies, DISC: Death-inducing signaling complex, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DR4: Death receptor 4, DR5: Death receptor 5, E1a: Adenovirus early region 1A, ECL: Enhanced chemiluminescence, EDTA: Ethylenediaminetetraacetic acid, ELISA: Enzyme-linked immunosorbent assay, etc.: Etcetera, FADD: Fas-associated protein with death domain, FBS: Fetal bovine serum, FITC: Fluorescein isothiocyanate, G1: Gap 1, G2: Gap 2, HPLC: High-performance liquid chromatography, HRP: Horseradish peroxidase, IAPs: Inhibitor of apoptosis proteins, IC50: Inhibitory concentration at half maximal inhibitory, IKK-α: Inhibitor of nuclear factor kappa-B kinase subunit alpha, IKK-β: Inhibitor of nuclear factor kappa-B kinase subunit beta, IKK-γ: Inhibitor of nuclear factor kappa-B kinase subunit gamma, IKK: IκB kinase, IkBα: Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, m: Meter, M: Mitotic, mm: Millimeter, mRNA: Messenger ribonucleic acid, NaCl: Sodium chloride, NaVO4: Sodium orthovanadate, NEMO: NF-Kappa-B essential modulator, NF-κB: Nuclear factor kappa-light chain-enhancer of activated B cells, NSCLC: Nonsmall cell lung carcinoma, PBS: Phosphate buffered saline, PGE2: Prostaglandin E2, PI: Propidium iodide, PMSF: Phenylmethylsulfonyl fluoride, pRB: Phosphorylated retinoblastoma, R. angustifolia : Ruta angustifolia L. Pers, Rb: Retinoblastoma, rpm: Rotation per minute, RPMI: Roswell Park Memorial Institute, S phase: Synthesis phase, SD: Standard deviation, SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Smac: Second mitochondria-derived activator of caspase, SPSS: Statistical Package for the Social Sciences, STAT3: Signal transducer and activation of transcription 3, tBID: Truncated BID, TNF: Tumor necrosis factor, TRADD: Tumor necrosis factor receptor type-1 associated death domain, TRAIL: TNF-related apoptosis- inducing ligand, USA: United States of America, v/v: Volume over volume.

Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells.

PubMed

Yao, Mu; Xie, Chanlu; Kiang, Mei-Yee; Teng, Ying; Harman, David; Tiffen, Jessamy; Wang, Qian; Sved, Paul; Bao, Shisan; Witting, Paul; Holst, Jeff; Dong, Qihan

2015-10-27

Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.

Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata.

PubMed

Rajagopal, Sriram; Kumar, R Ajaya; Deevi, Dhanvanthri S; Satyanarayana, Chitkala; Rajagopalan, R

2003-01-01

Andrographis paniculata plant extract is known to possess a variety of pharmacological activities. Andrographolide, the major constituent of the extract is implicated towards its pharmacological activity. We studied the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. Andrographolide treatment inhibited the in vitro proliferation of different tumor cell lines, representing various types of cancers. The compound exerts direct anticancer activity on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein p27 and decreased expression of cyclin-dependent kinase 4 (CDK4). Immunostimulatory activity of andrographolide is evidenced by increased proliferation of lymphocytes and production of interleukin-2. Andrographolide also enhanced the tumor necrosis factor-alpha production and CD marker expression, resulting in increased cytotoxic activity of lymphocytes against cancer cells, which may contribute for its indirect anticancer activity. The in vivo anticancer activity of the compound is further substantiated against B16F0 melanoma syngenic and HT-29 xenograft models. These results suggest that andrographolide is an interesting pharmacophore with anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent.

TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional activity.

PubMed

Tomasini, Richard; Seux, Mylène; Nowak, Jonathan; Bontemps, Caroline; Carrier, Alice; Dagorn, Jean-Charles; Pébusque, Marie-Josèphe; Iovanna, Juan L; Dusetti, Nelson J

2005-12-08

TP53INP1 is an alternatively spliced gene encoding two nuclear protein isoforms (TP53INP1alpha and TP53INP1beta), whose transcription is activated by p53. When overexpressed, both isoforms induce cell cycle arrest in G1 and enhance p53-mediated apoptosis. TP53INP1s also interact with the p53 gene and regulate p53 transcriptional activity. We report here that TP53INP1 expression is induced during experimental acute pancreatitis in p53-/- mice and in cisplatin-treated p53-/- mouse embryo fibroblasts (MEFs). We demonstrate that ectopic expression of p73, a p53 homologue, leads to TP53INP1 induction in p53-deficient cells. In turn, TP53INP1s alters the transactivation capacity of p73 on several p53-target genes, including TP53INP1 itself, demonstrating a functional association between p73 and TP53INP1s. Also, when overexpressed in p53-deficient cells, TP53INP1s inhibit cell growth and promote cell death as assessed by cell cycle analysis and colony formation assays. Finally, we show that TP53INP1s potentiate the capacity of p73 to inhibit cell growth, that effect being prevented when the p53 mutant R175H is expressed or when p73 expression is blocked by a siRNA. These results suggest that TP53INP1s are functionally associated with p73 to regulate cell cycle progression and apoptosis, independently from p53.

Effects of an inhibitor of tripeptidyl peptidase II (Ala-Ala-Phe-chloromethylketone) and its combination with an inhibitor of the chymotrypsin-like activity of the proteasome (PSI) on apoptosis, cell cycle and proteasome activity in U937 cells.

PubMed

Bury, M; Młynarczuk, I; Pleban, E; Hoser, G; Kawiak, J; Wójcik, C

2001-01-01

AAF-AMC is not a specific TPP II substrate, since it is also hydrolyzed by purified proteasomes. Moreover, AAF-cmk, claimed to be a specific TPP II inhibitor, also inhibits the chymotrypsin-like activity of the proteasome. While AAF-cmk itself is mildly cytostatic to U-937 cells and induces cell cycle block in G1, its combination with PSI does not induce an increase in the cytostatic/cytotoxic effects. This suggests that TPP II is possibly less important for cell metabolism than it was previously believed and it is less probable that it can be able to fully compensate for the loss of the proteasome function.

Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.

PubMed

Edgar, Bruce A; Zielke, Norman; Gutierrez, Crisanto

2014-03-01

In endoreplication cell cycles, known as endocycles, cells successively replicate their genomes without segregating chromosomes during mitosis and thereby become polyploid. Such cycles, for which there are many variants, are widespread in protozoa, plants and animals. Endocycling cells can achieve ploidies of >200,000 C (chromatin-value); this increase in genomic DNA content allows a higher genomic output, which can facilitate the construction of very large cells or enhance macromolecular secretion. These cells execute normal S phases, using a G1-S regulatory apparatus similar to the one used by mitotic cells, but their capability to segregate chromosomes has been suppressed, typically by downregulation of mitotic cyclin-dependent kinase activity. Endocycles probably evolved many times, and the various endocycle mechanisms found in nature highlight the versatility of the cell cycle control machinery.

6-Gingerol Mediates its Anti Tumor Activities in Human Oral and Cervical Cancer Cell Lines through Apoptosis and Cell Cycle Arrest.

PubMed

Kapoor, Vaishali; Aggarwal, Sadhna; Das, Satya N

2016-04-01

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells. Copyright © 2016 John Wiley & Sons, Ltd.

Energy management by enhanced glycolysis in G1-phase in human colon cancer cells in vitro and in vivo.

PubMed

Bao, Yan; Mukai, Kuniaki; Hishiki, Takako; Kubo, Akiko; Ohmura, Mitsuyo; Sugiura, Yuki; Matsuura, Tomomi; Nagahata, Yoshiko; Hayakawa, Noriyo; Yamamoto, Takehiro; Fukuda, Ryo; Saya, Hideyuki; Suematsu, Makoto; Minamishima, Yoji Andrew

2013-09-01

Activation of aerobic glycolysis in cancer cells is well known as the Warburg effect, although its relation to cell- cycle progression remains unknown. In this study, human colon cancer cells were labeled with a cell-cycle phase-dependent fluorescent marker Fucci to distinguish cells in G1-phase and those in S + G2/M phases. Fucci-labeled cells served as splenic xenograft transplants in super-immunodeficient NOG mice and exhibited multiple metastases in the livers, frozen sections of which were analyzed by semiquantitative microscopic imaging mass spectrometry. Results showed that cells in G1-phase exhibited higher concentrations of ATP, NADH, and UDP-N-acetylglucosamine than those in S and G2-M phases, suggesting accelerated glycolysis in G1-phase cells in vivo. Quantitative determination of metabolites in cells synchronized in S, G2-M, and G1 phases suggested that efflux of lactate was elevated significantly in G1-phase. By contrast, ATP production in G2-M was highly dependent on mitochondrial respiration, whereas cells in S-phase mostly exhibited an intermediary energy metabolism between G1 and G2-M phases. Isogenic cells carrying a p53-null mutation appeared more active in glycolysis throughout the cell cycle than wild-type cells. Thus, as the cell cycle progressed from G2-M to G1 phases, the dependency of energy production on glycolysis was increased while the mitochondrial energy production was reciprocally decreased. These results shed light on distinct features of the phase-specific phenotypes of metabolic systems in cancer cells. ©2013 AACR.

A mathematical model for late term cancer chemotherapy

NASA Astrophysics Data System (ADS)

Izard, Zac; Hirschbeck, Sarah; Volk, Christian; Shojania Feizabadi, Mitra

2006-03-01

A mathematical model for cancer treated with the ``on-off'' type where the drug is either active or inactive and when the chemotherapeutic treatment only affects the cycling cells is presented. This model is considered for late term chemotherapy when the total population of cells doesn't show a significant change. The size of the cycling cells as a function of time has been investigated.

Paeoniflorin inhibits cell growth and induces cell cycle arrest through inhibition of FoxM1 in colorectal cancer cells.

PubMed

Yue, Meng; Li, Shiquan; Yan, Guoqiang; Li, Chenyao; Kang, Zhenhua

2018-01-01

Paeoniflorin (PF) exhibits tumor suppressive functions in a variety of human cancers. However, the function of PF and molecular mechanism in colorectal cancer are elusive. In the present study, we investigated whether PF could exert its antiproliferative activity, anti-migration, and anti-invasive function in colorectal cancer cells. We found that PF inhibited cell growth and induced apoptosis and blocked cell cycle progression in the G0/G1 phase in colorectal cancer cells. Moreover, we found that PF suppressed cell migration and invasion in colorectal cancer cells. FoxM1 has been reported to play an important oncogenic role in human cancers. We also determine whether PF inhibited the expression of FoxM1, leading to its anti-cancer activity. We found that PF treatment in colorectal cancer cells resulted in down-regulation of FoxM1. The rescue experiments showed that overexpression of FoxM1 abrogated the tumor suppressive function induced by PF treatment. Notably, depletion of FoxM1 promoted the anti-tumor activity of PF in colorectal cancer cells. Therefore, inhibition of FoxM1 could participate in the anti-tumor activity of PF in colorectal cancer cells.

The cell cycle and acute kidney injury

PubMed Central

Price, Peter M.; Safirstein, Robert L.; Megyesi, Judit

2009-01-01

Acute kidney injury (AKI) activates pathways of cell death and cell proliferation. Although seemingly discrete and unrelated mechanisms, these pathways can now be shown to be connected and even to be controlled by similar pathways. The dependence of the severity of renal-cell injury on cell cycle pathways can be used to control and perhaps to prevent acute kidney injury. This review is written to address the correlation between cellular life and death in kidney tubules, especially in acute kidney injury. PMID:19536080

Integrative Analysis Reveals an Outcome-associated and Targetable Pattern of p53 and Cell Cycle Deregulation in Diffuse Large B-cell Lymphoma

PubMed Central

Monti, Stefano; Chapuy, Bjoern; Takeyama, Kunihiko; Rodig, Scott J; Hao, Yangsheng; Yeda, Kelly T.; Inguilizian, Haig; Mermel, Craig; Curie, Treeve; Dogan, Ahmed; Kutok, Jeffery L; Beroukim, Rameen; Neuberg, Donna; Habermann, Thomas; Getz, Gad; Kung, Andrew L; Golub, Todd R; Shipp, Margaret A

2013-01-01

Summary Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy and suggest targeted treatment approaches. PMID:22975378

Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma

PubMed Central

Hu, Lihua; Li, Zhi; Liu, Jingpeng; Wang, Chunling; Nawshad, Ali

2016-01-01

Objective During the development of oral squamous cell carcinoma (OSCC), the transformed epithelial cells undergo increased proliferation resulting in tumor growth and invasion. Interestingly, throughout all phases of differentiation and progression of OSCC, TGFβ1 induces cell cycle arrest/apoptosis, however; the role of TGFβ1 in promoting cancer cell proliferation has not been explored in detail. The purpose of this study was to identify the effect of TGFβ1 on OSCC cell proliferation. Methods Using both human OSCC samples and cell lines (UMSCC38 and UMSCC 11B), we employed biochemical experiments to show protein, mRNA, gene expression and protein-DNA interactions during OSCC progression. Results Our results showed that TGFβ1 increased OSCC cell proliferation by up-regulating the expression of ΔNp63 and c-Myc oncogenes. While the basal OSCC cell proliferation is sustained by activating ΔNp63, increased induction of c-Myc causes unregulated OSCC cell proliferation. Following induction of the cell cycle by ΔNp63 and c-Myc, cancer cells that halt c-Myc activity undergo EMT/invasion while those that continue to express ΔNp63/c-Myc undergo unlimited progression through the cell cycle. Conclusion We conclude that OSCC proliferation is manifested by the induction of c-Myc in response to TGFβ1 signaling, which is essential for OSCC growth. Our data highlights the potential role of TGFβ1 in the induction of cancer progression and invasion of OSCC. PMID:27567435

Anti-Cervical Cancer Role of Matrine, Oxymatrine and Sophora Flavescens Alkaloid Gels and its Mechanism.

PubMed

Zhou, Yu Jie; Guo, Ya Jie; Yang, Xiao Li; Ou, Zhou Luo

2018-01-01

Background: Cervical cancer is one of the leading severe malignancies throughout the world. Sophra flavescens alkaloid (SFA) gels, a compound Traditional Chinese Medicine, has been clinically used in China for many years. Its individual active ingredients are matrine and oxymatrine, which has been showed that they can restrain primary tumorigenesis, while the underlying molecular mechanisms of SFA gels in cervical cancer cells remain unclear. Methods: To detect the effect of SFA gels and its active ingredients, CCK-8 assay and colony assay were used on cervical cancer cells proliferation. Transwell assay was used to detect cancer cell migration. Apoptosis and cell cycle arrest were used to detect whether SFA gels effect the cervical cancer cells proliferation. Western blot was used to detect whether SFA gels regulate the cervical cancer cells via the suppression of AKT/mTOR signaling pathway. Results: SFA gels can restrain cervical cancer cell proliferation, inhibit metastasis, induce cell cycle arrest in G2/M phase, induce cellular apoptosis through stimulation of Bax and E-cadherin, and suppression of Bcl-2, cyclin A, MMP2. Further study shows that SFA gels may regulate the cervical cancer cells via the suppression of AKT/mTOR signaling pathway. Conclusions: SFA gels, like its active ingredients, can restrain cervical cancer cells proliferation, suppress cervical cancer cell migration, induce the apoptosis and cell cycle arrest in cervical cancer cells. SFA gels may be a potential anti-tumor therapeutic agent for treating cervical cancer.

Anti-Cervical Cancer Role of Matrine, Oxymatrine and Sophora Flavescens Alkaloid Gels and its Mechanism

PubMed Central

Zhou, Yu Jie; Guo, Ya Jie; Yang, Xiao Li; Ou, Zhou Luo

2018-01-01

Background: Cervical cancer is one of the leading severe malignancies throughout the world. Sophra flavescens alkaloid (SFA) gels, a compound Traditional Chinese Medicine, has been clinically used in China for many years. Its individual active ingredients are matrine and oxymatrine, which has been showed that they can restrain primary tumorigenesis, while the underlying molecular mechanisms of SFA gels in cervical cancer cells remain unclear. Methods: To detect the effect of SFA gels and its active ingredients, CCK-8 assay and colony assay were used on cervical cancer cells proliferation. Transwell assay was used to detect cancer cell migration. Apoptosis and cell cycle arrest were used to detect whether SFA gels effect the cervical cancer cells proliferation. Western blot was used to detect whether SFA gels regulate the cervical cancer cells via the suppression of AKT/mTOR signaling pathway. Results: SFA gels can restrain cervical cancer cell proliferation, inhibit metastasis, induce cell cycle arrest in G2/M phase, induce cellular apoptosis through stimulation of Bax and E-cadherin, and suppression of Bcl-2, cyclin A, MMP2. Further study shows that SFA gels may regulate the cervical cancer cells via the suppression of AKT/mTOR signaling pathway. Conclusions: SFA gels, like its active ingredients, can restrain cervical cancer cells proliferation, suppress cervical cancer cell migration, induce the apoptosis and cell cycle arrest in cervical cancer cells. SFA gels may be a potential anti-tumor therapeutic agent for treating cervical cancer. PMID:29721044

Achieving Precision Death with Cell-Cycle Inhibitors that Target DNA Replication and Repair.

PubMed

Lin, Aimee Bence; McNeely, Samuel C; Beckmann, Richard P

2017-07-01

All cancers are characterized by defects in the systems that ensure strict control of the cell cycle in normal tissues. The consequent excess tissue growth can be countered by drugs that halt cell division, and, indeed, the majority of chemotherapeutics developed during the last century work by disrupting processes essential for the cell cycle, particularly DNA synthesis, DNA replication, and chromatid segregation. In certain contexts, the efficacy of these classes of drugs can be impressive, but because they indiscriminately block the cell cycle of all actively dividing cells, their side effects severely constrain the dose and duration with which they can be administered, allowing both normal and malignant cells to escape complete growth arrest. Recent progress in understanding how cancers lose control of the cell cycle, coupled with comprehensive genomic profiling of human tumor biopsies, has shown that many cancers have mutations affecting various regulators and checkpoints that impinge on the core cell-cycle machinery. These defects introduce unique vulnerabilities that can be exploited by a next generation of drugs that promise improved therapeutic windows in patients whose tumors bear particular genomic aberrations, permitting increased dose intensity and efficacy. These developments, coupled with the success of new drugs targeting cell-cycle regulators, have led to a resurgence of interest in cell-cycle inhibitors. This review in particular focuses on the newer strategies that may facilitate better therapeutic targeting of drugs that inhibit the various components that safeguard the fidelity of the fundamental processes of DNA replication and repair. Clin Cancer Res; 23(13); 3232-40. ©2017 AACR . ©2017 American Association for Cancer Research.

A minimal mathematical model combining several regulatory cycles from the budding yeast cell cycle.

PubMed

Sriram, K; Bernot, G; Képès, F

2007-11-01

A novel topology of regulatory networks abstracted from the budding yeast cell cycle is studied by constructing a simple nonlinear model. A ternary positive feedback loop with only positive regulations is constructed with elements that activates the subsequent element in a clockwise fashion. A ternary negative feedback loop with only negative regulations is constructed with the elements that inhibit the subsequent element in an anticlockwise fashion. Positive feedback loop exhibits bistability, whereas the negative feedback loop exhibits limit cycle oscillations. The novelty of the topology is that the corresponding elements in these two homogeneous feedback loops are linked by the binary positive feedback loops with only positive regulations. This results in the emergence of mixed feedback loops in the network that displays complex behaviour like the coexistence of multiple steady states, relaxation oscillations and chaos. Importantly, the arrangement of the feedback loops brings in the notion of checkpoint in the model. The model also exhibits domino-like behaviour, where the limit cycle oscillations take place in a stepwise fashion. As the aforementioned topology is abstracted from the budding yeast cell cycle, the events that govern the cell cycle are considered for the present study. In budding yeast, the sequential activation of the transcription factors, cyclins and their inhibitors form mixed feedback loops. The transcription factors that involve in the positive regulation in a clockwise orientation generates ternary positive feedback loop, while the cyclins and their inhibitors that involve in the negative regulation in an anticlockwise orientation generates ternary negative feedback loop. The mutual regulation between the corresponding elements in the transcription factors and the cyclins and their inhibitors generates binary positive feedback loops. The bifurcation diagram constructed for the whole system can be related to the different events of the cell cycle in terms of dynamical system theory. The checkpoint mechanism that plays an important role in different phases of the cell cycle are accounted for by silencing appropriate feedback loops in the model.

In Vivo and In Vitro Suppression of Hepatocellular Carcinoma by EF24, a Curcumin Analog

PubMed Central

Wang, Luoluo; Tian, Lantian; Song, Ruipeng; Han, Tianwen; Pan, Shangha; Liu, Lianxin

2012-01-01

The synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) is a potent analog of curcumin that exhibits enhanced biological activity and bioavailability without increasing toxicity. EF24 exerts antitumor activity by arresting the cell cycle and inducing apoptosis, suppressing many types of cancer cells in vitro. The antiproliferative and antiangiogenic properties of EF24 provide theoretical support for its development and application to liver cancers. We investigated the in vitro and in vivo activities of EF24 on liver cancer to better understand its therapeutic effects and mechanisms. EF24 induced significant apoptosis and G2/M-phase cell cycle arrest in mouse liver cancer cell lines, Hepa1-6 and H22. The expression levels of G2/M cell cycle regulating factors, cyclin B1 and Cdc2, were significantly decreased, pp53, p53, and p21 were significantly increased in EF24-treated cells. In addition, EF24 treatment significantly reduced Bcl-2 concomitant with an increase in Bax, enhanced the release of cytochrome c from the mitochondria into the cytosol, resulting in an upregulation of cleaved-caspase-3, which promoted poly (ADP-ribose) polymerase cleavage. EF24-treated cells also displayed decreases in phosphorylated Akt, phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Our in vitro protein expression data were confirmed in vivo using a subcutaneous hepatocellular carcinoma (HCC) tumor model. This mouse HCC model confirmed that total body weight was unchanged following EF24 treatment, although tumor weight was significantly decreased. Using an orthotopic HCC model, EF24 significantly reduced the liver/body weight ratio and relative tumor areas compared to the control group. In situ detection of apoptotic cells and quantification of Ki-67, a biomarker of cell proliferation, all indicated significant tumor suppression with EF24 treatment. These results suggest that EF24 exhibits anti-tumor activity on liver cancer cells via mitochondria-dependent apoptosis and inducing cell cycle arrest coupled with antiangiogenesis. The demonstrated activities of EF24 support its further evaluation as a treatment for human liver cancers. PMID:23118928

In vivo and in vitro suppression of hepatocellular carcinoma by EF24, a curcumin analog.

PubMed

Liu, Haitao; Liang, Yingjian; Wang, Luoluo; Tian, Lantian; Song, Ruipeng; Han, Tianwen; Pan, Shangha; Liu, Lianxin

2012-01-01

The synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) is a potent analog of curcumin that exhibits enhanced biological activity and bioavailability without increasing toxicity. EF24 exerts antitumor activity by arresting the cell cycle and inducing apoptosis, suppressing many types of cancer cells in vitro. The antiproliferative and antiangiogenic properties of EF24 provide theoretical support for its development and application to liver cancers. We investigated the in vitro and in vivo activities of EF24 on liver cancer to better understand its therapeutic effects and mechanisms. EF24 induced significant apoptosis and G2/M-phase cell cycle arrest in mouse liver cancer cell lines, Hepa1-6 and H22. The expression levels of G2/M cell cycle regulating factors, cyclin B1 and Cdc2, were significantly decreased, pp53, p53, and p21 were significantly increased in EF24-treated cells. In addition, EF24 treatment significantly reduced Bcl-2 concomitant with an increase in Bax, enhanced the release of cytochrome c from the mitochondria into the cytosol, resulting in an upregulation of cleaved-caspase-3, which promoted poly (ADP-ribose) polymerase cleavage. EF24-treated cells also displayed decreases in phosphorylated Akt, phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Our in vitro protein expression data were confirmed in vivo using a subcutaneous hepatocellular carcinoma (HCC) tumor model. This mouse HCC model confirmed that total body weight was unchanged following EF24 treatment, although tumor weight was significantly decreased. Using an orthotopic HCC model, EF24 significantly reduced the liver/body weight ratio and relative tumor areas compared to the control group. In situ detection of apoptotic cells and quantification of Ki-67, a biomarker of cell proliferation, all indicated significant tumor suppression with EF24 treatment. These results suggest that EF24 exhibits anti-tumor activity on liver cancer cells via mitochondria-dependent apoptosis and inducing cell cycle arrest coupled with antiangiogenesis. The demonstrated activities of EF24 support its further evaluation as a treatment for human liver cancers.

Features of the Phosphatidylinositol Cycle and its Role in Signal Transduction.

PubMed

Epand, Richard M

2017-08-01

The phosphatidylinositol cycle (PI-cycle) has a central role in cell signaling. It is the major pathway for the synthesis of phosphatidylinositol and its phosphorylated forms. In addition, some lipid intermediates of the PI-cycle, including diacylglycerol and phosphatidic acid, are also important lipid signaling agents. The PI-cycle has some features that are important for the understanding of its role in the cell. As a cycle, the intermediates will be regenerated. The PI-cycle requires a large amount of metabolic energy. There are different steps of the cycle that occur in two different membranes, the plasma membrane and the endoplasmic reticulum. In order to complete the PI-cycle lipid must be transferred between the two membranes. The role of the Nir proteins in the process has recently been elucidated. The lipid intermediates of the PI-cycle are normally highly enriched with 1-stearoyl-2-arachidonoyl molecular species in mammals. This enrichment will be retained as long as the intermediates are segregated from other lipids of the cell. However, there is a significant fraction (>15 %) of lipids in the PI-cycle of normal cells that have other acyl chains. Phosphatidylinositol largely devoid of arachidonoyl chains are found in cancer cells. Phosphatidylinositol species with less unsaturation will not be as readily converted to phosphatidylinositol-3,4,5-trisphosphate, the lipid required for the activation of Akt with resulting effects on cell proliferation. Thus, the cyclical nature of the PI-cycle, its dependence on acyl chain composition and its requirement for lipid transfer between two membranes, explain many of the biological properties of this cycle.

In vitro short-term exposure to air pollution PM{sub 2.5-0.3} induced cell cycle alterations and genetic instability in a human lung cell coculture model

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

Abbas, Imane; EA4492-UCEIV, Université du Littoral-Côte d’Opale, Dunkerque; Lebanese Atomic Energy Commission – CNRS, Beirut

Although its adverse health effects of air pollution particulate matter (PM2.5) are well-documented and often related to oxidative stress and pro-inflammatory response, recent evidence support the role of the remodeling of the airway epithelium involving the regulation of cell death processes. Hence, the overarching goals of the present study were to use an in vitro coculture model, based on human AM and L132 cells to study the possible alteration of TP53-RB gene signaling pathways (i.e. cell cycle phases, gene expression of TP53, BCL2, BAX, P21, CCND1, and RB, and protein concentrations of their active forms), and genetic instability (i.e. LOHmore » and/or MSI) in the PM{sub 2.5-0.3}-exposed coculture model. PM{sub 2.5-0.3} exposure of human AM from the coculture model induced marked cell cycle alterations after 24 h, as shown by increased numbers of L132 cells in subG1 and S+G2 cell cycle phases, indicating apoptosis and proliferation. Accordingly, activation of the TP53-RB gene signaling pathways after the coculture model exposure to PM{sub 2.5-0.3} was reported in the L132 cells. Exposure of human AM from the coculture model to PM{sub 2.5-0.3} resulted in MS alterations in 3p chromosome multiple critical regions in L132 cell population. Hence, in vitro short-term exposure of the coculture model to PM{sub 2.5-0.3} induced cell cycle alterations relying on the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation and genetic instability. - Highlights: • Better knowledge on health adverse effects of air pollution PM{sub 2.5}. • Human alveolar macrophage and normal human epithelial lung cell coculture. • Molecular abnormalities from TP53-RB gene signaling pathway. • Loss of heterozygosity and microsatellite instability. • Pathologic changes in morphology and number of cells in relation to airway remodeling.« less

Aqueous extract from pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell show activity against breast cancer cell line MCF-7 and Ehrlich ascites tumor in Balb-C mice.

PubMed

Hilbig, Josiane; Policarpi, Priscila de Britto; Grinevicius, Valdelúcia Maria Alves de Souza; Mota, Nádia Sandrine Ramos Santos; Toaldo, Isabela Maia; Luiz, Marilde Terezinha Bordignon; Pedrosa, Rozangela Curi; Block, Jane Mara

2018-01-30

In Brazil many health disorders are treated with the consumption of different varieties of tea. Shell extracts of pecan nut (Carya illinoinensis), which have significant amounts of phenolic compounds in their composition, are popularly taken as tea to prevent diverse pathologies. Phenolic compounds from pecan nut shell extract have been associated with diverse biological effects but the effect on tumor cells has not been reported yet. The aim of the current work was to evaluate the relationship between DNA fragmentation, cell cycle arrest and apoptosis induced by pecan nut shell extract and its antitumor activity. Cytotoxicity, proliferation, cell death and cell cycle were evaluated in MCF-7 cells by MTT, colony assay, differential coloring and flow cytometry assays, respectively. DNA damage effects were evaluated through intercalation into CT-DNA and plasmid DNA cleavage. Tumor growth inhibition, survival time increase, apoptosis and cell cycle arrest were assessed in Ehrlich ascites tumor in Balb/C mice. The cytotoxic effect of pecan nut shell extracts, the induction of cell death by apoptosis and also the cell cycle arrest in MCF-7 cells have been demonstrated. The survival time in mice with Ehrlich ascites tumor increased by 67%. DNA damage was observed in the CT-DNA, plasmid DNA and comet assays. The mechanism involved in the antitumor effect of pecan nut shell extracts may be related to the activation of key proteins involved in apoptosis cell death (Bcl-XL, Bax and p53) and on the cell cycle regulation (cyclin A, cyclin B and CDK2). These results were attributed to the phenolic profile of the extract, which presented compounds such as gallic, 4-hydroxybenzoic, chlorogenic, vanillic, caffeic and ellagic acid, and catechin, epicatechin, epigallocatechin and epicatechin gallate. The results indicated that pecan nut shell extracts are effective against tumor cells growth and may be considered as an alternative to the treatment of cancer. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

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

Wang, Ruoxing; Guo, Yan-Lin, E-mail: yanlin.guo@usm.edu

2012-10-01

Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remainsmore » unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. -- Highlights: Black-Right-Pointing-Pointer Inhibition of Cdks slows down mESCs proliferation. Black-Right-Pointing-Pointer mESCs display remarkable recovery capacity from short-term cell cycle interruption. Black-Right-Pointing-Pointer Short-term cell cycle interruption does not compromise mESC self-renewal. Black-Right-Pointing-Pointer Oct4 and Nanog are up-regulated via de novo synthesis by cell cycle interruption.« less

Imaging Prostate Cancer (PCa) Phenotype and Evolution

DTIC Science & Technology

2016-10-01

inhibit growth of some but not all cell lines. 2. Keywords: Deferiprone, aconitase, metabolism, tricarboxylic acid cycle , magnetic resonance 3...TRAMP C2 and MycCaP cell proliferation, migration, and invasiveness. Determine if knockdown of m-acon and Deferiprone inhibit TCA cycle activity...migration and inhibits TCA cycle (metabolism). Similarly in vivo (Aim 2), we 6 Fig. 2: Effect of DFP on in vivo growth of MycCaP (left) and TRAMP C2

Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin.

PubMed

Einbond, Linda Saxe; Wu, Hsan-Au; Kashiwazaki, Ryota; He, Kan; Roller, Marc; Su, Tao; Wang, Xiaomei; Goldsberry, Sarah

2012-10-01

Studies indicate that extracts and purified components, including carnosic acid, from the herb rosemary display significant growth inhibitory activity on a variety of cancers. This paper examines the ability of rosemary/carnosic acid to inhibit the growth of human breast cancer cells and to synergize with curcumin. To do this, we treated human breast cancer cells with rosemary/carnosic acid and assessed effects on cell proliferation, cell cycle distribution, gene expression patterns, activity of the purified Na/K ATPase and combinations with curcumin. Rosemary/carnosic acid potently inhibits proliferation of ER-negative human breast cancer cells and induces G1 cell cycle arrest. Further, carnosic acid is selective for MCF7 cells transfected for Her2, indicating that Her2 may function in its action. To reveal primary effects, we treated ER-negative breast cancer cells with carnosic acid for 6h. At a low dose, 5 μg/ml (15 μM), carnosic acid activated the expression of 3 genes, induced through the presence of antioxidant response elements, including genes involved in glutathione biosynthesis (CYP4F3, GCLC) and transport (SLC7A11). At a higher dose, 20 μg/ml, carnosic acid activated the expression of antioxidant (AKR1C2, TNXRD1, HMOX1) and apoptosis (GDF15, PHLDA1, DDIT3) genes and suppressed the expression of inhibitor of transcription (ID3) and cell cycle (CDKN2C) genes. Carnosic acid exhibits synergy with turmeric/curcumin. These compounds inhibited the activity of the purified Na-K-ATPase which may contribute to this synergy. Rosemary/carnosic acid, alone or combined with curcumin, may be useful to prevent and treat ER-negative breast cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

PubMed

Yang, Shucai; Ma, Jing; Xiao, Jianbing; Lv, Xiaohong; Li, Xinlei; Yang, Huike; Liu, Ying; Feng, Sijia; Zhang, Yafang

2012-08-01

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Copyright © 2012 Wiley Periodicals, Inc.

Voluntary running exercise prevents β-cell failure in susceptible islets of the Zucker diabetic fatty rat.

PubMed

Delghingaro-Augusto, Viviane; Décary, Simon; Peyot, Marie-Line; Latour, Martin G; Lamontagne, Julien; Paradis-Isler, Nicolas; Lacharité-Lemieux, Marianne; Akakpo, Huguette; Birot, Olivier; Nolan, Christopher J; Prentki, Marc; Bergeron, Raynald

2012-01-15

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving β-cell function is uncertain. We evaluated the role of physical activity on β-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with β-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key β-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining β-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and β-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered β-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

PubMed Central

Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

2013-01-01

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells

PubMed Central

2012-01-01

Background Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. Methods Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. Results Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. Conclusion Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (−692 to −684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression negatively via the induction of the CDK inhibitor p21WAF1. PMID:22591439

Lanthanum resulted in unbalance of nutrient elements and disturbance of cell proliferation cycles in V. faba L. seedlings.

PubMed

Wang, Chengrun; Lu, Xianwen; Tian, Yuan; Cheng, Tao; Hu, Lingling; Chen, Fenfen; Jiang, Chuanjun; Wang, Xiaorong

2011-11-01

Effects of lanthanum (La) on mineral nutrients, cell cycles, and root lengthening have been little reported. The present work investigated these physiological responses in roots of Vicia faba seedlings cultivated in La3+-contained solutions for 15 days. The results showed that the increasing contents of La in the roots and leaves contributed to disbalances of contents of Ca, Fe, Cu, Zn, Mg, Mn, P, and K elements, and potential redistributions of some elements in the roots and leaves. These disbalances might be involved in the subsequent alteration of cell cycle phases in the root tips. Low-dose promotion and high-dose inhibition (Hormetic effects) were demonstrated as the dose responses of G0/G1-, S- or G2/M-phase ratios. The cell cycles were most probably arrested at G1/S interphase by La3+ in the root tips. The fact that the root lengths were not consistent with the changes of cell cycle phases suggested that the cell proliferation activities might be masked by other factors (e.g., cell expansion) under long-time exposure to La3+.

Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation

PubMed Central

Ankers, John M; Awais, Raheela; Jones, Nicholas A; Boyd, James; Ryan, Sheila; Adamson, Antony D; Harper, Claire V; Bridge, Lloyd; Spiller, David G; Jackson, Dean A; Paszek, Pawel; Sée, Violaine; White, Michael RH

2016-01-01

Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators. These interactions modulate the NF-κB response. In S-phase, the NF-κB response was delayed or repressed, while cell cycle progression was unimpeded. By contrast, activation of NF-κB at the G1/S boundary resulted in a longer cell cycle and more synchronous initial NF-κB responses between cells. These data identify new mechanisms by which the cellular response to stress is differentially controlled at different stages of the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.10473.001 PMID:27185527

Statistical analysis of lithium iron sulfide status cell cycle life and failure mode

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

Gay, E.C.; Battles, J.E.; Miller, W.E.

1983-08-01

A statistical model was developed for life cycle testing of electrochemical cell life cycle trials and verified experimentally. The Weibull distribution was selected to predict the end of life for a cell, based on a 20 percent loss of initial stabilized capacity or a decrease to less than 95 percent coulombic efficiency. Groups of 12 or more Li-alloy/FeS cells were cycled to determine the mean time to failure (MTTF) and also to identify the failure modes. The cells were all full size electric vehicle batteries with 150-350 A-hr capacity. The Weibull shape factors were determined and verified in prediction ofmore » the number of cell failures in two 10 cell modules. The short circuit failure in the cells with BN-felt and MgO powder separators were found to be caused by the formation of Li-Al protrusions that penetrated the BN-felt separators, and the extrusion of active material at the edge of the electrodes.« less

EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management

PubMed Central

Ok, Chi Young; Li, Ling; Young, Ken H

2015-01-01

Epstein–Barr virus (EBV) is a ubiquitous herpesvirus, affecting >90% of the adult population. EBV targets B-lymphocytes and achieves latent infection in a circular episomal form. Different latency patterns are recognized based on latent gene expression pattern. Latent membrane protein-1 (LMP-1) mimics CD40 and, when self-aggregated, provides a proliferation signal via activating the nuclear factor-kappa B, Janus kinase/signal transducer and activator of transcription, phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase pathways to promote cellular proliferation. LMP-1 also induces BCL-2 to escape from apoptosis and gives a signal for cell cycle progression by enhancing cyclin-dependent kinase 2 and phosphorylation of retinoblastoma (Rb) protein and by inhibiting p16 and p27. LMP-2A blocks the surface immunoglobulin-mediated lytic cycle reactivation. It also activates the Ras/PI3K/Akt pathway and induces Bcl-xL expression to promote B-cell survival. Recent studies have shown that ebv-microRNAs can provide extra signals for cellular proliferation, cell cycle progression and anti-apoptosis. EBV is well known for association with various types of B-lymphocyte, T-lymphocyte, epithelial cell and mesenchymal cell neoplasms. B-cell lymphoproliferative disorders encompass a broad spectrum of diseases, from benign to malignant. Here we review our current understanding of EBV-induced lymphomagenesis and focus on biology, diagnosis and management of EBV-associated B-cell lymphoproliferative disorders. PMID:25613729

Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

PubMed

Lo Re, Oriana; Panebianco, Concetta; Porto, Stefania; Cervi, Carlo; Rappa, Francesca; Di Biase, Stefano; Caraglia, Michele; Pazienza, Valerio; Vinciguerra, Manlio

2018-02-01

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. © 2017 Wiley Periodicals, Inc.

Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

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

Krueger, Sarah A.; Collis, Spencer J.; Joiner, Michael C.

2007-11-15

Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G{sub 2}-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 {mu}g/mL chloroquine, 15 {mu}M genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eightmore » cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G{sub 2}-phase cells entering mitosis, using histone H3 phosphorylation analysis. Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G{sub 2}-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Conclusion: Overcoming HRS is reliant on the function of the early G{sub 2}-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.« less

Isolated spinal cord contusion in rats induces chronic brain neuroinflammation, neurodegeneration, and cognitive impairment. Involvement of cell cycle activation.

PubMed

Wu, Junfang; Stoica, Bogdan A; Luo, Tao; Sabirzhanov, Boris; Zhao, Zaorui; Guanciale, Kelsey; Nayar, Suresh K; Foss, Catherine A; Pomper, Martin G; Faden, Alan I

2014-01-01

Cognitive dysfunction has been reported in patients with spinal cord injury (SCI), but it has been questioned whether such changes may reflect concurrent head injury, and the issue has not been addressed mechanistically or in a well-controlled experimental model. Our recent rodent studies examining SCI-induced hyperesthesia revealed neuroinflammatory changes not only in supratentorial pain-regulatory sites, but also in other brain regions, suggesting that additional brain functions may be impacted following SCI. Here we examined effects of isolated thoracic SCI in rats on cognition, brain inflammation, and neurodegeneration. We show for the first time that SCI causes widespread microglial activation in the brain, with increased expression of markers for activated microglia/macrophages, including translocator protein and chemokine ligand 21 (C-C motif). Stereological analysis demonstrated significant neuronal loss in the cortex, thalamus, and hippocampus. SCI caused chronic impairment in spatial, retention, contextual, and fear-related emotional memory-evidenced by poor performance in the Morris water maze, novel objective recognition, and passive avoidance tests. Based on our prior work implicating cell cycle activation (CCA) in chronic neuroinflammation after SCI or traumatic brain injury, we evaluated whether CCA contributed to the observed changes. Increased expression of cell cycle-related genes and proteins was found in hippocampus and cortex after SCI. Posttraumatic brain inflammation, neuronal loss, and cognitive changes were attenuated by systemic post-injury administration of a selective cyclin-dependent kinase inhibitor. These studies demonstrate that chronic brain neurodegeneration occurs after isolated SCI, likely related to sustained microglial activation mediated by cell cycle activation.

Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.

PubMed

Bandara, L R; Adamczewski, J P; Zamanian, M; Poon, R Y; Hunt, T; Thangue, N B

1992-01-01

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2017-11-01

SUBJECT TERMS cell cycle, Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY...screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor, cancer, cell-cycle inhibition, activation...modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography . 3.0 ACCOMPLISHMENTS Summary: We

12-Chloracetyl-PPD, a novel dammarane derivative, shows anti-cancer activity via delay the progression of cell cycle G2/M phase and reactive oxygen species-mediate cell apoptosis.

PubMed

Wang, Xu De; Sun, Yuan Yuan; Zhao, Chen; Qu, Fan Zhi; Zhao, Yu Qing

2017-03-05

(20R)-Dammarane-3β, 12β, 20, 25-tetrol (25-OH-PPD) is a ginsenoside isolated from Panax ginseng (C. A. Meyer). This compound exhibits anti-cancer activities on many human cancer cell lines. In this study, we investigated anti-cancer mechanisms of 12β-O-( L -Chloracetyl)-dammar-20(22)-ene-3β,25-diol(12-Chloracetyl-PPD), a modified 25-OH-PPD. We found that compound 12-Chloracetyl-PPD resulted in a concentration-dependent inhibition of viability in prostate, breast, and gastric cancer cells, without affecting the viability of normal cell (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2). In MDA-MB-435 and C4-2B cancer cells, 12-Chloracetyl-PPD induced G2/M cell cycle arrest, down-regulated mouse double minute 2 (MDM2) expression, up-regulated p53 expression, triggered apoptosis, and stimulated reactive oxygen species production. Apoptosis can be attenuated by the reactive oxygen species scavenger N-acetylcysteine. Our results suggested that compound 12-Chloracetyl-PPD showed obvious anti-cancer activity based on delaying cell cycle arrest and inducing cell apoptosis by reactive oxygen species production, which supported development of 12-Chloracetyl-PPD as a potential agent for cancer chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Representing perturbed dynamics in biological network models

NASA Astrophysics Data System (ADS)

Stoll, Gautier; Rougemont, Jacques; Naef, Felix

2007-07-01

We study the dynamics of gene activities in relatively small size biological networks (up to a few tens of nodes), e.g., the activities of cell-cycle proteins during the mitotic cell-cycle progression. Using the framework of deterministic discrete dynamical models, we characterize the dynamical modifications in response to structural perturbations in the network connectivities. In particular, we focus on how perturbations affect the set of fixed points and sizes of the basins of attraction. Our approach uses two analytical measures: the basin entropy H and the perturbation size Δ , a quantity that reflects the distance between the set of fixed points of the perturbed network and that of the unperturbed network. Applying our approach to the yeast-cell-cycle network introduced by Li [Proc. Natl. Acad. Sci. U.S.A. 101, 4781 (2004)] provides a low-dimensional and informative fingerprint of network behavior under large classes of perturbations. We identify interactions that are crucial for proper network function, and also pinpoint functionally redundant network connections. Selected perturbations exemplify the breadth of dynamical responses in this cell-cycle model.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats

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

Kim, Su-Jung; Chung, Yong-Koo; Chung, Tae-Wook

2009-01-09

Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1{beta} (IL-1{beta}) stimulation with increasing in vitro age. Tumor necrosis factor-{alpha} (TNF-{alpha})-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-{kappa}B and AP-1.more » These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.« less

Methotrexate increases expression of cell cycle checkpoint genes via Jun-N-terminal kinase activation

PubMed Central

Spurlock, Charles F.; Tossberg, John T.; Fuchs, Howard A.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. PMID:22183962

A Learning Cycle Approach To Introducing Osmosis.

ERIC Educational Resources Information Center

Lawson, Anton E.

2000-01-01

Presents an inquiry activity with a learning cycle approach to engage students in testing their own hypotheses about how molecules move through cell membranes. Offers student materials and teacher materials, including teaching tips for each phase of the learning cycle. (Contains 11 references.) (ASK)

Development and testing of a high cycle life 30 A-h sealed AgO-Zn battery

NASA Technical Reports Server (NTRS)

Bogner, R. S.

1972-01-01

A two-phase program was initiated to investigate design parameters and technology to develop an improved AgO-Zn battery. The basic performance goal was 100 charge/discharge cycles (22 h/2 h) at 50 percent depth of discharge following a six-month period of charged stand at room temperature. Phase 1, cell evaluation, involved testing 70 cells in five-cell groups. The major design variables were active material ratios, electrolyte concentrations, separator systems, and negative plate shape. Phase 1 testing showed that cycle life could be improved 10 percent to 20 percent by using greater ratios of zinc to silver oxide and higher electrolyte concentrations. Wedge-shaped negatives increased cycle life by nearly 100 percent. Phase 2 battery evaluation, which was initiated before the Phase 1 results were known completely, involved evaluation of six designs as 19-cell batteries. Only one battery exceeded 100 cycles following nine months charged stand.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost

PubMed Central

Ding, Xiang; Zhu, Hongqing; Hou, Yiling; Hou, Wanru; Zhang, Nan; Fu, Lei

2017-01-01

Background: The mechanism of the immunoregulatory activities of polysaccharide is still not clear. Materials and Methods: Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages. Results: These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups. Conclusion: An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways. SUMMARY Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide. PMID:28839373

Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

PubMed Central

Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

2011-01-01

Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

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

Tian, Junqiang; Doi, Hiroshi; Saar, Matthias

2013-12-01

Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome.more » The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.« less

Genetic alterations in Krebs cycle and its impact on cancer pathogenesis.

PubMed

Sajnani, Karishma; Islam, Farhadul; Smith, Robert Anthony; Gopalan, Vinod; Lam, Alfred King-Yin

2017-04-01

Cancer cells exhibit alterations in many cellular processes, including oxygen sensing and energy metabolism. Glycolysis in non-oxygen condition is the main energy production process in cancer rather than mitochondrial respiration as in benign cells. Genetic and epigenetic alterations of Krebs cycle enzymes favour the shift of cancer cells from oxidative phosphorylation to anaerobic glycolysis. Mutations in genes encoding aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, and citrate synthase are noted in many cancers. Abnormalities of Krebs cycle enzymes cause ectopic production of Krebs cycle intermediates (oncometabolites) such as 2-hydroxyglutarate, and citrate. These oncometabolites stabilize hypoxia inducible factor 1 (HIF1), nuclear factor like 2 (Nrf2), inhibit p53 and prolyl hydroxylase 3 (PDH3) activities as well as regulate DNA/histone methylation, which in turn activate cell growth signalling. They also stimulate increased glutaminolysis, glycolysis and production of reactive oxygen species (ROS). Additionally, genetic alterations in Krebs cycle enzymes are involved with increased fatty acid β-oxidations and epithelial mesenchymal transition (EMT) induction. These altered phenomena in cancer could in turn promote carcinogenesis by stimulating cell proliferation and survival. Overall, epigenetic and genetic changes of Krebs cycle enzymes lead to the production of oncometabolite intermediates, which are important driving forces of cancer pathogenesis and progression. Understanding and applying the knowledge of these mechanisms opens new therapeutic options for patients with cancer. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

PubMed Central

Laos, Maarja; Anttonen, Tommi; Kirjavainen, Anna; Hällström, Taija af; Laiho, Marikki; Pirvola, Ulla

2014-01-01

Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with the in vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs. PMID:25063730

Terrestrosin D, a steroidal saponin from Tribulus terrestris L., inhibits growth and angiogenesis of human prostate cancer in vitro and in vivo.

PubMed

Wei, Shihu; Fukuhara, Hideo; Chen, Guang; Kawada, Chiaki; Kurabayashi, Atsushi; Furihata, Mutsuo; Inoue, Keiji; Shuin, Taro

2014-01-01

The aim of this study was to investigate whether terrestrosin D (TED) inhibits the progression of castration-resistant prostate cancer and consider its mechanism. Cell cycle, mitochondrial membrane potential (ΔΨm) and apoptosis were determined by flow cytometry. Caspase-3 activity and vascular endothelial growth factor secretion were detected by a caspase-3 assay and human vascular endothelial growth factor kit, respectively. A PC-3 xenograft mouse model was used to evaluate the anticancer effect of TED in vivo. In vitro, TED strongly suppressed the growth of prostate cancer cells and endothelial cells in a dose-dependent manner. TED induced cell cycle arrest and apoptosis in PC-3 cells and human umbilical vascular endothelial cells (HUVECs). TED-induced apoptosis did not involve the caspase pathway. TED also decreased ΔΨm in PC-3 cells and HUVECs. In vivo, TED significantly suppressed tumor growth in nude mice bearing PC-3 cells, without any overt toxicity. Immunohistochemical analysis showed TED induced apoptotic cell death and inhibited angiogenesis in xenograft tumor cells. Cell cycle arrest and induction of apoptosis in cancer cells and endothelial cells might be plausible mechanisms of actions for the observed antitumor and antiangiogenic activities of TED. © 2014 S. Karger AG, Basel.

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest

PubMed Central

Rai, Urvashi; Najm, Fadi

2017-01-01

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle. PMID:28339487

Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology.

PubMed

Dutto, Ilaria; Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A; Prosperi, Ennio

2015-02-01

The cell cycle inhibitor p21(CDKN1A) is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main, well-known function of p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. In addition, p21 is involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. However, p21 appears to a have a dual-face behavior because, in addition to its tumor suppressor functions, it may act as an oncogene, depending on the cell type and on the cellular localization. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions have been analyzed in an impressive number of studies investigating the activity of several types of chemicals, in order to determine their possible harmful effects on human cells. Here, we review these studies in order to highlight the different roles p21 may play in the cell response to chemical exposure and to better evaluate the information provided by this biomarker.

Overexpression of the Ubiquilin-4 (UBQLN4) is Associated with Cell Cycle Arrest and Apoptosis in Human Normal Gastric Epithelial Cell Lines GES-1 Cells by Activation of the ERK Signaling Pathway

PubMed Central

Huang, Shengkai; Dong, Xin; Wang, Jia; Ding, Jie; Li, Yan; Li, Dongdong; Lin, Hong; Wang, Wenjie; Zhao, Mei

2018-01-01

Background Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. Material/Methods We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. Results Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. Conclusions These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells. PMID:29807370

Scalloped and Yorkie are required for cell cycle re-entry of quiescent cells after tissue damage.

PubMed

Meserve, Joy H; Duronio, Robert J

2015-08-15

Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. We used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. We discovered that Hippo signaling regulates compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage. © 2015. Published by The Company of Biologists Ltd.

Purine analogue ENERGI-F706 induces apoptosis of 786-O renal carcinoma cells via 5'-adenosine monophosphate-activated protein kinase activation.

PubMed

Hsu, Chao-Yu; Lin, Chun-Hsiang; Lin, Jiun-Tsai; Cheng, Yi-Fang; Chen, Han-Min; Kao, Shao-Hsuan

2015-09-01

Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGI‑F706 on the human renal carcinoma cell line 786‑O and the underlying mechanisms. The results revealed that ENERGI‑F706 (0.2‑0.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786‑O cells, ENERGI‑F706 exerted less suppressive effects on the viability of the human non‑tumorigenic renal cell line HK‑2. Flow cytometric analysis showed that ENERGI‑F706 contributed to cell cycle arrest at S‑phase and triggered apoptosis of 786‑O cells. Immunoblot analysis revealed that anti‑apoptotic B‑cell lymphoma 2 (Bcl‑2) levels were reduced and pro‑apoptotic Bcl‑2‑associated X protein levels were diminished. In addition, activation of caspase‑9, caspase‑3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786‑O cells in response to ENERGI‑F706. Effects of ENERGI‑F706 on AMPK cascades were investigated and the results showed that ENERGI‑F706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl‑2, cleavage of caspase‑3 and PARP as well as suppressed cell viability induced by ENERGI‑F706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGI‑F706 significantly suppressed the viability of 786‑O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGI‑F706 may be suitable for the treatment of renal cell carcinoma.

Antiproliferative activity of Greek propolis.

PubMed

Pratsinis, Harris; Kletsas, Dimitris; Melliou, Eleni; Chinou, Ioanna

2010-04-01

The butanolic extract and the isolated chemical constituents, mainly diterpenes and flavonoids, from Greek propolis have been tested for their cytostatic activities against human malignant and normal cell strains. The extract and the diterpenes were found to be the most active against HT-29 human colon adenocarcinoma cells, without affecting normal human cells. Manool, a diterpene isolated for the first time from Greek propolis, was the most active compound, arresting the cancer cells at the G(2)/M phase of the cell cycle.

6-Gingerol inhibits osteosarcoma cell proliferation through apoptosis and AMPK activation.

PubMed

Fan, Jingzhang; Yang, Xin; Bi, Zhenggang

2015-02-01

6-Gingerol, a major component of ginger, is demonstrated to possess a variety of pharmacological activities. Despite demonstration of its anti-cancer activity, the exact mechanism underlying the effects of 6-gingerol against sarcoma remains sketchy. In the present study, we investigated the anti-cancer effects of 6-gingerol on osteosarcoma cells. MTT assay was performed to determine cell viability. Phosphorylation and protein levels were determined by immunoblotting. Cell cycle was determined using flow cytometry. Quantitative polymerase chain reaction was employed to determine the changes in the messenger RNA (mRNA) expression of genes. Treatment with 6-gingerol resulted in a significant decrease in the viability of osteosarcoma cells in a dose-dependent fashion. In parallel, the number of cells arrested at the sub-G1 cell cycle phase was significantly increased. The results showed that 6-gingerol induced activation of caspase cascades and regulated cellular levels of Bcl2 and Bax. Moreover, 6-gingerol activated AMP-activated protein kinase (AMPK) signaling associated with the apoptotic pathways. Our findings suggest that 6-gingerol suppresses the growth of osteosarcoma cells. The anti-cancer activity is attributed to the activation of apoptotic signaling and the inhibition of anti-apoptotic signaling incorporating with 6-gingerol-induced AMPK activation. The study identifies a new molecular mechanism by which AMPK is involved in anti-cancer effects of 6-gingerol.

Phosphatidylcholine and the CDP-Choline Cycle

PubMed Central

Fagone, Paolo; Jackowski, Suzanne

2012-01-01

The CDP-choline pathway of phosphatidylcholine (PtdCho) biosynthesis was first described more than 50 years ago. Investigation of the CDP-choline pathway in yeast provides a basis for understanding the CDP-choline pathway in mammals. PtdCho is considered as an intermediate in a cycle of synthesis and degradation, and the activity of a CDP-choline cycle is linked to subcellular membrane lipid movement. The components of the mammalian CDP-choline pathway include choline transport, choline kinase, phosphocholine cytidylyltransferase, and choline phosphotransferase activities. The protein isoforms and biochemical mechanisms of regulation of the pathway enzymes are related to their cell and tissue-specific functions. Regulated PtdCho turnover mediated by phospholipases or neuropathy target esterase participates in the mammalian CDP-choline cycle. Knockout mouse models define the biological functions of the CDP-choline cycle in mammalian cells and tissues. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:23010477

HSF1 is activated as a consequence of lymphocyte activation and regulates a major proteostasis network in T cells critical for cell division during stress

PubMed Central

Gandhapudi, Siva K.; Murapa, Patience; Threlkeld, Zachary D.; Ward, Martin; Sarge, Kevin D.; Snow, Charles; Woodward, Jerold G.

2013-01-01

Heat Shock Transcription Factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is also evoked in response to a variety of cellular stressors including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated thatmurine HSF1 became activated to the DNA-binding form and trans-activated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1+/+ and HSF1−/− gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in non-stressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1−/− T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under non-febrile temperatures. HSF1−/− T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was not affected. Finally, B cell, and hematopoietic stem cell proliferation from HSF1−/− mice, but not HSF1+/+ mice were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions. PMID:24043900

Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

PubMed

Hwang, S-K; Jin, H; Kwon, J T; Chang, S-H; Kim, T H; Cho, C-S; Lee, K H; Young, M R; Colburn, N H; Beck, G R; Yang, H-S; Cho, M-H

2007-09-01

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

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.

Alkaline and non-aqueous proton-conducting pouch-cell batteries

DOEpatents

Young, Kwo-hsiung; Nei, Jean; Meng, Tiejun

2018-01-02

Provided are sealed pouch-cell batteries that are alkaline batteries or non-aqueous proton-conducing batteries. A pouch cell includes a flexible housing such as is used for pouch cell construction where the housing is in the form of a pouch, a cathode comprising a cathode active material suitable for use in an alkaline battery, an anode comprising an anode active material suitable for use in an alkaline battery, an electrolyte that is optionally an alkaline or proton-conducting electrolyte, and wherein the pouch does not include or require a safety vent or other gas absorbing or releasing system as the anode active material and the cathode active material do not increase the internal atmospheric pressure any more than 2 psig during cycling. The batteries provided function contrary to the art recognized belief that such battery systems were impossible due to unacceptable gas production during cycling.

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

Frémin, Christophe; Bessard, Anne; Ezan, Frédéric; Gailhouste, Luc; Régeard, Morgane; Le Seyec, Jacques; Gilot, David; Pagès, Gilles; Pouysségur, Jacques; Langouët, Sophie; Baffet, Georges

2009-03-01

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.

Functional Interaction between Phosducin-like Protein 2 and Cytosolic Chaperonin Is Essential for Cytoskeletal Protein Function and Cell Cycle Progression

PubMed Central

Stirling, Peter C.; Srayko, Martin; Takhar, Karam S.; Pozniakovsky, Andrei; Hyman, Anthony A.

2007-01-01

The C haperonin Containing Tcp1 (CCT) maintains cellular protein folding homeostasis in the eukaryotic cytosol by assisting the biogenesis of many proteins, including actins, tubulins, and regulators of the cell cycle. Here, we demonstrate that the essential and conserved eukaryotic phosducin-like protein 2 (PhLP2/PLP2) physically interacts with CCT and modulates its folding activity. Consistent with this functional interaction, temperature-sensitive alleles of Saccharomyces cerevisiae PLP2 exhibit cytoskeletal and cell cycle defects. We uncovered several high-copy suppressors of the plp2 alleles, all of which are associated with G1/S cell cycle progression but which do not appreciably affect cytoskeletal protein function or fully rescue the growth defects. Our data support a model in which Plp2p modulates the biogenesis of several CCT substrates relating to cell cycle and cytoskeletal function, which together contribute to the essential function of PLP2. PMID:17429077

Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle

PubMed Central

Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

2016-01-01

Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis. PMID:27213594

Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle.

PubMed

Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

2016-06-28

Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis.

Interaction of FeS 2 and Sulfur in Li-S Battery System

DOE PAGES

Sun, Ke; Cama, Christina A.; DeMayo, Rachel A.; ...

2016-09-09

Many transition metal sulfides are electronically conductive, electrochemically active and reversible in reactions with lithium. However, the application of transition metal sulfides as sulfur cathode additives in lithium-sulfur (Li-S) batteries has not been fully explored. In this study, Pyrite (FeS 2) is studied as a capacity contributing conductive additive in sulfur cathode for Li-S batteries. Electrochemically discharging the S-FeS 2 composite electrodes to 1.0 V activates the FeS 2 component, contributing to the improved Li-S cell discharge energy density. However, direct activation of the FeS 2 component in a fresh S-FeS 2 cell results in a significant shuttling effect inmore » the subsequent charging process, preventing further cell cycling. The slight FeS 2 solubility in electrolyte and its activation alone in S-FeS 2 cells are not the root causes of the severe shuttling effect. The observed severe shuttling effect is strongly correlated to the 1st charging of the activated S-FeS 2 electrode that promotes iron dissolution in electrolyte and the deposition of electronically conductive FeS on the anode SEI. Pre-cycling of the S-FeS 2 cell prior to the FeS 2 activation or the use of LiNO 3 electrolyte additive help to prevent the severe shuttling effect and allow the cell to cycle between 2.6 V to 1.0 V with an extra capacity contribution from the FeS2 components. However, a more effective method of anode pre-passivation is still needed to fully protect the lithium surface from FeS deposition and allow the S-FeS 2 electrode to maintain high energy density over extended cycles. A mechanism explaining the observed phenomena based on the experimental data is proposed and discussed« less

Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

PubMed

Lee, Min Ho; Cho, Yoonjung; Jung, Byung Chul; Kim, Sung Hoon; Kang, Yeo Wool; Pan, Cheol-Ho; Rhee, Ki-Jong; Kim, Yoon Suk

2015-08-14

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth. Copyright © 2015 Elsevier Inc. All rights reserved.

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer.

PubMed

La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Verrico, Annalisa; Miele, Andrea; Monti, Ludovica; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Ricci, Biancamaria; Soriani, Alessandra; Santoni, Angela; Caraglia, Michele; Porto, Stefania; Da Pozzo, Eleonora; Martini, Claudia; Brancale, Andrea; Marinelli, Luciana; Novellino, Ettore; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Bigogno, Chiara; Dondio, Giulio; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

2015-08-13

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer

PubMed Central

La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Verrico, Annalisa; Miele, Andrea; Monti, Ludovica; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Ricci, Biancamaria; Soriani, Alessandra; Santoni, Angela; Caraglia, Michele; Porto, Stefania; Pozzo, Eleonora Da; Martini, Claudia; Brancale, Andrea; Marinelli, Luciana; Novellino, Ettore; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Bigogno, Chiara; Dondio, Giulio; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

2015-01-01

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethox-yphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4–7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20–50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer. PMID:26132075

Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

PubMed

Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

2004-09-03

Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.

Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

PubMed Central

Bandara, L R; Buck, V M; Zamanian, M; Johnston, L H; La Thangue, N B

1993-01-01

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation. Images PMID:8223441

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.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

PubMed

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-04-01

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

PubMed Central

Le, Quy; Maizels, Nancy

2015-01-01

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome. PMID:26355458

Cytokinetics of adult rat SVZ after EAE.

PubMed

Sajad, Mir; Chawla, Raman; Zargan, Jamil; Umar, Sadiq; Sadaqat, Mir; Khan, Haider A

2011-01-31

Cytokinetics regulating cell cycle division can be modulated by several endogenous factors. EAE (experimental autoimmune encephalomyelitis) increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S phase labeling with 5-bromo-2-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after EAE. 20% of the SVZ cell population was proliferating in adjuvant control rats. However, EAE significantly increased them up to 27% and these cells had a cell cycle length (TC) of 15.6h, significantly (P<0.05) shorter than the 19 h TC in non EAE SVZ cells. Few TUNEL (+) cells were detected in the SVZ cells of adjuvant controls. EAE increased (P<0.05) TUNEL (+) nuclei in SVZ suggesting early stage progenitor cell death. Cell cycle phase analysis revealed that EAE substantially shortened the length of the G1 phase (9.6h) compared with the G1 phase of 12.25 h in adjuvant control SVZ cells (P<0.05). This reduction in G1 contributes to EAE-induced reduction of TC because no significant changes were detected on the length of S, G2 and M phases between the two groups. Our results show a surge in proliferating progenitor cells in the SVZ with concomitant increase in apoptotic cell death after EAE. Furthermore, increase in the SVZ proliferation contributes to EAE-induced neurogenesis and this increase is regulated by shortening the G1 phase. Our investigation suggests the activation of quiescent cells in SVZ to generate actively proliferating progenitors. Moreover, the increase in the cell death in proliferating population may contribute towards negative regulation of proliferative cell number and hence diminished regenerative capacity of CNS following EAE. Copyright © 2010 Elsevier B.V. All rights reserved.

Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells

PubMed Central

Navanesan, Suerialoasan; Abdul Wahab, Norhanom; Manickam, Sugumaran; Sim, Kae Shin

2015-01-01

Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death. PMID:26287817

Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization

PubMed Central

Witte, Kristen; Strickland, Devin; Glotzer, Michael

2017-01-01

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment. DOI: http://dx.doi.org/10.7554/eLife.26722.001 PMID:28682236

Degradation diagnosis of aged Li4Ti5O12/LiFePO4 batteries

NASA Astrophysics Data System (ADS)

Castaing, Rémi; Reynier, Yvan; Dupré, Nicolas; Schleich, Donald; Jouanneau Si Larbi, Séverine; Guyomard, Dominique; Moreau, Philippe

2014-12-01

Li4Ti5O12/LiFePO4 cells are cycled under 4 different conditions of discharge profile (galvanostatic or driving-based) and cycling rates (C/8 or 1C) during 4-5 months. All the cells exhibit capacity fade whose extent is not correlated with the aging condition. In order to understand aging phenomena, cells are disassembled at the end of cycle life and the recovered electrodes are analyzed using electrochemistry, electron microscopy, XRD and MAS-NMR. Positive and negative electrodes show no loss in active material and no change in electrochemical activity, active material structure and composite electrode structure. This rules out any irreversible electrode degradation. Lithium stoichiometry estimated by both XRD and electrochemistry is unexpectedly low in the positive electrode when the aging is stopped at full discharge. That indicates a loss of cyclable lithium or electrons leading to cell balancing evolution. That loss may have been caused by parasitic reactions occurring at both electrodes, in accordance with their rich surface chemistry as evidenced by MAS-NMR.

ERK reinforces actin polymerization to power persistent edge protrusion during motility.

PubMed

Mendoza, Michelle C; Vilela, Marco; Juarez, Jesus E; Blenis, John; Danuser, Gaudenz

2015-05-19

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. We tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular signal-regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell surface receptors, and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. Copyright © 2015, American Association for the Advancement of Science.

Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy

PubMed Central

Podo, Franca; Paris, Luisa; Cecchetti, Serena; Spadaro, Francesca; Abalsamo, Laura; Ramoni, Carlo; Ricci, Alessandro; Pisanu, Maria Elena; Sardanelli, Francesco; Canese, Rossella; Iorio, Egidio

2016-01-01

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20–50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods. PMID:27532027

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

PubMed

Chao, Xu; Zhou, Xiaojun; Zheng, Gang; Dong, Changhu; Zhang, Wei; Song, Xiaomei; Jin, Tianbo

2014-05-01

Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong's Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture. This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells. Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot. Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123 µmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins. Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.

The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines.

PubMed

Strakova, Nicol; Ehrmann, Jiri; Dzubak, Petr; Bouchal, Jan; Kolar, Zdenek

2004-06-01

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.

Curcumin analog EF24 induces apoptosis via ROS-dependent mitochondrial dysfunction in human colorectal cancer cells.

PubMed

He, Guodong; Feng, Chen; Vinothkumar, Rajamanickam; Chen, Weiqian; Dai, Xuanxuan; Chen, Xi; Ye, Qingqing; Qiu, Chenyu; Zhou, Huiping; Wang, Yi; Liang, Guang; Xie, Yubo; Wu, Wei

2016-12-01

Colorectal cancer is the most commonly diagnosed malignancy with high mortality rates worldwide. Improved therapeutic strategies with minimal adverse side effects are urgently needed. In this study, the anti-tumor effects of EF24, a novel analog of the natural compound curcumin, were evaluated in colorectal cancer cells. The anti-tumor activity of EF24 on human colon cancer lines (HCT-116, SW-620, and HT-29) was determined by measures of cell cycle arrest, apoptosis, and mitochondrial function. The contribution of ROS in the EF24-induced anti-tumor activity was evaluated by measures of H 2 O 2 and pretreatment with an ROS scavenger, NAC. The findings indicated that EF24 treatment dose-dependently inhibited cell viability and caused cell cycle arrest at G2/M phase in all the tested colon cancer cell lines. Furthermore, we demonstrated that EF24 treatment induced apoptosis effectively via enhancing intracellular accumulation of ROS in both HCT-116 and SW-620 cells, but with moderate effects in HT-29 cells. We found that EF24 treatment decreased the mitochondrial membrane potential in the colon cancer cells, leading to the release of mitochondrial cytochrome c. Also, EF24 induced activation of caspases 9 and 3, causing decreased Bcl-2 protein expression and Bcl-2/Bax ratio. Pretreatment with NAC, a ROS scavenger, abrogated the EF24-induced cell death, apoptosis, cell cycle arrest, and mitochondrial dysfunction, suggesting an upstream ROS generation which was responsible for the anticancer effects of EF24. Our findings support an anticancer mechanism by which EF24 enhanced ROS accumulation in colon cancer cells, thereby resulting in mitochondrial membrane collapse and activated intrinsic apoptotic signaling. Thus, EF24 could be a potential candidate for therapeutic application of colon cancer.

Krebs Cycle Moonlights in Caspase Regulation.

PubMed

Minis, Adi; Steller, Hermann

2016-04-04

In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and spatially restricted caspase activation during Drosophila sperm differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Growth cycle of Helicobacter pylori in gastric mucous layer.

PubMed

Nakazawa, Teruko

2002-12-01

Helicobacter pylori bacterium is characterized by its strong urease activity. Our studies on the role of H. pylori urease revealed; (i) it is essential for colonization, (ii) exogenous urea is required for acid resistance, (iii) the bacteria have the ability to move toward urea and sodium bicarbonate, (iv) urea hydrolysis accelerates chemotactic locomotion, and (v) decay of urease mRNA to accomplish the active center is pH-regulated; i.e., the mRNA is stabilized and destabilized under acidic and neutral conditions, respectively. Based on the above results, I propose the growth cycle of H. pylori in gastric mucous layer. H. pylori bacteria proliferate on the epithelial cell surface by utilizing nutrients derived from degraded cells. Proliferated bacteria leave the cell surface to pH-variable region where they encounter strong acid. Urease is activated with simultaneous opening of UreI channel so that urea is hydrolyzed to neutralize acid. Chemotaxis of H. pylori toward urea and sodium bicarbonate that are abundant on the cell surface is accelerated by urea hydrolysis so that the bacteria go back to the cell surface for the next round of proliferation. This growth cycle may allow the bacteria to infect persistently in the stomach.

Tivantinib (ARQ-197) exhibits anti-tumor activity with down-regulation of FAK in oral squamous cell carcinoma

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

Xi, Wei-Hong; Yang, Li-Yun; Cao, Zhong-Yi, E-mail: m18070383032@163.com

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and the 5 years survival rate of the patients is about 60% in the USA, due to acquired chemotherapeutic resistance and metastasis of the disease. In this study, we found that tivantinib, a selective MET inhibitor, suppresses OCSS cell proliferation and colony formation, however, anti-tumor activities induced by tivantinib are independent of the inhibition of MET signaling pathway. In addition, tivantinib cause G2/M cell cycle arrest and caspases-dependent apoptosis in OSCC cell lines. We also found that tivantinib dose-dependently suppressed the activation and expression of FAK. Inmore » all, these data suggested that tivantinib may be developed as a chemotherapeutic agent to effectively treat certain cancers including OSCC. - Highlights: • Tivantinib suppresses OSCC cell growth independent of the inhibition of HGF/MET signaling pathway. • Tivantinib blocks cell cycle and induces caspases-mediated apoptosis. • Tivantinib elicits its anti-tumor activity with the inhibition of FAK signaling pathway.« less

Thiocoraline alters neuroendocrine phenotype and activates the Notch pathway in MTC-TT cell line

PubMed Central

Tesfazghi, Sara; Eide, Jacob; Dammalapati, Ajitha; Korlesky, Colin; Wyche, Thomas P; Bugni, Tim S; Chen, Herbert; Jaskula-Sztul, Renata

2013-01-01

Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC. Thiocoraline is a potent Notch pathway activator and an inhibitor of medullary thyroid cancer cell line (MTC-TT) cell proliferation at low nanomolar concentrations. These results provide evidence for the use of thiocoraline as a potential treatment for intractable MTC. PMID:24403239

Cell cycle effects of L-sulforaphane, a major antioxidant from cruciferous vegetables: The role of the anaphase promoting complex.

PubMed

Shelley, Zhaoping; Royce, Simon G; Ververis, Katherine; Karagiannis, Tom C

2014-01-01

L-sulforaphane (LSF) is a natural isothiocyanate found in cruciferous vegetables particularly broccoli. LSF has been identified as a potent antioxidant and anti-cancer agent and is widely known to regulate phase II detoxifying enzymes and induce cell cycle arrest or apoptosis in malignant cells in vitro and in vivo. Previous studies have found significant G2/M cell cycle arrest in response to LSF in various model of cancer and results have mainly been attributed to increased cyclin B1 protein levels and increased p21expression. Using genome-wide mRNA-Seq analysis we provide insights into the molecular mechanisms of action of LSF to identify a key pathway in cell cycle progression - the role of the anaphase promoting complex (APC) pathway. We evaluated gene expression changes in human erythroleukemic K562 cells following treatment with 15 μM LSF for 48h and compared them to immortalized human keratinocytes, human microvascular endothelial cells (HMEC-1) cells and normal human umbilical endothelial cells (HUVEC). We identified disparate gene expression changes in response to LSF between malignant and normal cells and immortalized cell lines. The results highlight significant down-regulation of kinase CDK1 which is suggestive that the existence and activity of APC/CDC20 complex will be inhibited along with its associated down-stream degradation of key cell cycle regulators preventing cell cycle progression from mitotic exit.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1→S transition.

PubMed

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P; Nath, Utpal

2011-07-01

The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1→S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1→S arrest is discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis.

PubMed

Lee, Seung Joon; Langhans, Sigrid A

2012-01-26

Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin.

Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

PubMed Central

2012-01-01

Background Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Methods Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. Results We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. Conclusions We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin. PMID:22280307

Apoptosis induction and anti-cancer activity of LeciPlex formulations.

PubMed

Dhawan, Vivek V; Joshi, Ganesh V; Jain, Ankitkumar S; Nikam, Yuvraj P; Gude, Rajiv P; Mulherkar, Rita; Nagarsenker, Mangal S

2014-10-01

Cationic agents have been reported to possess anti-neoplastic properties against various cancer cell types. However, their complexes with lipids appear to interact differently with different cancer cells. The purpose of this study was to (i) design and generate novel cationic lecithin nanoparticles, (ii) assess and understand the mechanism underlying their putative cytotoxicity and (iii) test their effect on cell cycle progression in various cancer-derived cell lines. In addition, we aimed to evaluate the in vivo potential of these newly developed nanoparticles in oral anti-cancer delivery. Cationic lecithin nanoparticles were generated using a single step nanoprecipitation method and they were characterized for particle size, zeta potential, stability and in vitro release. Their cytotoxic potential was assessed using a sulforhodamine B assay, and their effect on cell cycle progression was evaluated using flow cytometry. The nanoparticle systems were also tested in vivo for their anti-tumorigenic potential. In contrast to cationic agents alone, the newly developed nanoformulations showed a specific toxicity against cancer cells. The mechanism of toxic cell death included apoptosis, S and G2/M cell cycle phase arrest, depending on the type of cationic agent and the cancer-derived cell line used. Both blank and drug-loaded systems exhibited significant anti-cancer activity, suggesting a synergistic anti-tumorigenic effect of the drug and its delivery system. Both in vitro and in vivo data indicate that cationic agents themselves exhibit broad anti-neoplastic activities. Complex formation of the cationic agents with phospholipids was found to provide specificity to the anti-cancer activity. These formulations thus possess potential for the design of effective anti-cancer delivery systems.

Anethum graveolens (dill) - A medicinal herb induces apoptosis and cell cycle arrest in HepG2 cell line.

PubMed

Mohammed, Furkhan Ahmed; Elkady, Ayman I; Syed, Fareeduddin Quadri; Mirza, Muqtadir Baig; Hakeem, Khalid Rehman; Alkarim, Saleh

2018-06-12

The medicinal herb, Anethum graveolens L. (dill) is one of the potent culinary herbs used as an alternative form of medicine worldwide. The unguent topical Oil from the aerial parts of A. graveolens was found to be effective in the management of uterus cancer in ethnomedicine has been reported. The incidence and mortality rates of Hepatocellular carcinoma (HCC) are steadily rising worldwide, especially, in underdeveloped and developing countries. Moreover, HCC develops rapidly in patients with chronic cirrhosis or hepatitis, where the solid tumours/malignancies coexist with the inflammation. Recent studies have shown that the medicinal herb, Anethum graveolens, holds anticancer potential, which could be a promising approach for the treatment of various tumours. In the current study, we have analysed the antiproliferative effect of ethyl acetate fraction of Dill Seeds (EAFD) on HepG2 cell line. Cell viability and proliferation were observed by MTT assay; Morphological changes were studied using fluorescent stains like Hoechst 33342, acridine orange/ethidium bromide and JC-1 dye. Further, the pro-apoptotic activity was demonstrated through Annexin-V-FITC/ PI assay and cell cycle analysis. Different concentrations (0.1, 0.2, 0.4, 0.6, 0.8 mg/ml) of EAFD were studied. EAFD markedly suppressed the proliferation of HepG2 cells in a dose and time-dependent manner. The phase contrast and fluorescence microscopy revealed the morphological alterations like disruption, shrinkage, detachment and blebbing of cell membrane accompanied by nuclear condensation after exposure to EAFD. Radical scavenging activity was evidenced by measurement of ROS levels post-treatment. Modulation of mitochondrial membrane potential was exhibited leading to the activation of caspases 3/7 and 9 which is a committed step towards apoptosis. Annexin V-FITC/ PI assay and cell cycle, later confirmed the apoptosis and cell cycle arrest in 'G2/M' phase through flow cytometric analysis. In conclusion, a significant apoptogenic effect was exhibited by EAFD against HepG2 cells in inducing apoptosis and cell cycle arrest. Our findings indicate that the medicinal herb- Anethum graveolens, holds potential in treating hepatocellular carcinoma effectively. Copyright © 2018 Elsevier B.V. All rights reserved.

p21 in cancer: intricate networks and multiple activities.

PubMed

Abbas, Tarek; Dutta, Anindya

2009-06-01

One of the main engines that drives cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21WAF1/Cip1) promotes cell cycle arrest in response to many stimuli. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals. This Review focuses on recent advances in our understanding of the regulation of p21 and its biological functions with emphasis on its p53-independent tumour suppressor activities and paradoxical tumour-promoting activities, and their implications in cancer.

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

Evidence for the presence of a mammalian-like cholinesterase in Paramecium primaurelia (Protista, Ciliophora) developmental cycle.

PubMed

Delmonte Corrado, M U; Politi, H; Trielli, F; Angelini, C; Falugi, C

1999-01-01

By histochemical and immunohistochemical methods, the presence of cholinergic-like molecules has previously been demonstrated in Paramecium primaurelia, and their functional role in mating-cell pairing was suggested. In this work, both true acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were electrophoretically investigated, and the presence of molecules immunologically related to BuChE was checked by immunoblotting. The AChE activity, shown in the membrane protein fraction of mating-competent cells and in the cytoplasmic fraction of immature cells, is due to a 260-kDa molecular form, similar to the membrane-bound tetrameric form present in human erythrocytes. This AChE activity does not appear in either the cytoplasmic fraction of mating-competent cells or in the membrane protein fraction of immature cells. No evidence was found for the presence or the activity of BuChE-like molecules. The role of AChE in P. primaurelia developmental cycle is discussed.

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

PubMed

Zhang, Yali; Guo, Zonglou; Xu, Lihong

2014-03-01

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy.

PubMed

Baithalu, Rubina Kumari; Singh, S K; Gupta, Chhavi; Raja, Anuj K; Saxena, Abhishake; Kumar, Yogendra; Singh, R; Agarwal, S K

2013-08-01

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3β-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3β-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23μm) and LLC (range 25-55μm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3β-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Yeast for virus research

PubMed Central

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

Temporal remodeling of the cell cycle accompanies differentiation in the Drosophila germline.

PubMed

Hinnant, Taylor D; Alvarez, Arturo A; Ables, Elizabeth T

2017-09-01

Development of multicellular organisms relies upon the coordinated regulation of cellular differentiation and proliferation. Growing evidence suggests that some molecular regulatory pathways associated with the cell cycle machinery also dictate cell fate; however, it remains largely unclear how the cell cycle is remodeled in concert with cell differentiation. During Drosophila oogenesis, mature oocytes are created through a series of precisely controlled division and differentiation steps, originating from a single tissue-specific stem cell. Further, germline stem cells (GSCs) and their differentiating progeny remain in a predominantly linear arrangement as oogenesis proceeds. The ability to visualize the stepwise events of differentiation within the context of a single tissue make the Drosophila ovary an exceptional model for study of cell cycle remodeling. To describe how the cell cycle is remodeled in germ cells as they differentiate in situ, we used the Drosophila Fluorescence Ubiquitin-based Cell Cycle Indicator (Fly-FUCCI) system, in which degradable versions of GFP::E2f1 and RFP::CycB fluorescently label cells in each phase of the cell cycle. We found that the lengths of the G1, S, and G2 phases of the cell cycle change dramatically over the course of differentiation, and identified the 4/8-cell cyst as a key developmental transition state in which cells prepare for specialized cell cycles. Our data suggest that the transcriptional activator E2f1, which controls the transition from G1 to S phase, is a key regulator of mitotic divisions in the early germline. Our data support the model that E2f1 is necessary for proper GSC proliferation, self-renewal, and daughter cell development. In contrast, while E2f1 degradation by the Cullin 4 (Cul4)-containing ubiquitin E3 ligase (CRL4) is essential for developmental transitions in the early germline, our data do not support a role for E2f1 degradation as a mechanism to limit GSC proliferation or self-renewal. Taken together, these findings provide further insight into the regulation of cell proliferation and the acquisition of differentiated cell fate, with broad implications across developing tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Nontraditional, Safe, High Voltage Rechargeable Cells of Long Cycle Life.

PubMed

Braga, Maria Helena; M Subramaniyam, Chandrasekar; Murchison, Andrew J; Goodenough, John B

2018-05-23

A room-temperature all-solid-state rechargeable battery cell containing a tandem electrolyte consisting of a Li + -glass electrolyte in contact with a lithium anode and a plasticizer in contact with a conventional, low cost oxide host cathode was charged to 5 V versus lithium with a charge/discharge cycle life of over 23,000 cycles at a rate of 153 mA·g -1 of active material. A larger positive electrode cell with 329 cycles had a capacity of 585 mAh·g -1 at a cutoff of 2.5 V and a current of 23 mA·g -1 of the active material; the capacity rose with cycle number over the 329 cycles tested during 13 consecutive months. Another cell had a discharge voltage from 4.5 to 3.7 V over 316 cycles at a rate of 46 mA·g -1 of active material. Both the Li + -glass electrolyte and the plasticizer contain electric dipoles that respond to the internal electric fields generated during charge by a redistribution of mobile cations in the glass and by extraction of Li + from the active cathode host particles. The electric dipoles remain oriented during discharge to retain an internal electric field after a discharge. The plasticizer accommodates to the volume changes in the active cathode particles during charge/discharge cycling and retains during charge the Li + extracted from the cathode particles at the plasticizer/cathode-particle interface; return of these Li + to the active cathode particles during discharge only involves a displacement back across the plasticizer/cathode interface and transport within the cathode particle. A slow motion at room temperature of the electric dipoles in the Li + -glass electrolyte increases with time the electric field across the EDLC of the anode/Li + -glass interface to where Li + from the glass electrolyte is plated on the anode without being replenished from the cathode, which charges the Li + -glass electrolyte negative and consequently the glass side of the Li + -glass/plasticizer EDLC. Stripping back the Li + to the Li + -glass during discharge is enhanced by the negative charge in the Li + -glass. Since the Li + -glass is not reduced on contact with metallic lithium, no passivating interface layer contributes to a capacity fade; instead, the discharge capacity increases with cycle number as a result of dipole polarization in the Li + -glass electrolyte leading to a capacity increase of the Li + -glass/plasticizer EDLC. The storage of electric power by both faradaic electrochemical extraction/insertion of Li + in the cathode and electrostatic stored energy in the EDLCs provides a safe and fast charge and discharge with a long cycle life and a greater capacity than can be provided by the cathode host extraction/insertion reaction. The cell can be charged to a high voltage versus a lithium anode because of the added charge of the EDLCs.

Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage.

PubMed

Karimian, Ansar; Ahmadi, Yasin; Yousefi, Bahman

2016-06-01

An appropriate control over cell cycle progression depends on many factors. Cyclin-dependent kinase (CDK) inhibitor p21 (also known as p21(WAF1/Cip1)) is one of these factors that promote cell cycle arrest in response to a variety of stimuli. The inhibitory effect of P21 on cell cycle progression correlates with its nuclear localization. P21 can be induced by both p53-dependent and p53-independent mechanisms. Some other important functions attributed to p21 include transcriptional regulation, modulation or inhibition of apoptosis. These functions are largely dependent on direct p21/protein interactions and also on p21 subcellular localizations. In addition, p21 can play a role in DNA repair by interacting with proliferating cell nuclear antigen (PCNA). In this review, we will focus on the multiple functions of p21 in cell cycle regulation, apoptosis and gene transcription after DNA damage and briefly discuss the pathways and factors that have critical roles in p21 expression and activity. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

PubMed Central

Antosova, Barbora; Smolikova, Jana; Borkovcova, Romana; Strnad, Hynek; Lachova, Jitka; Machon, Ondrej; Kozmik, Zbynek

2013-01-01

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency. PMID:24205179

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21*

PubMed Central

Basu, Nirmalya; Saha, Sayanti; Khan, Imran; Ramachandra, Subbaraya G.; Visweswariah, Sandhya S.

2014-01-01

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c−/−, mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence. PMID:24217248

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae.

PubMed

Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

2016-10-26

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit.

PubMed

Rock, Jeremy M; Amon, Angelika

2011-09-15

In budding yeast, a Ras-like GTPase signaling cascade known as the mitotic exit network (MEN) promotes exit from mitosis. To ensure the accurate execution of mitosis, MEN activity is coordinated with other cellular events and restricted to anaphase. The MEN GTPase Tem1 has been assumed to be the central switch in MEN regulation. We show here that during an unperturbed cell cycle, restricting MEN activity to anaphase can occur in a Tem1 GTPase-independent manner. We found that the anaphase-specific activation of the MEN in the absence of Tem1 is controlled by the Polo kinase Cdc5. We further show that both Tem1 and Cdc5 are required to recruit the MEN kinase Cdc15 to spindle pole bodies, which is both necessary and sufficient to induce MEN signaling. Thus, Cdc15 functions as a coincidence detector of two essential cell cycle oscillators: the Polo kinase Cdc5 synthesis/degradation cycle and the Tem1 G-protein cycle. The Cdc15-dependent integration of these temporal (Cdc5 and Tem1 activity) and spatial (Tem1 activity) signals ensures that exit from mitosis occurs only after proper genome partitioning.

Benzo[a]pyrene-7,8-dihydrodiol promotes checkpoint activation and G2/M arrest in human bronchoalveolar carcinoma H358 cells.

PubMed

Caino, M Cecilia; Oliva, Jose L; Jiang, Hao; Penning, Trevor M; Kazanietz, Marcelo G

2007-03-01

Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens that require metabolic activation inside cells. The proximate carcinogens PAH-diols can be converted to o-quinones by aldo-keto reductases (AKRs) or to diol-epoxides by cytochrome P450 (P450) enzymes. We assessed the effect of benzo[a]pyrene-7,8-dihydrodiol (BPD) on proliferation in p53-null bronchoalveolar carcinoma H358 cells. BPD treatment led to a significant inhibition of proliferation and arrest in G2/M in H358 cells. The relative contribution of the AKR and P450 pathways to cell cycle arrest was assessed. Overexpression of AKR1A1 did not affect cell proliferation or cell cycle progression, and benzo[a]pyrene-7,8-dione did not cause any noticeable effect on cell growth, suggesting that AKR1A1 metabolic products were not involved in the antiproliferative effect of BPD. On the other hand, blockade of P450 induction or inhibition of P450 activity greatly impaired the effect of BPD. Moreover, P450 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly enhanced the antiproliferative effect of BPD. Mechanistic studies revealed that BPD caused a DNA damage response, Chk1 activation, and accumulation of phospho-Cdc2 (Tyr15) in H358 cells, effects that were impaired by an ataxia-telangectasia mutated (ATM)/ATM-related (ATR) inhibitor. Similar results were observed in human bronchoepithelial BEAS-2B cells, arguing for analogous mechanisms in tumorigenic and immortalized nontumorigenic cells lacking functional p53. Our data suggest that a p53-independent pathway operates in lung epithelial cells in response to BPD that involves P450 induction and subsequent activation of the ATR/ATM/Chk1 damage check-point pathway and cell cycle arrest in G2/M.

In vitro and in vivo anti-tumour activities of echinoside A and ds-echinoside A from Pearsonothuria graeffei.

PubMed

Zhao, Qin; Xue, Yong; Wang, Jing-feng; Li, Hui; Long, Teng-teng; Li, Zhaojie; Wang, Yu-ming; Dong, Ping; Xue, Chang-hu

2012-03-15

Echinoside A (EA) and ds-echinoside A (DSEA) are triterpene glycosides isolated from the sea cucumber Pearsonothuria graeffei. DSEA, the desulfurisation product of EA, has the following structure: β-D-xylopyranosyl-holost-8(9),11(12)-diene-3β,17α-diol. In the present study, we examined the anti-tumour activities-in particular, the structure-activity relationships-of EA and DSEA in vitro and in vivo. Both EA and DSEA exhibited an inhibitory effect on cell proliferation, along with apoptosis-inducing activity, in HepG2 cells. Moreover, they significantly arrested the cell cycle in the G₀/G₁ phase. A reverse transcriptase-polymerase chain reaction assay revealed that EA and DSEA significantly increased the expression of the cell-cycle-related genes, namely, p16, p21 and c-myc, and decreased that of cyclin D₁. Western blotting analysis demonstrated that they down-regulated the expression of Bcl-2, and enhanced mitochondria cytochrome c release, caspase-3 activation, and poly(adenosine diphosphate ribose) polymerase, cleavage. Nuclear factor kappa B (NF-κB) expression was significantly decreased by DSEA, but was unaffected by EA. EA and DSEA (2.5 mg kg⁻¹) treatment of mice bearing H22 hepatocarcinoma tumours reduced the tumour weight by 49.8% and 55.0%, respectively. EA and DSEA exhibit marked anti-cancer activity in HepG2 cells, by blocking cell-cycle progression and inducing apoptosis through the mitochondrial pathway. DSEA-induced apoptosis was more potent than EA-induced apoptosis. Furthermore, the two triterpene glycosides derived from P. graeffei may induce apoptosis of HepG2 cells in an NF-κB-dependent or NF-κB-independent manner, depending on their structure. Copyright © 2011 Society of Chemical Industry.

Never in mitosis gene A related kinase-6 attenuates pressure overload-induced activation of the protein kinase B pathway and cardiac hypertrophy.

PubMed

Bian, Zhouyan; Liao, Haihan; Zhang, Yan; Wu, Qingqing; Zhou, Heng; Yang, Zheng; Fu, Jinrong; Wang, Teng; Yan, Ling; Shen, Difei; Li, Hongliang; Tang, Qizhu

2014-01-01

Cardiac hypertrophy appears to be a specialized form of cellular growth that involves the proliferation control and cell cycle regulation. NIMA (never in mitosis, gene A)-related kinase-6 (Nek6) is a cell cycle regulatory gene that could induce centriole duplication, and control cell proliferation and survival. However, the exact effect of Nek6 on cardiac hypertrophy has not yet been reported. In the present study, the loss- and gain-of-function experiments were performed in Nek6 gene-deficient (Nek6-/-) mice and Nek6 overexpressing H9c2 cells to clarify whether Nek6 which promotes the cell cycle also mediates cardiac hypertrophy. Cardiac hypertrophy was induced by transthoracic aorta constriction (TAC) and then evaluated by echocardiography, pathological and molecular analyses in vivo. We got novel findings that the absence of Nek6 promoted cardiac hypertrophy, fibrosis and cardiac dysfunction, which were accompanied by a significant activation of the protein kinase B (Akt) signaling in an experimental model of TAC. Consistent with this, the overexpression of Nek6 prevented hypertrophy in H9c2 cells induced by angiotonin II and inhibited Akt signaling in vitro. In conclusion, our results demonstrate that the cell cycle regulatory gene Nek6 is also a critical signaling molecule that helps prevent cardiac hypertrophy and inhibits the Akt signaling pathway.

Emodin Increases Expression of Insulin-Like Growth Factor Binding Protein 1 through Activation of MEK/ERK/AMPKα and Interaction of PPARγ and Sp1 in Lung Cancer.

PubMed

Tang, Qing; Wu, JingJing; Zheng, Fang; Hann, Swei Sunny; Chen, YuQing

2017-01-01

Emodin has anti-neoplastic activities on multiple tumors. However, the molecular mechanisms underlying this effect still remain to be fully understood. Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays and flow cytometry, respectively. Cell invasion and migration were examined by transwell invasion and wound healing assays. Western blot analysis was performed to examine the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPKα), extracellular signaling-regulated kinase 1/2 (ERK1/2), peroxisome proliferators-activated receptor gamma (PPARγ), insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and the transcription factor Sp1. QRT-PCR was used to examine the mRNA levels of the IGFBP1 gene. Small interfering RNAs (siRNAs) were used to knockdown PPARγ and IGFBP1 genes. Exogenously expression of IGFBP1 and Sp1 was determined by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings. We showed that emodin induced cell cycle arrest of NSCLC cells. Emodin increased PPARγ protein and luciferase reporter activity, which were abolished by inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK and AMPK. Silencing of PPARγ abrogated emodin-inhibited cell growth and cell cycle arrest. Furthermore, emodin elevated IGFBP1 mRNA, protein, and promoter activity through activation of PPARγ. Intriguingly, overexpressed Sp1 attenuated emodin-induced IGFBP1 expression, which was not observed in cells with silenced PPARγ gene. Moreover, silencing of IGFBP1 gene blunted emodin-induced inhibition of cell growth and cell cycle arrest. On the contrary, overexpressed IGFBP1 enhanced emodin-induced phosphorylation of AMPKα and ERK1/2, and restored emodin-inhibited growth in cells with silenced endogenous IGFBP1 gene. Emodin also inhibited growth of lung xenograft tumors and Sp1, and increased IGFBP1 and PPARγ protein expressions In vivo. Collectively, our results show that emodin inhibits growth of non-small-cell lung cancer (NSCLC) cells through ERK and AMPKα-mediated induction of PPARγ, followed by reduction of Sp1. This in turn induces IGFBP1 gene expression. Thus, the signaling cascades, positive feedback loop and cooperative interplay between transcription factors-induced the expression of IGFBP1 gene contribute to the overall responses of emodin. This study provides a novel mechanism by which emodin inhibits growth of human lung cancer cells. © 2017 The Author(s) Published by S. Karger AG, Basel.

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.

The Cak1p Protein Kinase Is Required at G(1)/S and G(2)/M in the Budding Yeast Cell Cycle

PubMed Central

Sutton, A.; Freiman, R.

1997-01-01

The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G(2) to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G(2) to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G(1) into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function. PMID:9286668

Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells

PubMed Central

van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

2014-01-01

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation. PMID:24695638

Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

PubMed

van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

2014-01-01

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

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.

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress

PubMed Central

Laggner, Maria; Pollreisz, Andreas; Schmidinger, Gerald; Schmidt-Erfurth, Ursula; Chen, Ying-Ting

2017-01-01

Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC’s stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H2DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation. PMID:28700649

Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP-1/p53 regulatory pathway.

PubMed

Luo, Hao; Liang, Hairong; Chen, Jiajia; Xu, Yongchun; Chen, Yuting; Xu, Longmei; Yun, Lin; Liu, Jiaxian; Yang, Hui; Liu, Linhua; Peng, Jianming; Liu, Zhidong; Tang, Lin; Chen, Wen; Tang, Huanwen

2017-09-01

Hydroquinone (HQ), one of the most important metabolites derived from benzene, induces cell cycle arrest and apoptosis. Poly(ADP-ribose) polymerase-1 (PARP-1) participates in various biological processes, including DNA repair and cell cycle regulation. To explore whether PARP-1 regulatory pathway mediated HQ-induced cell cycle arrest and apoptosis, we assessed the effect of PARP-1 suppression on induction of apoptosis analyzed by FACSCalibur flow cytometer in PARP-1 deficientTK6 cells (TK6-shPARP-1). We observed an increase in the fraction of cells in G1 phase by 7.6% and increased apoptosis by 4.5% in PARP-1-deficient TK6 cells (TK6-shPARP-1) compared to those negative control cells (TK6-shNC cells) in response to HQ treatment. Furthermore, HQ might activate the extrinsic pathways of apoptosis via up-regulation of Fas expression, followed by caspase-3 activation, apoptotic body, and sub G1 accumulation. Enhanced p53 expression was observed in TK6-shPARP-1 cells than in TK6-shNC cells after HQ treatment. In contrast, Fas expression was lower in TK6-shPARP-1 cells than in TK6-shNC cells. Therefore, we conclude that HQ may activate apoptotic signals via Fas up-regulation and p53-mediated apoptosis in TK6-shNC cells. The reduction of PARP-1 expression further intensified up-regulation of p53 in TK6-shPARP-1 cells, resulting in an increased G1→S phase cell arrest and apoptosis in TK6-shPARP-1 cells compared to TK6-shNC cells. © 2017 Wiley Periodicals, Inc.

Physangulidine A, a withanolide from Physalis angulata, perturbs the cell cycle and induces cell death by apoptosis in prostate cancer cells.

PubMed

Reyes-Reyes, E Merit; Jin, Zhuang; Vaisberg, Abraham J; Hammond, Gerald B; Bates, Paula J

2013-01-25

Recently, our group reported the discovery of three new withanolides, physangulidines A-C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G(2)/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.

Anti-colorectal cancer effects of tripolinolate A from Tripolium vulgare.

PubMed

Chen, Lu; Wang, Wen-Ling; Song, Teng-Fei; Xie, Xin; Ye, Xue-Wei; Liang, Ying; Huang, Hao-Cai; Yan, Shi-Lun; Lian, Xiao-Yuan; Zhang, Zhi-Zhen

2017-08-01

Tripolinolate A (TLA) is recently identified as a new compound from a halophyte plant Tripolium vulgare and has been shown to have significant in vitro activity against the proliferation of colorectal cancer and glioma cells. This study was designed to further investigate the effects of TLA on the proliferation of human normal cells, and the apoptosis and cell cycle in colorectal cancer cells, and the growth of tumors in the colorectal cancer-bearing animals. The data obtained from this study demonstrated that: 1) TLA had much less cytotoxicity in the human normal cells than the colorectal cancer cells; 2) TLA remarkably induced apoptosis in the human colorectal cancer cells and blocked cell cycle at G 2 /M phase, and 3) TLA had significant anti-colorectal cancer activity in the tumor-bearing animals. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL1.

PubMed

Heyman, Jefri; Polyn, Stefanie; Eekhout, Thomas; De Veylder, Lieven

2017-09-01

The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis ( Arabidopsis thaliana ), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/C CCS52A1 and APC/C CCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/C CCS52A1 and APC/C CCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/C CCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation.

PubMed

Wee, Ping; Wang, Zhixiang

2017-01-01

Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis.

Fungal-Induced Cell Cycle Impairment, Chromosome Instability and Apoptosis via Differential Activation of NF-κB

PubMed Central

Ben-Abdallah, Mariem; Sturny-Leclère, Aude; Avé, Patrick; Louise, Anne; Moyrand, Frédérique; Weih, Falk; Janbon, Guilhem; Mémet, Sylvie

2012-01-01

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis. PMID:22396644

Fungal-induced cell cycle impairment, chromosome instability and apoptosis via differential activation of NF-κB.

PubMed

Ben-Abdallah, Mariem; Sturny-Leclère, Aude; Avé, Patrick; Louise, Anne; Moyrand, Frédérique; Weih, Falk; Janbon, Guilhem; Mémet, Sylvie

2012-01-01

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis.

Modeling Cancer Cell Growth Dynamics In vitro in Response to Antimitotic Drug Treatment

PubMed Central

Lorz, Alexander; Botesteanu, Dana-Adriana; Levy, Doron

2017-01-01

Investigating the role of intrinsic cell heterogeneity emerging from variations in cell-cycle parameters and apoptosis is a crucial step toward better informing drug administration. Antimitotic agents, widely used in chemotherapy, target exclusively proliferative cells and commonly induce a prolonged mitotic arrest followed by cell death via apoptosis. In this paper, we developed a physiologically motivated mathematical framework for describing cancer cell growth dynamics that incorporates the intrinsic heterogeneity in the time individual cells spend in the cell-cycle and apoptosis process. More precisely, our model comprises two age-structured partial differential equations for the proliferative and apoptotic cell compartments and one ordinary differential equation for the quiescent compartment. To reflect the intrinsic cell heterogeneity that governs the growth dynamics, proliferative and apoptotic cells are structured in “age,” i.e., the amount of time remaining to be spent in each respective compartment. In our model, we considered an antimitotic drug whose effect on the cellular dynamics is to induce mitotic arrest, extending the average cell-cycle length. The prolonged mitotic arrest induced by the drug can trigger apoptosis if the time a cell will spend in the cell cycle is greater than the mitotic arrest threshold. We studied the drug’s effect on the long-term cancer cell growth dynamics using different durations of prolonged mitotic arrest induced by the drug. Our numerical simulations suggest that at confluence and in the absence of the drug, quiescence is the long-term asymptotic behavior emerging from the cancer cell growth dynamics. This pattern is maintained in the presence of small increases in the average cell-cycle length. However, intermediate increases in cell-cycle length markedly decrease the total number of cells and can drive the cancer population to extinction. Intriguingly, a large “switch-on/switch-off” increase in the average cell-cycle length maintains an active cell population in the long term, with oscillating numbers of proliferative cells and a relatively constant quiescent cell number. PMID:28913178

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

PubMed

Bordonaro, M; Mariadason, J M; Aslam, F; Heerdt, B G; Augenlicht, L H

1999-10-01

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.