COMSAT's destructive physical analysis of aerospace nickel-cadmium cells for NASA/Goddard Space Flight Center

NASA Technical Reports Server (NTRS)

Robbins, Kathleen M. B.; Rao, Gopalakrishna M.; Yi, Thomas Y.

1993-01-01

Over the past 5 years, COMSAT has performed numerous destructive physical analyses (DPA's) on NASA-Goddard-supplied nickel-cadmium (Ni/Cd) cells. The samples included activated but uncycled cells, wet stored cells, cycled cells, and anomalous cells. The DPA's provided visual, morphological, and chemical analyses of the cell components. The DPA data for the analyzed cells are presented. For the cells investigated, the leading cause of poor performance, as determined by DPA, has been poor negative electrode utilization, which resulted in negative-electrode-limiting operation.

Origination of asexual plantlets in three species of Crassulaceae.

PubMed

Guo, Jiansheng; Liu, Hailiang; He, Yangyang; Cui, Xianghuan; Du, Xiling; Zhu, Jian

2015-03-01

During asexual plant reproduction, cells from different organs can be reprogrammed to produce new individuals, a process that requires the coordination of cell cycle reactivation with the acquisition of other cellular morphological characteristics. However, the factors that influence the variety of asexual reproduction have not yet been determined. Here, we report on plantlet formation in Kalanchoe daigremontiana, Graptopetalum paraguayense, and Crassula portulacea (Crassulaceae) and analyse the effect of initiating cells on asexual reproduction in these three species. Additionally, the roles of WUSCHEL (WUS) and CUP-SHAPED COTYLEDON 1 (CUC1) in the asexual reproduction of these species were analysed through qRT-PCR. Our results indicated that pre-existing stem cell-like cells at the sites of asexual reproduction were responsible for the formation of plantlets. These cells were arrested in different phases of the cell cycle and showed different cell morphological characteristics and cell counts. The accumulation of auxin and cytokinin at the sites of asexual plantlet formation indicated their important functions, particularly for cell cycle reactivation. These differences may influence the pattern and complexity of asexual reproduction in these Crassulaceae species. Additionally, the dynamic expression levels of CUC1 and WUS may indicate that CUC1 functions in the formation of callus and shoot meristems; whereas, WUS was only associated with shoot induction.

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components.

PubMed

García-Cruz, Karla V; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R

2016-07-29

Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. XAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

PubMed

Long, Lin; He, Jian-Zhong; Chen, Ye; Xu, Xiu-E; Liao, Lian-Di; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

2018-05-07

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

From coin cells to 400 mAh pouch cells: Enhancing performance of high-capacity lithium-ion cells via modifications in electrode constitution and fabrication

NASA Astrophysics Data System (ADS)

Trask, Stephen E.; Li, Yan; Kubal, Joseph J.; Bettge, Martin; Polzin, Bryant J.; Zhu, Ye; Jansen, Andrew N.; Abraham, Daniel P.

2014-08-01

In this article we describe efforts to improve performance and cycle life of cells containing Li1.2Ni0.15Mn0.55Co0.1O2-based positive and graphite-based negative electrodes. Initial work to identify high-performing materials, compositions, fabrication variables, and cycling conditions is conducted in coin cells. The resulting information is then used for the preparation of double-sided electrodes, assembly of pouch cells, and electrochemical testing. We report the cycling performance of cells with electrodes prepared under various conditions. Our data indicate that cells with positive electrodes containing 92 wt.% Li1.2Ni0.15Mn0.55Co0.1O2, 4 wt.% carbons (no graphite), and 4 wt.% PVdF (92-4-4) show ∼20% capacity fade after 1000 cycles in the 2.5-4.4 V range, significantly better than our baseline cells that show the same fade after only 450 cycles. Our analyses indicate that the major contributors to cell energy fade are capacity loss and impedance rise. Therefore incorporating approaches that minimize capacity fade and impedance rise, such as electrode coatings and electrolyte additives, can significantly enhance calendar and cycle life of this promising cell chemistry.

Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

PubMed Central

Scotti, Claudia; Sommi, Patrizia; Pasquetto, Maria Valentina; Cappelletti, Donata; Stivala, Simona; Mignosi, Paola; Savio, Monica; Chiarelli, Laurent Roberto; Valentini, Giovanna; Bolanos-Garcia, Victor M.; Merrell, Douglas Scott; Franchini, Silvia; Verona, Maria Luisa; Bolis, Cristina; Solcia, Enrico; Manca, Rachele; Franciotta, Diego; Casasco, Andrea; Filipazzi, Paola; Zardini, Elisabetta; Vannini, Vanio

2010-01-01

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application. PMID:21085483

From coin cells to 400 mAh pouch cells: Enhancing performance of high-capacity lithium-ion cells via modifications in electrode constitution and fabrication

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

Trask, Stephen E.; Li, Yan; Kubal, Joseph J.

2014-08-01

In this article we describe efforts to improve performance and cycle life of cells containing Li1.2Ni0.15Mn0.55Co0.1O2-based positive and graphite-based negative electrodes. Initial work to identify high-performing materials, compositions, fabrication variables, and cycling conditions is conducted in coin cells. The resulting information is then used for the preparation of double-sided electrodes, assembly of pouch cells, and electrochemical testing. We report the cycling performance of cells with electrodes prepared under various conditions. Our data indicate that cells with positive electrodes containing 92 wt% Li1.2Ni0.15Mn0.55Co0.1O2, 4 wt% carbons (no graphite), and 4 wt% PVdF (92-4-4) show ~20% capacity fade after 1000 cycles inmore » the 2.5-4.4V range, significantly better than our baseline cells that show the same fade after only 450 cycles. Our analyses indicate that the major contributors to cell energy fade are capacity loss and impedance rise. Therefore incorporating approaches that minimize capacity fade and impedance rise, such as electrode coatings and electrolyte additives, can significantly enhance calendar and cycle life of this promising cell chemistry.« less

Cell cycle pathway dysregulation in human keratinocytes during chronic exposure to low arsenite.

PubMed

Al-Eryani, Laila; Waigel, Sabine; Jala, Venkatakrishna; Jenkins, Samantha F; States, J Christopher

2017-09-15

Arsenic is naturally prevalent in the earth's crust and widely distributed in air and water. Chronic low arsenic exposure is associated with several cancers in vivo, including skin cancer, and with transformation in vitro of cell lines including immortalized human keratinocytes (HaCaT). Arsenic also is associated with cell cycle dysregulation at different exposure levels in multiple cell lines. In this work, we analyzed gene expression in HaCaT cells to gain an understanding of gene expression changes contributing to transformation at an early time point. HaCaT cells were exposed to 0 or 100nM NaAsO 2 for 7weeks. Total RNA was purified and analyzed by microarray hybridization. Differential expression with fold change≥|1.5| and p-value≤0.05 was determined using Partek Genomic Suite™ and pathway and network analyses using MetaCore™ software (FDR≤0.05). Cell cycle analysis was performed using flow cytometry. 644 mRNAs were differentially expressed. Cell cycle/cell cycle regulation pathways predominated in the list of dysregulated pathways. Genes involved in replication origin licensing were enriched in the network. Cell cycle assay analysis showed an increase in G2/M compartment in arsenite-exposed cells. Arsenite exposure induced differential gene expression indicating dysregulation of cell cycle control, which was confirmed by cell cycle analysis. The results suggest that cell cycle dysregulation is an early event in transformation manifested in cells unable to transit G2/M efficiently. Further study at later time points will reveal additional changes in gene expression related to transformation processes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Gabrielson, Marike; Reizer, Edwin; Stål, Olle

An increasing body of evidence is pointing towards mitochondrial regulation of the cell cycle. In a previous study of HER2-positive tumours we could demonstrate a common loss in the gene encoding for the mitochondrial transporter SLC25A43 and also a significant relation between SLC25A43 protein expression and S-phase fraction. Here, we investigated the consequence of suppressed SLC25A43 expression on cell cycle progression and proliferation in breast epithelial cells. In the present study, we suppressed SLC25A43 using siRNA in immortalised non-cancerous breast epithelial MCF10A cells and HER2-positive breast cancer cells BT-474. Viability, apoptosis, cell proliferation rate, cell cycle phase distribution, and nuclearmore » Ki-67 and p21, were assessed by flow cytometry. Cell cycle related gene expressions were analysed using real-time PCR. We found that SLC25A43 knockdown in MCF10A cells significantly inhibited cell cycle progression during G{sub 1}-to-S transition, thus significantly reducing the proliferation rate and fraction of Ki-67 positive MCF10A cells. In contrast, suppressed SLC25A43 expression in BT-474 cells resulted in a significantly increased proliferation rate together with an enhanced G{sub 1}-to-S transition. This was reflected by an increased fraction of Ki-67 positive cells and reduced level of nuclear p21. In line with our previous results, we show a role for SLC25A43 as a regulator of cell cycle progression and proliferation through a putative mitochondrial checkpoint. These novel data further strengthen the connection between mitochondrial function and the cell cycle, both in non-malignant and in cancer cells. - Highlights: • Proposed cell cycle regulation through the mitochondrial transporter SLC25A43. • SLC25A43 alters cell proliferation rate and cell cycle progression. • Suppressed SLC25A43 influences transcription of cell cycle regulatory genes.« less

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

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

PubMed

Morse, David; Daoust, Philip; Benribague, Siham

2016-12-01

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

A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

NASA Astrophysics Data System (ADS)

Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

2016-03-01

We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

PubMed

Bhattacharya, A; Schmitz, U; Wolkenhauer, O; Schönherr, M; Raatz, Y; Kunz, M

2013-06-27

WEE1 kinase has been described as a major gate keeper at the G2 cell cycle checkpoint and to be involved in tumour progression in different malignant tumours. Here we analysed the expression levels of WEE1 in a series of melanoma patient samples and melanoma cell lines using immunoblotting, quantitative real-time PCR and immunohistochemistry. WEE1 expression was significantly downregulated in patient samples of metastatic origin as compared with primary melanomas and in melanoma cell lines of high aggressiveness as compared with cell lines of low aggressiveness. Moreover, there was an inverse correlation between the expression of WEE1 and WEE1-targeting microRNA miR-195. Further analyses showed that transfection of melanoma cell lines with miR-195 indeed reduced WEE1 mRNA and protein expression in these cells. Reporter gene analysis confirmed direct targeting of the WEE1 3' untranslated region (3'UTR) by miR-195. Overexpression of miR-195 in SK-Mel-28 melanoma cells was accompanied by WEE1 reduction and significantly reduced stress-induced G2-M cell cycle arrest, which could be restored by stable overexpression of WEE1. Moreover, miR-195 overexpression and WEE1 knockdown, respectively, increased melanoma cell proliferation. miR-195 overexpression also enhanced migration and invasiveness of melanoma cells. Taken together, the present study shows that WEE1 expression in malignant melanoma is directly regulated by miR-195. miR-195-mediated downregulation of WEE1 in metastatic lesions may help to overcome cell cycle arrest under stress conditions in the local tissue microenvironment to allow unrestricted growth of tumour cells.

Nickel-Hydrogen Cell Testing Experience, NASA/Goddard Space Flight Center

NASA Technical Reports Server (NTRS)

Rao, Gopalakrishna M.

1999-01-01

The objectives of the project were to test the Nickel-Hydrogen Cell to: (1) verify the Aerospace Cell Flight Worthiness, (2) Elucidate the Aerospace Cell Thermal Behavior, (3) Develop the Aerospace Battery Assembly Design(s) and In-orbit Battery Management plan(s) and (4) Understand the Aerospace Cell Failure Mechanism. The tests included the LEO and GEO Life cycle tests, Calorimetric Analysis, Destructive Physical analysis, and special tests. Charts show the Mission Profile Cycling Data, Stress Cycling Data. The test data complies with the mission requirements, validating the flight worthiness of batteries. The nominal stress and mission profile cycling performance test shows the charge voltage as high as 1.60V and recharge ratio greater than 1.05. It is apparent that the electrochemical signatures alone do not provide conclusive proof for Nickel precharge. The researchers recommend a gas and positive plate analyses for further confirmation.

Rapid Assessment of Genotoxicity by Flow Cytometric Detection of Cell Cycle Alterations.

PubMed

Bihari, Nevenka

2017-01-01

Flow cytometry is a convenient method for the determination of genotoxic effects of environmental pollution and can reveal genotoxic compounds in unknown environmental mixtures. It is especially suitable for the analyses of large numbers of samples during monitoring programs. The speed of detection is one of the advantages of this technique which permits the acquisition of 10 4 -10 5 cells per sample in 5 min. This method can rapidly detect cell cycle alterations resulting from DNA damage. The outcome of such an analysis is a diagram of DNA content across the cell cycle which indicates cell proliferation, G 2 arrests, G 1 delays, apoptosis, and ploidy.Here, we present the flow cytometric procedure for rapid assessment of genotoxicity via detection of cell cycle alterations. The described protocol simplifies the analysis of genotoxic effects in marine environments and is suitable for monitoring purposes. It uses marine mussel cells in the analysis and can be adapted to investigations on a broad range of marine invertebrates.

[The effect of 3-aminobenzamide on the mitotic cycle of Chinese hamster cells cultured on a medium with 5-bromodeoxyuridine following ionizing radiation action].

PubMed

Kirillova, T V; Rozanov, Iu M; Spivak, I M

1992-01-01

A specific inhibitor of poly(ADP-ribose)polymerase-3-aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-K1 cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU cells), and increase their radiosensitivity; 2) induce G2 delay in BDU cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2 delay in X-irradiated BDU cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure.

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

PubMed Central

Handberg-Thorsager, Mette; Vervoort, Michel

2017-01-01

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos. PMID:29231816

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

PubMed Central

Voet, Thierry; Kumar, Parveen; Van Loo, Peter; Cooke, Susanna L.; Marshall, John; Lin, Meng-Lay; Zamani Esteki, Masoud; Van der Aa, Niels; Mateiu, Ligia; McBride, David J.; Bignell, Graham R.; McLaren, Stuart; Teague, Jon; Butler, Adam; Raine, Keiran; Stebbings, Lucy A.; Quail, Michael A.; D’Hooghe, Thomas; Moreau, Yves; Futreal, P. Andrew; Stratton, Michael R.; Vermeesch, Joris R.; Campbell, Peter J.

2013-01-01

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis. PMID:23630320

Cell cycle-coupled expansion of AR activity promotes cancer progression.

PubMed

McNair, C; Urbanucci, A; Comstock, C E S; Augello, M A; Goodwin, J F; Launchbury, R; Zhao, S G; Schiewer, M J; Ertel, A; Karnes, J; Davicioni, E; Wang, L; Wang, Q; Mills, I G; Feng, F Y; Li, W; Carroll, J S; Knudsen, K E

2017-03-23

The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

PubMed

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells

PubMed Central

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival. PMID:25616580

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

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

Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

PubMed

Cen, Yan-Hui; Guo, Wen-Wen; Luo, Bin; Lin, Yong-Da; Zhang, Qing-Mei; Zhou, Su-Fang; Luo, Guo-Rong; Xiao, Shao-Wen; Xie, Xiao-Xun

2012-10-01

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.

Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

PubMed Central

Trojanowski, Damian; Ginda, Katarzyna; Pióro, Monika; Hołówka, Joanna; Skut, Partycja; Jakimowicz, Dagmara

2015-01-01

ABSTRACT It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography. PMID:25691599

The B-MYB Transcriptional Network Guides Cell Cycle Progression and Fate Decisions to Sustain Self-Renewal and the Identity of Pluripotent Stem Cells

PubMed Central

Zhan, Ming; Riordon, Daniel R.; Yan, Bin; Tarasova, Yelena S.; Bruweleit, Sarah; Tarasov, Kirill V.; Li, Ronald A.; Wersto, Robert P.; Boheler, Kenneth R.

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity. PMID:22936984

The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

PubMed

Zhan, Ming; Riordon, Daniel R; Yan, Bin; Tarasova, Yelena S; Bruweleit, Sarah; Tarasov, Kirill V; Li, Ronald A; Wersto, Robert P; Boheler, Kenneth R

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

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.

Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity

PubMed Central

Lammers, Twan; Peschke, Peter; Ehemann, Volker; Debus, Jürgen; Slobodin, Boris; Lavi, Sara; Huber, Peter

2007-01-01

Background PP2Cα is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFβ-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Cα in cell growth and in radio- and chemosensitivity, wild type and PP2Cα siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Cα in tumorigenesis. Results It was found that knockdown of PP2Cα did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Cα siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Cα siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased. Conclusion Based on these findings, we conclude that PP2Cα is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Cα may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer. PMID:17941990

Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation1[OPEN

PubMed Central

Yu, Peng; Eggert, Kai; von Wirén, Nicolaus; Li, Chunjian; Hochholdinger, Frank

2015-01-01

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box proteinS-Phase Kinase-Associated Protein 2B-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses. PMID:26198256

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.

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

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

PubMed

Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

1999-08-01

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis

NASA Technical Reports Server (NTRS)

Takahashi, T.; Nowakowski, R. S.; Caviness, V. S. Jr

1996-01-01

Neurons of neocortical layers II-VI in the dorsomedial cortex of the mouse arise in the pseudostratified ventricular epithelium (PVE) through 11 cell cycles over the six embryonic days 11-17 (E11-E17). The present experiments measure the proportion of daughter cells that leave the cycle (quiescent or Q fraction or Q) during a single cell cycle and the complementary proportion that continues to proliferate (proliferative or P fraction or P; P = 1 - Q). Q and P for the PVE become 0.5 in the course of the eighth cycle, occurring on E14, and Q rises to approximately 0.8 (and P falls to approximately 0.2) in the course of the 10th cycle occurring on E16. This indicates that early in neuronogenesis, neurons are produced relatively slowly and the PVE expands rapidly but that the reverse happens in the final phase of neuronogenesis. The present analysis completes a cycle of analyses that have determined the four fundamental parameters of cell proliferation: growth fraction, lengths of cell cycle, and phases Q and P. These parameters are the basis of a coherent neuronogenetic model that characterizes patterns of growth of the PVE and mathematically relates the size of the initial proliferative population to the neuronal population of the adult neocortex.

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

Changes in endometrial natural killer cell expression of CD94, CD158a and CD158b are associated with infertility.

PubMed

McGrath, Emma; Ryan, Elizabeth J; Lynch, Lydia; Golden-Mason, Lucy; Mooney, Eoghan; Eogan, Maeve; O'Herlihy, Colm; O'Farrelly, Cliona

2009-04-01

Cycle-dependent fluctuations in natural killer (NK) cell populations in endometrium and circulation may differ, contributing to unexplained infertility. NK cell phenotypes were determined by flow cytometry in endometrial biopsies and matched blood samples. While circulating and endometrial T cell populations remained constant throughout the menstrual cycle in fertile and infertile women, circulating NK cells in infertile women increased during the secretory phase. However, increased expression of CD94, CD158b (secretory phase), and CD158a (proliferative phase) by endometrial NK cells from infertile women was observed. These changes were not reflected in the circulation. In infertile women, changes in circulating NK cell percentages are found exclusively during the secretory phase and not in endometrium; cycle-related changes in NK receptor expression are observed only in infertile endometrium. While having exciting implications for understanding NK cell function in fertility, our data emphasize the difficulty in attaching diagnostic or prognostic significance to NK cell analyses in individual patients.

Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2.

PubMed

Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra

2016-01-01

Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells. © 2015 John Wiley & Sons Ltd.

RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

PubMed

Yu, W; Sanders, B G; Kline, K

1997-01-01

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

A transient plasticity study and low cycle fatigue analysis of the Space Station Freedom photovoltaic solar array blanket

NASA Technical Reports Server (NTRS)

Armand, Sasan C.; Liao, Mei-Hwa; Morris, Ronald W.

1990-01-01

The Space Station Freedom photovoltaic solar array blanket assembly is comprised of several layers of materials having dissimilar elastic, thermal, and mechanical properties. The operating temperature of the solar array, which ranges from -75 to +60 C, along with the material incompatibility of the blanket assembly components combine to cause an elastic-plastic stress in the weld points of the assembly. The weld points are secondary structures in nature, merely serving as electrical junctions for gathering the current. The thermal mechanical loading of the blanket assembly operating in low earth orbit continually changes throughout each 90 min orbit, which raises the possibility of fatigue induced failure. A series of structural analyses were performed in an attempt to predict the fatigue life of the solar cell in the Space Station Freedom photovoltaic array blanket. A nonlinear elastic-plastic MSC/NASTRAN analysis followed by a fatigue calculation indicated a fatigue life of 92,000 to 160,000 cycles for the solar cell weld tabs. Additional analyses predict a permanent buckling phenomenon in the copper interconnect after the first loading cycle. This should reduce or eliminate the pulling of the copper interconnect on the joint where it is welded to the silicon solar cell. It is concluded that the actual fatigue life of the solar array blanket assembly should be significantly higher than the calculated 92,000 cycles, and thus the program requirement of 87,500 cycles (orbits) will be met. Another important conclusion that can be drawn from the overall analysis is that, the strain results obtained from the MSC/NASTRAN nonlinear module are accurate to use for low-cycle fatigue analysis, since both thermal cycle testing of solar cells and analysis have shown higher fatigue life than the minimum program requirement of 87,500 cycles.

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

PubMed

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

2008-01-01

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

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

PubMed

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

2014-03-01

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

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.

S-phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex.

PubMed

Turrero García, Miguel; Chang, YoonJeung; Arai, Yoko; Huttner, Wieland B

2016-02-15

The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

The transcription factor Foxg1 regulates telencephalic progenitor proliferation cell autonomously, in part by controlling Pax6 expression levels

PubMed Central

2011-01-01

Background The transcription factor Foxg1 is an important regulator of telencephalic cell cycles. Its inactivation causes premature lengthening of telencephalic progenitor cell cycles and increased neurogenic divisions, leading to severe hypoplasia of the telencephalon. These proliferation defects could be a secondary consequence of the loss of Foxg1 caused by the abnormal expression of several morphogens (Fibroblast growth factor 8, bone morphogenetic proteins) in the telencephalon of Foxg1 null mutants. Here we investigated whether Foxg1 has a cell autonomous role in the regulation of telencephalic progenitor proliferation. We analysed Foxg1+/+↔Foxg1-/- chimeras, in which mutant telencephalic cells have the potential to interact with, and to have any cell non-autonomous defects rescued by, normal wild-type cells. Results Our analysis showed that the Foxg1-/- cells are under-represented in the chimeric telencephalon and the proportion of them in S-phase is significantly smaller than that of their wild-type neighbours, indicating that their under-representation is caused by a cell autonomous reduction in their proliferation. We then analysed the expression of the cell-cycle regulator Pax6 and found that it is cell-autonomously downregulated in Foxg1-/- dorsal telencephalic cells. We went on to show that the introduction into Foxg1-/- embryos of a transgene designed to reverse Pax6 expression defects resulted in a partial rescue of the telencephalic progenitor proliferation defects. Conclusions We conclude that Foxg1 exerts control over telencephalic progenitor proliferation by cell autonomous mechanisms that include the regulation of Pax6, which itself is known to regulate proliferation cell autonomously in a regional manner. PMID:21418559

Characterization of the Th Profile of the Bovine Endometrium during the Oestrous Cycle and Early Pregnancy

PubMed Central

Oliveira, Lilian J.; Mansourri-Attia, Nadéra; Fahey, Alan G.; Browne, John; Forde, Niamh; Roche, James F.; Lonergan, Patrick; Fair, Trudee

2013-01-01

Despite extensive research in the area of cow fertility, the extent to which the maternal immune system is modulated during pregnancy in cattle remains unclear. Therefore, the objective of the current study was to characterize the presence and response profile of B, T-helper (LTh), T- cytotoxic (LTc), gamma delta-T (γδT) and natural killer (NK) lymphocytes in terms of cell number, distribution and cytokine expression in bovine endometrial tissue to pregnancy. Endometrial tissue samples were collected from beef heifers on Days 5, 7, 13 and 16 of the estrous cycle or pregnancy. Samples were analysed by immunofluorescence to identify the presence and abundance of B-B7 (B-cells), CD4 (LTh), CD8 (LTc), γδT cell receptor (TCR) and CD335/NKp46 (NK cells) -positive immune cells. Quantitative real time PCR (QPCR) was carried out to analyse mRNA relative abundance of FOXP3 (a marker of regulatory T (Treg) cells) and a panel of immune factors, including MHC-I, LIF, Interleukins 1, 2, 6, 8, 10, 11,12A, IFNa and IFNG. Results indicate that B-B7+ cells are quite populous in bovine endometrial tissue, CD4+ and CD8+ -cells are present in moderate numbers and γδTCR+ and CD335+ cells are present in low numbers. Pregnancy affected the total number and distribution pattern of the NK cell population, with the most significant variation observed on Day 16 of pregnancy. Neither B lymphocytes nor T lymphocyte subsets were regulated temporally during the oestrous cycle or by pregnancy prior to implantation. mRNA transcript abundance of the immune factors LIF, IL1b, IL8 and IL12A, IFNa and IFNG, expression was regulated temporally during the estrous cycle and LIF, IL1b, IL-10, IL11, IL12A were also temporally regulated during pregnancy. In conclusion, the endometrial immune profile of the oestrous cycle favours a Th2 environment in anticipation of pregnancy and the presence of an embryo acts to fine tune this environment. PMID:24204576

Cell cycle dynamics in a response/signalling feedback system with a gap.

PubMed

Gong, Xue; Buckalew, Richard; Young, Todd; Boczko, Erik

2014-01-01

We consider a dynamical model of cell cycles of n cells in a culture in which cells in one specific phase (S for signalling) of the cell cycle produce chemical agents that influence the growth/cell cycle progression of cells in another phase (R for responsive). In the case that the feedback is negative, it is known that subpopulations of cells tend to become clustered in the cell cycle; while for a positive feedback, all the cells tend to become synchronized. In this paper, we suppose that there is a gap between the two phases. The gap can be thought of as modelling the physical reality of a time delay in the production and action of the signalling agents. We completely analyse the dynamics of this system when the cells are arranged into two cell cycle clusters. We also consider the stability of certain important periodic solutions in which clusters of cells have a cyclic arrangement and there are just enough clusters to allow interactions between them. We find that the inclusion of a small gap does not greatly alter the global dynamics of the system; there are still large open sets of parameters for which clustered solutions are stable. Thus, we add to the evidence that clustering can be a robust phenomenon in biological systems. However, the gap does effect the system by enhancing the stability of the stable clustered solutions. We explain this phenomenon in terms of contraction rates (Floquet exponents) in various invariant subspaces of the system. We conclude that in systems for which these models are reasonable, a delay in signalling is advantageous to the emergence of clustering.

Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts.

PubMed

Markopoulos, Georgios S; Roupakia, Eugenia; Tokamani, Maria; Vartholomatos, George; Tzavaras, Theodore; Hatziapostolou, Maria; Fackelmayer, Frank O; Sandaltzopoulos, Raphael; Polytarchou, Christos; Kolettas, Evangelos

2017-10-01

Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL-1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G 1 /S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL-1 showed that a distinct set of 15 miRNAs were significantly up-regulated in senescent cells including hsa-let-7d-5p, hsa-let-7e-5p, hsa-miR-23a-3p, hsa-miR-34a-5p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-503-5p, hsa-miR-574-3p, hsa-miR-574-5p and hsa-miR-4454. Importantly, pathway analysis of miRNA target genes down-regulated during replicative senescence in a public RNA-seq data set revealed a significant high number of genes regulating cell cycle progression, both G 1 /S and G 2 /M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative-stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down-regulation of cell cycle effectors correlated with the up-regulation of miR-221 upon both replicative and oxidative stress-induced senescence. Transient expression of miR-221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up-regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Inorganic separator technology program

NASA Technical Reports Server (NTRS)

Smatko, J. S.; Weaver, R. D.; Kalhammer, F. R.

1973-01-01

Testing and failure analyses of silver zinc cells with largely inorganic separators were performed. The results showed that the wet stand and cycle life objective of the silver-zinc cell development program were essentially accomplished and led to recommendations for cell composition, design, and operation that should yield further improvement in wet and cycle life. A series of advanced inorganic materials was successfully developed and formulated into rigid and semiflexible separator samples. Suitable screening tests for evaluation of largely inorganic separators were selected and modified for application to the separator materials. The results showed that many of these formulations are potentially superior to previously used materials and permitted selection of three promising materials for further evaluation in silver-zinc cells.

Extracellular guanosine and GTP promote expression of differentiation markers and induce S-phase cell-cycle arrest in human SH-SY5Y neuroblastoma cells.

PubMed

Guarnieri, S; Pilla, R; Morabito, C; Sacchetti, S; Mancinelli, R; Fanò, G; Mariggiò, M A

2009-04-01

SH-SY5Y neuroblastoma cells, a model for studying neuronal differentiation, are able to differentiate into either cholinergic or dopaminergic/adrenergic phenotypes depending on media conditions. Using this system, we asked whether guanosine (Guo) or guanosine-5'-triphosphate (GTP) are able to drive differentiation towards one particular phenotype. Differentiation was determined by evaluating the frequency of cells bearing neurites and assessing neurite length after exposure to different concentrations of Guo or GTP for different durations. After 6 days, 0.3 mM Guo or GTP induced a significant increase in the number of cells bearing neurites and increased neurite length. Western blot analyses confirmed that purines induced differentiation; cells exposed to purines showed increases in the levels of GAP43, MAP2, and tyrosine hydroxylase. Proliferation assays and cytofluorimetric analyses indicated a significant anti-proliferative effect of purines, and a concentration-dependent accumulation of cells in S-phase, starting after 24 h of purine exposure and extending for up to 6 days. A transcriptional profile analysis using gene arrays showed that an up-regulation of cyclin E2/cdk2 evident after 24 h was responsible for S-phase entry, and a concurrent down-regulation of cell-cycle progression-promoting cyclin B1/B2 prevented S-phase exit. In addition, patch-clamp recordings revealed that 0.3 mM Guo or GTP, after 6 day incubation, significantly decreased Na(+) currents. In conclusion, we showed Guo- and GTP-induced cell-cycle arrest in neuroblastoma cells and suggest that this makes these cells more responsive to differentiation processes that favor the dopaminergic/adrenergic phenotype.

Impaired mitotic progression and preimplantation lethality in mice lacking OMCG1, a new evolutionarily conserved nuclear protein.

PubMed

Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten; Nacerddine, Karim; Babinet, Charles; Cohen-Tannoudji, Michel

2005-07-01

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1-/- embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo.

Impaired Mitotic Progression and Preimplantation Lethality in Mice Lacking OMCG1, a New Evolutionarily Conserved Nuclear Protein†

PubMed Central

Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten; Nacerddine, Karim; Babinet, Charles; Cohen-Tannoudji, Michel

2005-01-01

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1−/− embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo. PMID:15988037

Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions

PubMed Central

Sunter, Jack D.; Benz, Corinna; Andre, Jane; Whipple, Sarah; McKean, Paul G.; Gull, Keith; Ginger, Michael L.; Lukeš, Julius

2015-01-01

ABSTRACT The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure – the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms. PMID:26148511

S‐phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex

PubMed Central

Turrero García, Miguel; Chang, YoonJeung; Arai, Yoko

2016-01-01

ABSTRACT The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper‐layer neurons are produced. Based on cumulative 5‐ethynyl‐2′‐deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S‐phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self‐renewal to those of neuron production. Hence, S‐phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal. J. Comp. Neurol. 524:456–470, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:25963823

p57KIP2 regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex

PubMed Central

Mairet-Coello, Georges; Tury, Anna; Van Buskirk, Elise; Robinson, Kelsey; Genestine, Matthieu; DiCicco-Bloom, Emanuel

2012-01-01

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57KIP2, an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57KIP2 regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27KIP1 controlled IPC proliferation exclusively. Furthermore, p57KIP2 deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27KIP1 increased IPC proliferation at E16.5. Consequently, loss of p57KIP2 increased primarily layer 5-6 neuron production, whereas loss of p27KIP1 increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57KIP2 and p27KIP1 control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation. PMID:22223678

The life-cycle of Emiliania huxleyi: A brief review and a study of relative ploidy levels analysed by flow cytometry

NASA Astrophysics Data System (ADS)

Green, J. C.; Course, P. A.; Tarran, G. A.

1996-10-01

Emiliania huxleyi exists in several principal forms including the familiar coccolith-bearing C-cell, non-motile naked N-cells, and scale-bearing swarmers (S-cells), but the relationships between these cells are unclear. Flow cytometric analyses have been undertaken on whole cells using fluorochrome staining of the DNA in order to determine the relative DNA content and the relative GC content of the S- and C-cells of selected clones. Results showed that the DNA complement of the S-cells was half that of the C-cells and the two cell types are, therefore, haploid and diploid relative to each other. The S-cells may, therefore, represent a gametic stage, though processes such as sexual fusion and meiosis have not been observed.

Use of pressure cycling technology for cell lysis and recovery of bacterial and fungal communities from soil

USDA-ARS?s Scientific Manuscript database

Current molecular methodologies, specifically DNA-based approaches, provide access to previously hidden soil biodiversity and are routinely employed in environmental studies of microbial ecology. Selection of cell lysis methodology is critical to community analyses due to the inability of any singul...

Synthesis and anticancer activity of novel curcumin-quinolone hybrids.

PubMed

Raghavan, Saiharish; Manogaran, Prasath; Gadepalli Narasimha, Krishna Kumari; Kalpattu Kuppusami, Balasubramanian; Mariyappan, Palanivelu; Gopalakrishnan, Anjana; Venkatraman, Ganesh

2015-09-01

A number of new curcumin-quinolone hybrids were synthesised from differently substituted 3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most potent compound 14, was analysed for its mode of action using various cell biology experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle progression in S and G2/M phase. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

PubMed Central

Adam, Julie; Yang, Ming; Bauerschmidt, Christina; Kitagawa, Mitsuhiro; O’Flaherty, Linda; Maheswaran, Pratheesh; Özkan, Gizem; Sahgal, Natasha; Baban, Dilair; Kato, Keiko; Saito, Kaori; Iino, Keiko; Igarashi, Kaori; Stratford, Michael; Pugh, Christopher; Tennant, Daniel A.; Ludwig, Christian; Davies, Benjamin; Ratcliffe, Peter J.; El-Bahrawy, Mona; Ashrafian, Houman; Soga, Tomoyoshi; Pollard, Patrick J.

2013-01-01

Summary The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target. PMID:23643539

A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

PubMed

Adam, Julie; Yang, Ming; Bauerschmidt, Christina; Kitagawa, Mitsuhiro; O'Flaherty, Linda; Maheswaran, Pratheesh; Özkan, Gizem; Sahgal, Natasha; Baban, Dilair; Kato, Keiko; Saito, Kaori; Iino, Keiko; Igarashi, Kaori; Stratford, Michael; Pugh, Christopher; Tennant, Daniel A; Ludwig, Christian; Davies, Benjamin; Ratcliffe, Peter J; El-Bahrawy, Mona; Ashrafian, Houman; Soga, Tomoyoshi; Pollard, Patrick J

2013-05-30

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing.

PubMed

Gao, Shuai; Yan, Liying; Wang, Rui; Li, Jingyun; Yong, Jun; Zhou, Xin; Wei, Yuan; Wu, Xinglong; Wang, Xiaoye; Fan, Xiaoying; Yan, Jie; Zhi, Xu; Gao, Yun; Guo, Hongshan; Jin, Xiao; Wang, Wendong; Mao, Yunuo; Wang, Fengchao; Wen, Lu; Fu, Wei; Ge, Hao; Qiao, Jie; Tang, Fuchou

2018-06-01

The development of the digestive tract is critical for proper food digestion and nutrient absorption. Here, we analyse the main organs of the digestive tract, including the oesophagus, stomach, small intestine and large intestine, from human embryos between 6 and 25 weeks of gestation as well as the large intestine from adults using single-cell RNA-seq analyses. In total, 5,227 individual cells are analysed and 40 cell types clearly identified. Their crucial biological features, including developmental processes, signalling pathways, cell cycle, nutrient digestion and absorption metabolism, and transcription factor networks, are systematically revealed. Moreover, the differentiation and maturation processes of the large intestine are thoroughly investigated by comparing the corresponding transcriptome profiles between embryonic and adult stages. Our work offers a rich resource for investigating the gene regulation networks of the human fetal digestive tract and adult large intestine at single-cell resolution.

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.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed Central

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-01-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression. PMID:11105756

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-11-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

Cardiomyocyte cell cycle control and growth estimation in vivo--an analysis based on cardiomyocyte nuclei.

PubMed

Walsh, Stuart; Pontén, Annica; Fleischmann, Bernd K; Jovinge, Stefan

2010-06-01

Adult mammalian cardiomyocytes are traditionally viewed as being permanently withdrawn from the cell cycle. Whereas some groups have reported none, others have reported extensive mitosis in adult myocardium under steady-state conditions. Recently, a highly specific assay of 14C dating in humans has suggested a continuous generation of cardiomyocytes in the adult, albeit at a very low rate. Mice represent the most commonly used animal model for these studies, but their short lifespan makes them unsuitable for 14C studies. Herein, we investigate the cellular growth pattern for murine cardiomyocyte growth under steady-state conditions, addressed with new analytical and technical strategies, and we furthermore relate this to gene expression patterns. The observed levels of DNA synthesis in early life were associated with cardiomyocyte proliferation. Mitosis was prolonged into early life, longer than the most conservative previous estimates. DNA synthesis in neonatal life was attributable to bi-nucleation, therefore suggesting that cardiomyocytes withdraw from the cell cycle shortly after birth. No cell cycle activity was observed in adult cardiomyocytes and significant polyploidy was observed in cardiomyocyte nuclei. Gene analyses identified 32 genes whose expression was predicted to be particular to day 3-4 neonatal myocytes, compared with embryonic or adult cells. These cell cycle-associated genes are crucial to the understanding of the mechanisms of bi-nucleation and physiological cellular growth in the neonatal period.

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.

Evolution of the genetic machinery of the visual cycle: a novelty of the vertebrate eye?

PubMed

Albalat, Ricard

2012-05-01

The discovery in invertebrates of ciliary photoreceptor cells and ciliary (c)-opsins established that at least two of the three elements that characterize the vertebrate photoreceptor system were already present before vertebrate evolution. However, the origin of the third element, a series of biochemical reactions known as the "retinoid cycle," remained uncertain. To understand the evolution of the retinoid cycle, I have searched for the genetic machinery of the cycle in invertebrate genomes, with special emphasis on the cephalochordate amphioxus. Amphioxus is closely related to vertebrates, has a fairly prototypical genome, and possesses ciliary photoreceptor cells and c-opsins. Phylogenetic and structural analyses of the amphioxus sequences related with the vertebrate machinery do not support a function of amphioxus proteins in chromophore regeneration but suggest that the genetic machinery of the retinoid cycle arose in vertebrates due to duplications of ancestral nonvisual genes. These results favor the hypothesis that the retinoid cycle machinery was a functional innovation of the primitive vertebrate eye.

Photoperiod length paces the temporal orchestration of cell cycle and carbon-nitrogen metabolism in Crocosphaera watsonii.

PubMed

Dron, Anthony; Rabouille, Sophie; Claquin, Pascal; Talec, Amélie; Raimbault, Virginie; Sciandra, Antoine

2013-12-01

We analysed the effect of photoperiod length (PPL) (16:8 and 8:16 h of light-dark regime, named long and short PPL, respectively) on the temporal orchestration of the two antagonistic, carbon and nitrogen acquisitions in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii strain WH8501 growing diazotrophically. Carbon and nitrogen metabolism were monitored at high frequency, and their patterns were compared with the cell cycle progression. The oxygen-sensitive N2 fixation process occurred mainly during the dark period, where photosynthesis cannot take place, inducing a light-dark cycle of cellular C : N ratio. Examination of circadian patterns in the cell cycle revealed that cell division occurred during the midlight period, (8 h and 4 h into the light in the long and short PPL conditions, respectively), thus timely separated from the energy-intensive diazotrophic process. Results consistently show a nearly 5 h time lag between the end of cell division and the onset of N2 fixation. Shorter PPLs affected DNA compaction of C. watsonii cells and also led to a decrease in the cell division rate. Therefore, PPL paces the growth of C. watsonii: a long PPL enhances cell division while a short PPL favours somatic growth (biomass production) with higher carbon and nitrogen cell contents. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Localization of early germ cells in a stony coral, Euphyllia ancora: potential implications for a germline stem cell system in coral gametogenesis

NASA Astrophysics Data System (ADS)

Shikina, Shinya; Chung, Yi-Jou; Wang, Hsiang-Ming; Chiu, Yi-Ling; Shao, Zih-Fang; Lee, Yan-Horn; Chang, Ching-Fong

2015-06-01

Most corals exhibit annual or multiple gametogenic cycles. Thus far, coral gametogenesis has been studied in many species and locations during the past three decades; however, currently, only a few papers exist that describe the origin of germ cells, such as germline stem cells (GSCs), which support the continuous production of gametes in every reproductive cycle. To address this issue, in this study, we focused on and identified piwi gene, which has been used as a marker of germline cells, including GSCs, in various metazoans, in a scleractinian coral, Euphyllia ancora. Reverse-transcription PCR and Western blotting analyses revealed that E. ancora piwi-like ( Eapiwi) is expressed in mesentery tissues where the sites of gametogenesis are located for both sexes. Immunohistochemistry with a specific antibody against Eapiwi revealed strong immunoreactivity in the spermatogonia in males and in the oogonia and early oocytes in females, demonstrating that Eapiwi could be used as an early germ cell marker in E. ancora. Subsequent immunohistochemical analyses regarding the spatial and temporal distribution patterns of early germ cells in mesentery tissues revealed that early germ cells were present throughout the year in the mesentery tissue we examined, regardless of the sexual reproductive cycle. In particular, small numbers of early germ cells were observed in specific sites of mesentery tissues with fully matured gonads in both sexes. These early germ cells were not released together with mature gametes during the spawning period and remained in the mesentery tissues. These results suggested that these early germ cells most likely serve as a reservoir of germline cells and that some of these cells would produce differentiated germ cells for the upcoming sexual reproduction period; hence, these cells would function as GSCs. Our data provide new information for understanding continuous gamete production in corals.

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.

Life, performance and safety of Grace rechargeable lithium-titanium disulfide cells

NASA Astrophysics Data System (ADS)

Zuckerbrod, D.; Giovannoni, R. T.; Grossman, K. R.

The development of rechargeable Li-TiS2 cells is discussed. This proprietary process produces thin, flexible TiS2 cathodes with a life in excess of 500 cycles. TiS2 utilization of 93 percent is typically achieved at a C/5 discharge rate. A life of 200 cycles has been achieved for AA-size cells at a C/5 discharge rate and 60 cycles at the C rate. The practical energy density is 115 Wh/kg. Safety testing is underway. Vibration and high altitude did not cause venting. Crushing did not result in ignition or temperature rise. Forced overcharge and overdischarge did not result in skin temperatures above 40 C. The peak skin temperature during the short-circuit test was 120 C. Safety analyses point to the need for careful control of electrolyte volume and the benefits of a fusible separator. Grace is developing such a separator, which would shut down the electrochemical cell reaction at a temperature of 130 C.

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

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.

Integrative functional transcriptomic analyses implicate specific molecular pathways in pulmonary toxicity from exposure to aluminum oxide nanoparticles.

PubMed

Li, Xiaobo; Zhang, Chengcheng; Bian, Qian; Gao, Na; Zhang, Xin; Meng, Qingtao; Wu, Shenshen; Wang, Shizhi; Xia, Yankai; Chen, Rui

2016-09-01

Gene expression profiling has developed rapidly in recent years and it can predict and define mechanisms underlying chemical toxicity. Here, RNA microarray and computational technology were used to show that aluminum oxide nanoparticles (Al2O3 NPs) were capable of triggering up-regulation of genes related to the cell cycle and cell death in a human A549 lung adenocarcinoma cell line. Gene expression levels were validated in Al2O3 NPs exposed A549 cells and mice lung tissues, most of which showed consistent trends in regulation. Gene-transcription factor network analysis coupled with cell- and animal-based assays demonstrated that the genes encoding PTPN6, RTN4, BAX and IER play a role in the biological responses induced by the nanoparticle exposure, which caused cell death and cell cycle arrest in the G2/S phase. Further, down-regulated PTPN6 expression demonstrated a core role in the network, thus expression level of PTPN6 was rescued by plasmid transfection, which showed ameliorative effects of A549 cells against cell death and cell cycle arrest. These results demonstrate the feasibility of using gene expression profiling to predict cellular responses induced by nanomaterials, which could be used to develop a comprehensive knowledge of nanotoxicity.

Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

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

Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.

2006-09-10

Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis.more » Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.« less

The sow endometrium at different stages of the oestrous cycle: immunohistochemical study on the distribution of SWC3-expressing cells (granulocytes, monocytes and macrophages).

PubMed

Kaeoket, K; Dalin, A M; Magnusson, U; Persson, E

2001-10-01

Uterine samples from sows taken immediately after slaughter at late di-oestrus, pro-oestrus, oestrus, early di-oestrus and di-ocstrus, were analysed by immunohistochemistry with an avidinbiotin-peroxidase method using a monoclonal antibody (anti-SWC3) to granulocyte, monocyte and macrophage populations. The endometrium was then examined by light microscopy. In the surface and glandular epithelium, the largest numbers of SWC3-expressing cells (P < or = 0.01 and P < or = 0.05) were found at oestrus, and at pro-oestrus and oestrus, respectively. The numbers of SWC3-expressing cells in the epithelium were positively correlated with the plasma levels of oestradiol-17beta. In the connective tissue of the subepithelial and glandular layers, no significant effect of the oestrous Cycle stage was found on the number of SWC3-expressing cells. The present study showed a variation in the distribution of SWC3-expressing cells in the sow endometrium, especially in the surface and glandular epithelium, during different stages of the oestrous cycle.

Global isoform-specific transcript alterations and deregulated networks in clear cell renal cell carcinoma

PubMed Central

Hamilton, Michael J.; Girke, Thomas; Martinez, Ernest

2018-01-01

Extensive genome-wide analyses of deregulated gene expression have now been performed for many types of cancer. However, most studies have focused on deregulation at the gene-level, which may overlook the alterations of specific transcripts for a given gene. Clear cell renal cell carcinoma (ccRCC) is one of the best-characterized and most pervasive renal cancers, and ccRCCs are well-documented to have aberrant RNA processing. In the present study, we examine the extent of aberrant isoform-specific RNA expression by reporting a comprehensive transcript-level analysis, using the new kallisto-sleuth-RATs pipeline, investigating coding and non-coding differential transcript expression in ccRCC. We analyzed 50 ccRCC tumors and their matched normal samples from The Cancer Genome Altas datasets. We identified 7,339 differentially expressed transcripts and 94 genes exhibiting differential transcript isoform usage in ccRCC. Additionally, transcript-level coexpression network analyses identified vasculature development and the tricarboxylic acid cycle as the most significantly deregulated networks correlating with ccRCC progression. These analyses uncovered several uncharacterized transcripts, including lncRNAs FGD5-AS1 and AL035661.1, as potential regulators of the tricarboxylic acid cycle associated with ccRCC progression. As ccRCC still presents treatment challenges, our results provide a new resource of potential therapeutics targets and highlight the importance of exploring alternative methodologies in transcriptome-wide studies.

Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

PubMed Central

Miga, Karen H.; Sekulic, Nikolina; Soni, Gautam V.; Kim, Dong Hyun; Wong, Adeline K.; Lee, Ah Young; Nguyen, Kristen; Dekker, Cees; Ren, Bing; Black, Ben E.

2017-01-01

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat–containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1–containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2–4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases. PMID:28235947

[Effect of taspine derivatives on human liver cancer SMMC7721].

PubMed

Zhang, Yan-min; Wang, Nan; Dai, Bing-ling; He, Lang-chong

2011-07-01

To analyse the inhibition effect of taspine derivatives on human Liver cancer SMMC7721 cell and its mechanism. The effects of five taspine derivatives on SMMC7721 cell growth were determined by MTT. The flow cytometry was used to determine the cell cycle. The effects of Tas-D1 on the EGF and VEGF in SMMC7721 cell were determined by ELISA. The mRNA level of EGF and VEGF in SMMC7721 cell was determined by RT-PCR. The MTT assay demonstrated that the taspine derivative Tas-D1 significantly inhibited the growth of SMMC7721 cell in a dose-dependent manner. Cell was stopped at S phase by Tas-D1. Tas-D1 inhibited the expression of EGF and VEGF and their mRNA in a dose-dependent manner (P<0.05). The taspine derivative Tas-D1 can inhibit the growth of human Liver cancer SMMC7721 cell and change cell cycle, which may be related to the inhibition of EGF and VEGF expression.

The geometry of proliferating dicot cells.

PubMed

Korn, R W

2001-02-01

The distributions of cell size and cell cycle duration were studied in two-dimensional expanding plant tissues. Plastic imprints of the leaf epidermis of three dicot plants, jade (Crassula argentae), impatiens (Impatiens wallerana), and the common begonia (Begonia semperflorens) were made and cell outlines analysed. The average, standard deviation and coefficient of variance (CV = 100 x standard deviation/average) of cell size were determined with the CV of mother cells less than the CV for daughter cells and both are less than that for all cells. An equation was devised as a simple description of the probability distribution of sizes for all cells of a tissue. Cell cycle durations as measured in arbitrary time units were determined by reconstructing the initial and final sizes of cells and they collectively give the expected asymmetric bell-shaped probability distribution. Given the features of unequal cell division (an average of 11.6% difference in size of daughter cells) and the size variation of dividing cells, it appears that the range of cell size is more critically regulated than the size of a cell at any particular time.

Knockdown of hTERT and concurrent treatment with interferon-gamma inhibited proliferation and invasion of human glioblastoma cell lines

PubMed Central

George, Joseph; Banik, Naren L.; Ray, Swapan K.

2011-01-01

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. Telomerase and hTERT are remarkably upregulated in majority of cancers including glioblastoma. Interferon-gamma (IFN-γ) modulates several cellular activities including cell cycle and multiplication through transcriptional regulation. The present investigation was designed to unravel the molecular mechanisms of the inhibition of cell proliferation, migration, and invasion of human glioblastoma SNB-19 and LN-18 cell lines after knockdown of hTERT using a plasmid vector based siRNA and concurrent treatment with IFN-γ. We observed more than 80% inhibition of cell proliferation, migration, and invasion of both cell lines after the treatment with combination of hTERT siRNA and IFN-γ. Our studies also showed accumulation of apoptotic cells in subG1 phase and an increase in cell population in G0/G1 with a reduction in G2/M phase indicating cell cycle arrest in G0/G1 phase for apoptosis. Semiquantitative and real-time RT-PCR analyses demonstrated significant downregulation of c- Myc and upregulation of p21 Waf1 and p27 Kip1. Western blotting confirmed the downregulation of the molecules involved in cell proliferation, migration, and invasion and also showed upregulation of cell cycle inhibitors. In conclusion, our study demonstrated that knockdown of hTERT siRNA and concurrent treatment with IFN-γ effectively inhibited cell proliferation, migration, and invasion in glioblastoma cells through downregulation of the molecules involved in these processes and cell cycle inhibition. Therefore, the combination of hTERT siRNA and IFN-γ offers a potential therapeutic strategy for controlling growth of human glioblastoma cells. PMID:20394835

Accelerated test plan for nickel cadmium spacecraft batteries

NASA Technical Reports Server (NTRS)

Hennigan, T. J.

1973-01-01

An accelerated test matrix is outlined that includes acceptance, baseline and post-cycling tests, chemical and physical analyses, and the data analysis procedures to be used in determining the feasibility of an accelerated test for sealed, nickel cadmium cells.

Live Imaging-Based Model Selection Reveals Periodic Regulation of the Stochastic G1/S Phase Transition in Vertebrate Axial Development

PubMed Central

Kurokawa, Hiroshi; Sakaue-Sawano, Asako; Imamura, Takeshi; Miyawaki, Atsushi; Iimura, Tadahiro

2014-01-01

In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode. PMID:25474567

Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling

PubMed Central

Zhao, Jianzhi; Li, Hanjun; Zhou, Rujiang; Ma, Gang; Dekker, Joseph D.; Tucker, Haley O.; Yao, Zhengju; Guo, Xizhi

2015-01-01

Hair follicle stem cells (HFSCs) in the bugle circularly generate outer root sheath (ORS) through linear proliferation within limited cycles during anagen phases. However, the mechanisms controlling the pace of HFSC proliferation remain unclear. Here we revealed that Foxp1, a transcriptional factor, was dynamically relocated from the nucleus to the cytoplasm of HFSCs in phase transitions from anagen to catagen, coupled with the rise of oxidative stress. Mass spectrum analyses revealed that the S468 phosphorylation of Foxp1 protein was responsive to oxidative stress and affected its nucleocytoplasmic translocation. Foxp1 deficiency in hair follicles led to compromised ROS accrual and increased HFSC proliferation. And more, NAC treatment profoundly elongated the anagen duration and HFSC proliferation in Foxp1-deficient background. Molecularly, Foxp1 augmented ROS levels through suppression of Trx1-mediated reductive function, thereafter imposing the cell cycle arrest by modulating the activity of p19/p53 pathway. Our findings identify a novel role for Foxp1 in controlling HFSC proliferation with cellular dynamic location in response to oxidative stress during hair cycling. PMID:26171970

Optimal design of solid oxide fuel cell, ammonia-water single effect absorption cycle and Rankine steam cycle hybrid system

NASA Astrophysics Data System (ADS)

Mehrpooya, Mehdi; Dehghani, Hossein; Ali Moosavian, S. M.

2016-02-01

A combined system containing solid oxide fuel cell-gas turbine power plant, Rankine steam cycle and ammonia-water absorption refrigeration system is introduced and analyzed. In this process, power, heat and cooling are produced. Energy and exergy analyses along with the economic factors are used to distinguish optimum operating point of the system. The developed electrochemical model of the fuel cell is validated with experimental results. Thermodynamic package and main parameters of the absorption refrigeration system are validated. The power output of the system is 500 kW. An optimization problem is defined in order to finding the optimal operating point. Decision variables are current density, temperature of the exhaust gases from the boiler, steam turbine pressure (high and medium), generator temperature and consumed cooling water. Results indicate that electrical efficiency of the combined system is 62.4% (LHV). Produced refrigeration (at -10 °C) and heat recovery are 101 kW and 22.1 kW respectively. Investment cost for the combined system (without absorption cycle) is about 2917 kW-1.

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.

Design Principles for Nickel/Hydrogen Cells and Batteries

NASA Technical Reports Server (NTRS)

Thaller, Lawrence H.; Manzo, Michelle A.; Gonzalez-Sanabria, Olga D.

1987-01-01

Individual-pressure-vessel (IPV) nickel/hydrogen cells and bipolar batteries developed for use as energy-storage subsystems for satelite applications. Design principles applied draw upon extensive background in separator technology, alkaline-fuel-cell technology and several alkaline-cell technology areas. Principals are rather straightforward applications of capillary-force formalisms, coupled with slowly developing data base resulting from careful post-test analyses. Based on preconceived assumptions relative to how devices work and how to be designed so they display longer cycle lives at deep discharge.

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

PubMed Central

Moch, Marcin; Windoffer, Reinhard; Schwarz, Nicole; Pohl, Raphaela; Omenzetter, Andreas; Schnakenberg, Uwe; Herb, Fabian; Chaisaowong, Kraisorn; Merhof, Dorit; Ramms, Lena; Fabris, Gloria; Hoffmann, Bernd; Merkel, Rudolf; Leube, Rudolf E.

2016-01-01

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells. PMID:27007410

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.

Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication.

PubMed

Camara, Johanna E; Breier, Adam M; Brendler, Therese; Austin, Stuart; Cozzarelli, Nicholas R; Crooke, Elliott

2005-08-01

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

Genomic signal processing: from matrix algebra to genetic networks.

PubMed

Alter, Orly

2007-01-01

DNA microarrays make it possible, for the first time, to record the complete genomic signals that guide the progression of cellular processes. Future discovery in biology and medicine will come from the mathematical modeling of these data, which hold the key to fundamental understanding of life on the molecular level, as well as answers to questions regarding diagnosis, treatment, and drug development. This chapter reviews the first data-driven models that were created from these genome-scale data, through adaptations and generalizations of mathematical frameworks from matrix algebra that have proven successful in describing the physical world, in such diverse areas as mechanics and perception: the singular value decomposition model, the generalized singular value decomposition model comparative model, and the pseudoinverse projection integrative model. These models provide mathematical descriptions of the genetic networks that generate and sense the measured data, where the mathematical variables and operations represent biological reality. The variables, patterns uncovered in the data, correlate with activities of cellular elements such as regulators or transcription factors that drive the measured signals and cellular states where these elements are active. The operations, such as data reconstruction, rotation, and classification in subspaces of selected patterns, simulate experimental observation of only the cellular programs that these patterns represent. These models are illustrated in the analyses of RNA expression data from yeast and human during their cell cycle programs and DNA-binding data from yeast cell cycle transcription factors and replication initiation proteins. Two alternative pictures of RNA expression oscillations during the cell cycle that emerge from these analyses, which parallel well-known designs of physical oscillators, convey the capacity of the models to elucidate the design principles of cellular systems, as well as guide the design of synthetic ones. In these analyses, the power of the models to predict previously unknown biological principles is demonstrated with a prediction of a novel mechanism of regulation that correlates DNA replication initiation with cell cycle-regulated RNA transcription in yeast. These models may become the foundation of a future in which biological systems are modeled as physical systems are today.

A microPIXE investigation of the interaction of cells of Schizosaccharomyces pombe with the culture medium

NASA Astrophysics Data System (ADS)

Rombouts, P. M. M.; Gomez-Morilla, I.; Grime, G. W.; Webb, R. P.; Cuenca, L.; Rodriguez, R.; Browton, M.; Wardell, N.; Underwood, B.; Kirkby, N. F.; Kirkby, K. J.

2007-07-01

Schizosaccharomyces pombe ( S. pombe) is a eucaryotic cell type similar to mammalian cells but much more simple. As it also executes its cell cycle rapidly it is very useful for investigating basic processes in cells. In this paper we report a feasibility study of the applicability of microPIXE to investigate the interaction between S. pombe cells and the surrounding culture medium. Cells were cultured in various growth medium prior to preparation for analysis. 1 μl drops of medium and cells were spotted onto polypropylene foils held in contact with a polished copper block previously cooled in liquid nitrogen. The samples were dehydrated by freeze-drying. Micro PIXE analysis was carried out with the IBC microbeam facility using a beam of 2.5 MeV protons focused to 1-2 μm diameter. Initially no elemental contrast was observed between the cells and the medium, but by modifying the dilution of the cell suspension, the cells could be distinguished from the surrounding medium through an increased concentration of P and reduced concentration of Cl. The distribution of Na in the medium around the cells showed evidence of the action of the Na pump. Sporulation appears to be induced in the cells by adding Cu to the growth medium and the uptake of Cu by the cells could be clearly observed. This study shows that it is possible to analyse the mass transport of elements in and out of cells In the future this will enable concentration gradients to be analysed and allow the rate of production or consumption of individual cells to be calculated. By observing these patterns for individual cells (not populations) at various known points in the cell cycle, fundamental data can be derived.

Regulatory Response to Carbon Starvation in Caulobacter crescentus

PubMed Central

Britos, Leticia; Abeliuk, Eduardo; Taverner, Thomas; Lipton, Mary; McAdams, Harley; Shapiro, Lucy

2011-01-01

Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells. PMID:21494595

Quantitative characterization of the imaging limits of diffuse low-grade oligodendrogliomas.

PubMed

Gerin, Chloé; Pallud, Johan; Deroulers, Christophe; Varlet, Pascale; Oppenheim, Catherine; Roux, Francois-Xavier; Chrétien, Fabrice; Thomas, Stephen R; Grammaticos, Basile; Badoual, Mathilde

2013-10-01

Supratentorial diffuse low-grade gliomas in adults extend beyond maximal visible MRI-defined abnormalities, and a gap exists between the imaging signal changes and the actual tumor margins. Direct quantitative comparisons between imaging and histological analyses are lacking to date. However, they are of the utmost importance if one wishes to develop realistic models for diffuse glioma growth. In this study, we quantitatively compared the cell concentration and the edema fraction from human histological biopsy samples (BSs) performed inside and outside imaging abnormalities during serial imaging-based stereotactic biopsy of diffuse low-grade gliomas. The cell concentration was significantly higher in BSs located inside (1189 ± 378 cell/mm(2)) than outside (740 ± 124 cell/mm(2)) MRI-defined abnormalities (P = .0003). The edema fraction was significantly higher in BSs located inside (mean, 45% ± 23%) than outside (mean, 5 %± 9%) MRI-defined abnormalities (P < .0001). At borders of the MRI-defined abnormalities, 20% of the tissue surface area was occupied by edema and only 3% by tumor cells. The cycling cell concentration was significantly higher in BSs located inside (10 ± 12 cell/mm(2)), compared with outside (0.5 ± 0.9 cell/mm(2)), MRI-defined abnormalities (P = .0001). We showed that the margins of T2-weighted signal changes are mainly correlated with the edema fraction. In 62.5% of patients, the cycling tumor cell fraction (defined as the ratio of the cycling tumor cell concentration to the total number of tumor cells) was higher at the limits of the MRI-defined abnormalities than closer to the center of the tumor. In the remaining patients, the cycling tumor cell fraction increased towards the center of the tumor.

Diatom life cycles and ecology in the Cretaceous.

PubMed

Jewson, David H; Harwood, David M

2017-06-01

The earliest known diatom fossils with well-preserved siliceous frustules are from Lower Cretaceous neritic marine deposits in Antarctica. In this study, we analyzed the cell wall structure to establish whether their cell and life cycles were similar to modern forms. At least two filamentous species (Basilicostephanus ornatus and Archepyrgus melosiroides) had girdle band structures that functioned during cell division in a similar way to present day Aulacoseira species. Also, size analyses of cell diameter indicated that the cyclic process of size decline and size restoration used to time modern diatom life cycles was present in five species from the Lower Cretaceous (B. ornatus, A. melosiroides, Gladius antiquus, Ancylopyrgus reticulatus, Kreagra forfex) as well as two species from Upper Cretaceous deposits (Trinacria anissimowii and Eunotogramma fueloepi) from the Southwest Pacific. The results indicate that the "Diatom Sex Clock" was present from an early evolutionary stage. Other ecological adaptations included changes in mantle height and coiling. Overall, the results suggest that at least some of the species in these early assemblages are on a direct ancestral line to modern forms. © 2017 Phycological Society of America.

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase

NASA Technical Reports Server (NTRS)

George, K.; Wu, H.; Willingham, V.; Furusawa, Y.; Kawata, T.; Cucinotta, F. A.; Dicello, J. F. (Principal Investigator)

2001-01-01

PURPOSE: To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. MATERIALS AND METHODS: Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. RESULTS: There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. CONCLUSION: The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase.

PubMed

George, K; Wu, H; Willingham, V; Furusawa, Y; Kawata, T; Cucinotta, F A

2001-02-01

To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Salicylic acid antagonizes abscisic acid inhibition of shoot growth and cell cycle progression in rice

NASA Astrophysics Data System (ADS)

Meguro, Ayano; Sato, Yutaka

2014-04-01

We analysed effects of abscisic acid (ABA, a negative regulatory hormone), alone and in combination with positive or neutral hormones, including salicylic acid (SA), on rice growth and expression of cell cycle-related genes. ABA significantly inhibited shoot growth and induced expression of OsKRP4, OsKRP5, and OsKRP6. A yeast two-hybrid assay showed that OsKRP4, OsKRP5, and OsKRP6 interacted with OsCDKA;1 and/or OsCDKA;2. When SA was simultaneously supplied with ABA, the antagonistic effect of SA completely blocked ABA inhibition. SA also blocked ABA inhibition of DNA replication and thymidine incorporation in the shoot apical meristem. These results suggest that ABA arrests cell cycle progression by inducing expression of OsKRP4, OsKRP5, and OsKRP6, which inhibit the G1/S transition, and that SA antagonizes ABA by blocking expression of OsKRP genes.

Anti-prostate cancer activity of a beta-carboline alkaloid enriched extract from Rauwolfia vomitoria.

PubMed

Bemis, D L; Capodice, J L; Gorroochurn, P; Katz, A E; Buttyan, R

2006-11-01

The tropical shrub, Rauwolfia vomitoria, is a medicinal plant used traditionally to treat a variety of ailments. A bioactive beta-carboline alkaloid, alstonine, present in this extract was previously shown to have anti-cancer activity against cancer cell lines. This study considers the potential anti-prostate cancer activity of this extract in vitro and in vivo. Rauwolfia vomitoria extract standardized for beta-carboline alkaloids was tested for ability to influence the growth and survival of the human LNCaP prostate cancer cell line. A WST-1 assay was used to measure cell growth, and cell cycle analyses were conducted with flow cytometry. Western blot detection of PARP cleavage and accumulation of cells containing sub-genomic DNA indicated induction of apoptosis. Pathway specific microarray analyses were utilized to identify the effect of Rauwolfia extract on the expression of 225 genes. Mice xenografted with LNCaP cells were treated with the extract or placebo control, and tumor growth was measured for 5 weeks. The effects of the extract on xenografted tumor cell proliferation and apoptosis were measured by in situ BrdU incorporation and TUNEL staining. Rauwolfia extract decreased in vitro cell growth in a dose-dependent manner (p<0.001) and induced the accumulation of G1 phase cells. PARP cleavage demonstrated that apoptosis was induced only at the highest concentration tested (500 microg/ml) which was confirmed by detection of cells containing sub-genomic DNA. The expression of genes associated with DNA damage signaling pathway was up-regulated by Rauwolfia treatment, including that of GADD153 and MDG. The expression of a few cell cycle genes (p21, cyclin D1 and E2F1) was also modulated. These alterations were confirmed by RT-PCR. Tumor volumes were decreased by 60%, 70% and 58% in the groups fed the 75, 37.5 or 7.5 mg/kg Rauwolfia, respectively (Kruskal-Wallis test, p<0.001). The Rauwolfia vomitoria extract significantly suppressed the growth and cell cycle progression of LNCaP cells, in vitro and in vivo.

TRANSCRIPTOME ANALYSES REVEAL DIFFERENTIAL GENE EXPRESSION PATTERNS BETWEEN THE LIFE-CYCLE STAGES OF EMILIANIA HUXLEYI (HAPTOPHYTA) AND REFLECT SPECIALIZATION TO DIFFERENT ECOLOGICAL NICHES(1).

PubMed

Rokitta, Sebastian D; de Nooijer, Lennart J; Trimborn, Scarlett; de Vargas, Colomban; Rost, Björn; John, Uwe

2011-08-01

Coccolithophores, especially the abundant, cosmopolitan species Emiliania huxleyi (Lohmann) W. W. Hay et H. P. Mohler, are one of the main driving forces of the oceanic carbonate pump and contribute significantly to global carbon cycling, due to their ability to calcify. A recent study indicates that termination of diploid blooms by viral infection induces life-cycle transition, and speculation has arisen about the role of the haploid, noncalcifying stage in coccolithophore ecology. To explore gene expression patterns in both life-cycle stages, haploid and diploid cells of E. huxleyi (RCC 1217 and RCC 1216) were acclimated to limiting and saturating photon flux densities. Transcriptome analyses were performed to assess differential genomic expression related to different ploidy levels and acclimation light intensities. Analyses indicated that life-cycle stages exhibit different properties of regulating genome expression (e.g., pronounced gene activation and gene silencing in the diploid stage), proteome maintenance (e.g., increased turnover of proteins in the haploid stage), as well as metabolic processing (e.g., pronounced primary metabolism and motility in the haploid stage and calcification in the diploid stage). Furthermore, higher abundances of transcripts related to endocytotic and digestive machinery were observed in the diploid stage. A qualitative feeding experiment indicated that both life-cycle stages are capable of particle uptake (0.5 μm diameter) in late-stationary growth phase. Results showed that the two life-cycle stages represent functionally distinct entities that are evolutionarily shaped to thrive in the environment they typically inhabit. © 2011 Phycological Society of America.

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

Analysis and performance assessment of a new solar-based multigeneration system integrated with ammonia fuel cell and solid oxide fuel cell-gas turbine combined cycle

NASA Astrophysics Data System (ADS)

Siddiqui, Osamah; Dincer, Ibrahim

2017-12-01

In the present study, a new solar-based multigeneration system integrated with an ammonia fuel cell and solid oxide fuel cell-gas turbine combined cycle to produce electricity, hydrogen, cooling and hot water is developed for analysis and performance assessment. In this regard, thermodynamic analyses and modeling through both energy and exergy approaches are employed to assess and evaluate the overall system performance. Various parametric studies are conducted to study the effects of varying system parameters and operating conditions on the energy and exergy efficiencies. The results of this study show that the overall multigeneration system energy efficiency is obtained as 39.1% while the overall system exergy efficiency is calculated as 38.7%, respectively. The performance of this multigeneration system results in an increase of 19.3% in energy efficiency as compared to single generation system. Furthermore, the exergy efficiency of the multigeneration system is 17.8% higher than the single generation system. Moreover, both energy and exergy efficiencies of the solid oxide fuel cell-gas turbine combined cycle are determined as 68.5% and 55.9% respectively.

Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition

PubMed Central

Fu, Changlong; Zheng, Chunsong; Lin, Jie; Ye, Jinxia; Mei, Yangyang; Pan, Caibin; Wu, Guangwen; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2017-01-01

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose- and time-dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin-dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis. PMID:28358416

Pause, play, repeat

PubMed Central

Sansó, Miriam; Fisher, Robert P

2013-01-01

Cyclin-dependent kinases (CDKs) play a central role in governing eukaryotic cell division. It is becoming clear that the transcription cycle of RNA polymerase II (RNAP II) is also regulated by CDKs; in metazoans, the cell cycle and transcriptional CDK networks even share an upstream activating kinase, which is itself a CDK. From recent chemical-genetic analyses we know that CDKs and their substrates control events both early in transcription (the transition from initiation to elongation) and late (3′ end formation and transcription termination). Moreover, mutual dependence on CDK activity might couple the “beginning” and “end” of the cycle, to ensure the fidelity of mRNA maturation and the efficient recycling of RNAP II from sites of termination to the transcription start site (TSS). As is the case for CDKs involved in cell cycle regulation, different transcriptional CDKs act in defined sequence on multiple substrates. These phosphorylations are likely to influence gene expression by several mechanisms, including direct, allosteric effects on the transcription machinery, co-transcriptional recruitment of proteins needed for mRNA-capping, splicing and 3′ end maturation, dependent on multisite phosphorylation of the RNAP II C-terminal domain (CTD) and, perhaps, direct regulation of RNA-processing or histone-modifying machinery. Here we review these recent advances, and preview the emerging challenges for transcription-cycle research. PMID:23756342

Biological Effects of the Pim Kinase Inhibitor, SGI-1776, in Multiple Myeloma

PubMed Central

Cervantes-Gomez, Fabiola; Chen, Lisa S.; Orlowski, Robert Z.; Gandhi, Varsha

2013-01-01

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138+ cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner and effect on cell death and proliferation, induction of autophagy, as wells as changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138+ cells (<10%) as assessed by Annexin-V/PI. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, as assessed by acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266 25-70%; MM.1S 8-52%) and CD138+ cells (19-21%). Immunoblot analyses of autophagy LC3b marker and translation initiation proteins (phospho p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138+ myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy. PMID:23988451

Biological effects of the Pim kinase inhibitor, SGI-1776, in multiple myeloma.

PubMed

Cervantes-Gomez, Fabiola; Chen, Lisa S; Orlowski, Robert Z; Gandhi, Varsha

2013-09-01

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138(+) cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner, and effect on cell death and proliferation, induction of autophagy, and changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138(+) cells (< 10%) assessed using Annexin-V/propidium iodide. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, assessed using acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266, 25%-70%; MM.1S, 8%-52%) and CD138(+) cells (19%-21%). Immunoblot analyses of the autophagy LC3b marker and translation initiation proteins (phospho-p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138(+) myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy. Copyright © 2013 Elsevier Inc. All rights reserved.

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

PubMed

Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

2015-06-01

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.

Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms.

PubMed

Yu, Si; Wang, Lijiao; Cao, Zhixing; Gong, Daoyin; Liang, Qianyi; Chen, Hanting; Fu, Huizhu; Wang, Wenwen; Tang, Xue; Xie, Zihao; He, Yang; Peng, Cheng; Li, Yuzhi

2018-06-01

Polyphyllin Ι is a steroidal saponin isolated from the rhizoma of Paris polyphylla. In the present study, we aimed to investigate the anticancer effects of polyphyllin Ι in colorectal cancer and to elucidate the potential underlying molecular mechanisms. Using, CCK8 assay, flow cytometry, laser confocal microscope analysis and western blot, the anticancer effects of the polyphyllin Ι were analysed in colorectal cells. Our results indicate that polyphyllin Ι significantly decreased cell viability of HCT 116 cells and induced autophagy. Furthermore, we found that polyphyllin Ι induced autophagy in an ROS-dependent cell death and not related with PI3 K/AKT/mTOR pathway. We also provide evidence that excessive ROS triggered by polyphyllin Ι could induce G2/M phase arrest via regulating cycle proteins expression of cell cycle regulators, such as p21 and cyclinB1. In conclusion, polyphyllin Ι exhibit anticancer effect through ROS-dependent autophagy and induces G2/M arrest in colorectal cancer.

The BTK Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukaemia Survival, Proliferation and BCR Signalling: A New Therapeutic Approach

PubMed Central

Sivina, Mariela; Kreitman, Robert J.; Arons, Evgeny; Ravandi, Farhad; Burger, Jan A.

2014-01-01

B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins (A, G, and M) and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL. PMID:24697238

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

PubMed

Tsai, Tsung-Chang; Huang, Hui-Pei; Chang, Kai-Ting; Wang, Chau-Jong; Chang, Yun-Ching

2017-04-01

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL -1 ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017. © 2016 Wiley Periodicals, Inc.

Perfluoroalkyl-substituted ethylene carbonates: Novel electrolyte additives for high-voltage lithium-ion batteries

NASA Astrophysics Data System (ADS)

Zhu, Ye; Casselman, Matthew D.; Li, Yan; Wei, Alexander; Abraham, Daniel P.

2014-01-01

A new family of polyfluoroalkyl-substituted ethylene carbonates is synthesized and tested as additives in lithium-ion cells containing EC:EMC + LiPF6-based electrolyte. The influence of these compounds is investigated in Li1.2Ni0.15Mn0.55Co0.1O2//graphite cells via a combination of galvanostatic cycling and electrochemical impedance spectroscopy (EIS) tests. Among the four additives studied in this work (4-(trifluoromethyl)-1,3-dioxolan-2-one (TFM-EC), 4-(perfluorobutyl)-1,3-dioxolan-2-one (PFB-EC), 4-(perfluorohexyl)-1,3-dioxolan-2-one (PFH-EC), and 4-(perfluorooctyl)-1,3-dioxolan-2-one (PFO-EC)), small amounts (0.5 wt%) of PFO-EC is found to be most effective in lessening cell performance degradation during extended cycling. Linear sweep voltammetry (LSV), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy are used to further characterize the effects of PFO-EC on the positive and negative electrodes. LSV data from the electrolyte, and XPS analyses of electrodes harvested after cycling, suggest that PFO-EC is oxidized on the cathode forming surface films that slow electrode/cell impedance rise. Differential capacity (dQ/dV) plots from graphite//Li cells suggest that PFO-EC is involved in solid electrolyte interphase (SEI) formation. Raman data from anodes after cycling suggest that structural disordering of graphite is reduced by the addition of PFO-EC, which may explain the improved cell capacity retention.

Synthesis, characterization, cytotoxicity, cell cycle analysis of 3-(4-methoxyphenyl)-1-(pyridin-2-ylmethyl)thiourea and quantum chemical analyses

NASA Astrophysics Data System (ADS)

Mushtaque, Md.; Avecilla, Fernando; Khan, Md. Shahzad; Hafeez, Zubair Bin; Rezvi, M. Moshahid A.; Srivastava, Anurag

2017-08-01

Thiourea derivative,3-(4-methoxyphenyl)-1-(pyridin-2-ylmethyl)thiourea, was synthesized. The structure of the synthesized compound (3) was elucidated by IR, UV-visible, 1H NMR, mass Spectrometry, and X-ray single crystal structure. The computational quantum chemical studies like, IR, UV, NBO analysis were performed by DFT with Becke-3-Lee-Yang- Parr (B3LYP) exchange-correlation functional in combination with 6-311++G(d,p) basis sets. It was observed experimentally and theoretically that compound (3) exhibited syn-anti-conformation around sulphur atom. The DNA-binding constant Kb was found 3.3 × 106 Lmol-1. The docking energy of compound (3) with 1BNA was found -6.2 kcal/mol. MTT-assay against HepG2 (IC50 = 140.39) and Siha (IC50 = 119.87 μM) cell lines revealed that compound (3) wasnon-toxic up to140.39 μM against HepG2 and 119.87 μM against Siha cells respectively. It was also found that compound (3) is non-toxic against normal human cell line HEK-293(IC50 = 148.67 μM). Cell cycle analyses displayed that treated HepG2 cells at 40 μM and 80 μM showed 65% and 70% arrest in G0/G1with respect to untreated controls (60%) and Siha cells at the same concentration displayed 59% and 65% arrest with respect to G0/G1 as compared to untreated control (45%).

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

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

Chetty, Chandramu; Dontula, Ranadheer; Ganji, Purnachandra Nagaraju

2012-01-13

Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reductionmore » in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.« less

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

PubMed

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

2017-09-01

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

Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells.

PubMed

Derjuga, A; Richard, C; Crosato, M; Wright, P S; Chalifour, L; Valdez, J; Barraso, A; Crissman, H A; Nishioka, W; Bradbury, E M; Th'ng, J P

2001-10-12

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mm butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1.

Photoactive platinum diimine complexes showing induced cancer cell death by apoptosis.

PubMed

Zhang, Zhigang; Dai, Ruihui

2017-02-01

Photoinduced cytotoxicity mediated by a triphenylenamine-modified platinum diimine complex in human breast adenocarcinoma cells has been studied by cell viability assay. The triphenylenamine-modified platinum diimine complex showed more potent cytotoxicity in light than its carboxylate-modified analogue. To gain insights into the mechanism of photodynamic activity of this class of platinum diimine complexes, flow cytometric analyses were performed. The results suggest that upon irradiation the two platinum diimine complexes studied could induce cell cycle arrest in G 2 /M or S phase, and both of them could induce cancer cell death by apoptosis.

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

Transcriptome analyses of the Giardia lamblia life cycle

PubMed Central

Birkeland, Shanda R.; Preheim, Sarah P.; Davids, Barbara J.; Cipriano, Michael J.; Palm, Daniel; Reiner, David S.; Svärd, Staffan G.; Gillin, Frances D.; McArthur, Andrew G.

2010-01-01

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia. PMID:20570699

Molecular Genetic Traits Influencing Maize Endosperm Development and Value: Closeout Report for DOE Grant DE-FG02-96ER20242

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

Brian A. Larkins

2012-09-12

Development of the endosperm in cereal grasses entails different phases characterized by cell division, endoreduplication, accumulation of storage metabolites and cell death, which need to be carried out in an orderly fashion. While correct regulation of the cell cycle plays an essential role in endosperm development, the key regulatory factors and how the cell cycle interfaces with other pathways in this developmental context are largely unknown. We investigated the cyclin-dependent kinase (CDK)-retinoblastoma pathway and how it controls the cell cycle and coordinates it with other processes during maize endosperm development. Retinoblastoma-related (RBR) proteins may be inactivated through CDK-mediated phosphorylation, butmore » the identity of the responsible kinase in maize is unknown. We have previously shown that down-regulation of CDKA;1 severely inhibits the endoreduplication cell cycle and suggested that CDK may be an up-stream regulator of the retinoblastoma pathway. We discovered two types of maize RBR genes, RBR1 and RBR3, which differ in terms of structure, regulation and function. Phylogenetic analyses indicate that these genes may be distinctive features of the Poaceae. We found that RBR3 plays a positive rather than a negative role in DNA replication, cell transformation, and the expression of the minichromosome maintenance (MCM)2-7 family of DNA replication factors. These features are a paradigm shift in RBR gene function and appear to be unique within the RBR gene family. They suggest the existence in maize and related cereal crops of specific RBR/E2F-dependent pathways impinging on the cell cycle and development. RBR1 was down-regulated in transgenic endosperm using RNAi approaches. This resulted in the de-repression of a number of down-stream E2F targets, including RBR3, the MCM2-7 gene family, DNA methyltransferase (MET)1, CDKB;1, and the recently identified RBR4 gene. It also increased endosperm ploidy levels, stimulated the production of a larger number of cells, reduced the average cell size, and promoted programmed cell death. To test whether CDKA;1 inhibits RBR1 (through phosphorylation) in the pathway that leads to DNA synthesis and endoreduplication, the two CDKA;1 and RBR1 down-regulated mutants were crossed and their progeny analyzed. Our results indicate that CDKA;1 controls endoreduplication through an RBR1-dependent pathway. However, the ability of RBR1 to repress gene expression programs is independent from CDKA1, suggesting the presence of two differently regulated RBR1 activities in developing endosperm. One type of RBR1 activity controls E2F-dependent gene expression and is largely independent from CDKA;1, while another suppresses endoreduplication and can be inhibited by CDKA;1. In addition, RBR1 is part of a regulatory feedback loop that impinges on CDK activity. Together, these results indicate that the CDKA;1-RBR1 pathway integrates and controls different processes associated with endosperm development. Genome-wide analyses of the transcriptome, metabolome, and epigenetic mechanisms to understand how the cell cycle is coordinated with other pathways at a systems biology level are currently underway.« less

Calcium, BOBs, QEDs, microdomains and a cellular decision: control of mitotic cell division in sand dollar blastomeres.

PubMed

Silver, R B

1996-08-01

The role of Ca2+ in controlling cell processes (e.g. mitosis) presents an enigma in its ubiquity and selectivity. Intracellular free Ca2+ (Ca2+i) is an essential regulator of specific biochemical and physiological aspects of mitosis (e.g. nuclear envelope breakdown (NEB)). Changes in Ca2+i concentrations during mitosis in second cell-cycle sand dollar (Echinaracnius parma) blastomeres were imaged as Ca(2+)-dependent luminescence of the photoprotein aequorin with multi-spectral analytical video microscopy. Photons of this luminescence were seen as bright observable blobs (BOBs). Spatiotemporal patterns of BOBs were followed through one or more cell cycles to detect directly changes in Ca2+i, and were seen to change in a characteristic fashion prior to NEB, the onset of anaphase chromosome movement, and during cytokinesis. These patterns were observed from one cell cycle to the next in a single cell, from cell to cell, and from egg batch to egg batch. In both mitosis and synaptic transmission increases in Ca2+i concentration occurs in discrete, short-lived, highly localized pulses we name quantum emission domains (QEDs) within regions we named microdomains. Signal and statistical optical analyses of spatiotemporal BOB patterns show that many BOBs are linked by constant displacements in space-time (velocity). Linked BOBs are thus nonrandom and are classified as QEDS. Analyses of QED patterns demonstrated that the calcium signals required for NEB are nonrandom, and are evoked by an agent(s) generated proximal to a Ca2+i-QED; models of waves, diffusible agonists and Ca(2+)-activated Ca2+ release do not fit pre-NEB cell data. Spatial and temporal resolution of this multispectral approach significantly exceeds that reported for other methods, and avoids the perturbations associated with many fluorescent Ca2+ reporters that interfere with cells being studied (Ca(2+)-buffering, UV toxicity, etc.). Spatiotemporal patterns of Ca2+i-QED can control so many different processes, i.e. specific frequencies used to control particular processes. Predictive and structured patterns of calcium signals (e.g. a language expressed in Ca2+) may selectively regulate specific Ca(2+)-dependent cellular processes.

Organic solar cells and physics education

NASA Astrophysics Data System (ADS)

Csernovszky, Zoltán; Horváth, Ákos

2018-07-01

This paper explains the operational principles of a home-made organic solar cell with the representation of an electron-cycle on an energy-level diagram. We present test data for a home-made organic solar cell which operates as a galvanic cell and current source in an electrical circuit. To determine the maximum power of the cell, the optimal current was estimated with a linear approximation. Using different light sources and dyes, the electrical properties of organic solar cells were compared. The solar cells were studied by looking at spectrophotometric data from different sensitizer dyes, generated by a do-it-yourself diffraction grating spectroscope. The sensitizer dyes of solar cells were tested by the diffraction grating spectroscope. The data were analysed on a light-intensity‑wavelength diagram to discover which photons were absorbed and to understand the colours of the fruits containing these dyes. In terms of theoretical applications, the paper underlines the analogous nature of organic solar cells, a conventional single p‑n junction solar cell and the light-dependent reactions of photosynthesis, using energy-level diagrams of electron-cycles. To conclude, a classification of photon‑electron interactions in molecular systems and crystal lattices is offered, to show the importance of organic solar cells.

BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells.

PubMed

Guarini, Anna; Chiaretti, Sabina; Tavolaro, Simona; Maggio, Roberta; Peragine, Nadia; Citarella, Franca; Ricciardi, Maria Rosaria; Santangelo, Simona; Marinelli, Marilisa; De Propris, Maria Stefania; Messina, Monica; Mauro, Francesca Romana; Del Giudice, Ilaria; Foà, Robert

2008-08-01

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

The response of Asterochloris erici (Ahmadjian) Skaloud et Peksa to desiccation: a proteomic approach

USDA-ARS?s Scientific Manuscript database

The study of desiccation tolerance of lichens, and of their photobionts in particular, has frequently focused on the antioxidant system that protects the cell against photo-oxidative stress during dehydration/rehydration cycles. Thus, in this work we carried out proteomic and transcript analyses of ...

The bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) blocks hairy cell leukaemia survival, proliferation and B cell receptor signalling: a new therapeutic approach.

PubMed

Sivina, Mariela; Kreitman, Robert J; Arons, Evgeny; Ravandi, Farhad; Burger, Jan A

2014-07-01

B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, as well as B cell migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited also CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL. © 2014 John Wiley & Sons Ltd.

CUDC-907 Promotes Bone Marrow Adipocytic Differentiation Through Inhibition of Histone Deacetylase and Regulation of Cell Cycle.

PubMed

Ali, Dalia; Alshammari, Hassan; Vishnubalaji, Radhakrishnan; Chalisserry, Elna Paul; Hamam, Rimi; Alfayez, Musaad; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

2017-03-01

The role of bone marrow adipocytes (BMAs) in overall energy metabolism and their effects on bone mass are currently areas of intensive investigation. BMAs differentiate from bone marrow stromal cells (BMSCs); however, the molecular mechanisms regulating BMA differentiation are not fully understood. In this study, we investigated the effect of CUDC-907, identified by screening an epigenetic small-molecule library, on adipocytic differentiation of human BMSCs (hBMSCs) and determined its molecular mechanism of action. Human bone marrow stromal cells exposed to CUDC-907 (500 nM) exhibited enhanced adipocytic differentiation (∼2.9-fold increase, P < 0.005) compared with that of control cells. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis, cell cycle, and DNA replication. Chromatin immune precipitation combined with quantitative polymerase chain reaction showed significant increase in H3K9ac epigenetic marker in the promoter regions of AdipoQ, FABP4, PPARγ, KLF15, and CEBPA in CUDC-907-treated hBMSCs. Follow-up experiments corroborated that the inhibition of histone deacetylase (HDAC) activity enhanced adipocytic differentiation, while the inhibition of PI3K decreased adipocytic differentiation. In addition, CUDC-907 arrested hBMSCs in the G0-G1 phase of the cell cycle and reduced the number of S-phase cells. Our data reveal that HDAC, PI3K, and cell cycle genes are important regulators of BMA formation and demonstrate that adipocyte differentiation of hBMSCs is associated with complex changes in a number of epigenetic and genetic pathways, which can be targeted to regulate BMA formation.

Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections.

PubMed

Craig, Evan; Huyghues-Despointes, Charles-Eugene; Yu, Chun; Handy, Emma L; Sello, Jason K; Kima, Peter E

2017-05-01

In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.

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

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

PubMed

Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

PubMed

Wang, Yiqiang; Hedblom, Andreas; Koerner, Steffi K; Li, Mailin; Jernigan, Finith E; Wegiel, Barbara; Sun, Lijun

2016-12-01

A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC 50 : 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC 50 : 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC 50 : 0.55μM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10μM, indicating specificity towards cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gene Expression in Mammalian Cells After Exposure to 95 MeV Argon Ions

NASA Astrophysics Data System (ADS)

Arenz, A.; Hellweg, C. E.; Baumstark-Khan, C.

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) which present a significant hazard to space flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable radiation exposure. High linear energy transfer (LET) radiation has an increased relative biological effectiveness (RBE) as compared to X-rays for cell death induction, gene mutation, genomic instability, and carcinogenesis. The tumour suppressor gene p53 plays a crucial role in maintaining the integrity of the genome. The p53 protein acts as a transcription factor that mediates cell cycle arrest and apoptosis by binding to DNA and activating transcription of specific genes. It is also though to be involved in damage repair by transcriptional activation of the newly identified p53 dependent ribonuclease subunit R2 (p53R2) that is directly involved in the p53 cell cycle checkpoint for repair of damaged DNA. In that case it is responsible for nucleotide delivery for DNA repair synthesis. DNA damages of cultured human cells (e.g. MCF-7, AGS, A549) exposed to accelerated argon ions at the French heavy ion facility GANIL were analysed for expression levels of certain damage- and apoptosis-relevant genes. RNA was extracted from cells exposed to different particle fluences after various recovery times. A real-time QRT-PCR assay was applied, which employs both relative and absolute quantification of a candidate mRNA biomarker. The expressions of different DNA damage inducible genes (e.g. p53R2, GADD45, p21) were analysed. A reproducible up-regulation representing a twofold to fourfold change in p53R2 gene expression level was confirmed for X-irradiated and Ar-ion exposed cells dependent on dose. Kinetics of p53R2 gene expression modulations shows a response lasting up to 24 hours after irradiation.

Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

NASA Astrophysics Data System (ADS)

Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

1991-03-01

It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

The influence of high glucose on the Cip/Kip family expression profiles in HRECs.

PubMed

Tian, Jingyi; Ma, Hongjie; Luo, Yan; Hu, Andina; Lin, Shaofen; Li, Tao; Guo, Kai; Li, Jing; Cai, Meng; Tang, Shibo

2013-12-01

Neovascularization is the main characteristic of the proliferative stage of diabetic retinopathy. It has been proven that cell cycle regulation is involved in angiogenesis. The cell cycle regulators, Cip/Kip protein family, belong to the cyclin-dependent kinase inhibitors, are versatile proteins, and except for their function in cell cycle regulation, they also participate in transcription, apoptosis and migration. The expression profiles of the Cip/Kip family in human retina microvascular endothelial cells (HRECs) under normal or high glucose conditions has not been described before. This study was undertaken to determine the expression profiles of the Cip/Kip family proteins, e.g., proteins which are influenced by high glucose and in what manner. Western blot and immunofluorescence analyses were used to investigate the protein expression profiles. Only p21(cip1) and p27(kip1) were detected in HRECs, and they were located in the nucleus. P21(cip1) protein abundance was higher than p27(kip1) in HRECs. Incubation of HRECs in medium containing 30 mM D-glucose for 48 h resulted in downregulation of p21(cip1) protein expression, but had no influence on p27(kip1) protein levels or p21(cip1) mRNA abundance. These results were accompanied by cell cycle G1 phase exit and a lower cell survival rate. Our data show for the first time that high glucose changes the Cip/Kip family expression profiles in HRECs, which may be the foundation for the investigation of the role of the Cip/Kip family in the pathogenesis of diabetic retinopathy.

Design solutions for the solar cell interconnect fatigue fracture problem

NASA Technical Reports Server (NTRS)

Mon, G. R.; Ross, R. G., Jr.

1982-01-01

Mechanical fatigue of solar cell interconnects is a major failure mechanism in photovoltaic arrays. A comprehensive approach to the reliability design of interconnects, together with extensive design data for the fatigue properties of copper interconnects, has been published. This paper extends the previous work, developing failure prediction (fatigue) data for additional interconnect material choices, including aluminum and a variety of copper-Invar and copper-steel claddings. An improved global fatigue function is used to model the probability-of-failure statistics of each material as a function of level and number of cycles of applied strain. Life-cycle economic analyses are used to evaluate the relative merits of each material choce. The copper-Invar clad composites demonstrate superior performance over pure copper. Aluminum results are disappointing.

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.

Interference of peritoneal dialysis fluids with cell cycle mechanisms.

PubMed

Büchel, Janine; Bartosova, Maria; Eich, Gwendolyn; Wittenberger, Timo; Klein-Hitpass, Ludger; Steppan, Sonja; Hackert, Thilo; Schaefer, Franz; Passlick-Deetjen, Jutta; Schmitt, Claus P

2015-01-01

Peritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements. PMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays. The number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10(-35)), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%). In conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis. Copyright © 2015 International Society for Peritoneal Dialysis.

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.

Interference of Peritoneal Dialysis Fluids with Cell Cycle Mechanisms

PubMed Central

Büchel, Janine; Bartosova, Maria; Eich, Gwendolyn; Wittenberger, Timo; Klein-Hitpass, Ludger; Steppan, Sonja; Hackert, Thilo; Schaefer, Franz; Passlick-Deetjen, Jutta; Schmitt, Claus P.

2015-01-01

♦ Introduction: Peritoneal dialysis fluids (PDF) differ with respect to osmotic and buffer compound, and pH and glucose degradation products (GDP) content. The impact on peritoneal membrane integrity is still insufficiently described. We assessed global genomic effects of PDF in primary human peritoneal mesothelial cells (PMC) by whole genome analyses, quantitative real-time polymerase chain reaction (RT-PCR) and functional measurements. ♦ Methods: PMC isolated from omentum of non-uremic patients were incubated with conventional single chamber PDF (CPDF), lactate- (LPDF), bicarbonate- (BPDF) and bicarbonate/lactate-buffered double-chamber PDF (BLPDF), icodextrin (IPDF) and amino acid PDF (APDF), diluted 1:1 with medium. Affymetrix GeneChip U133Plus2.0 (Affymetrix, CA, USA) and quantitative RT-PCR were applied; cell viability was assessed by proliferation assays. ♦ Results: The number of differentially expressed genes compared to medium was 464 with APDF, 208 with CPDF, 169 with IPDF, 71 with LPDF, 45 with BPDF and 42 with BLPDF. Out of these genes 74%, 73%, 79%, 72%, 47% and 57% were downregulated. Gene Ontology (GO) term annotations mainly revealed associations with cell cycle (p = 10-35), cell division, mitosis, and DNA replication. One hundred and eighteen out of 249 probe sets detecting genes involved in cell cycle/division were suppressed, with APDF-treated PMC being affected the most regarding absolute number and degree, followed by CPDF and IPDF. Bicarbonate-containing PDF and BLPDF-treated PMC were affected the least. Quantitative RT-PCR measurements confirmed microarray findings for key cell cycle genes (CDK1/CCNB1/CCNE2/AURKA/KIF11/KIF14). Suppression was lowest for BPDF and BLPDF, they upregulated CCNE2 and SMC4. All PDF upregulated 3 out of 4 assessed cell cycle repressors (p53/BAX/p21). Cell viability scores confirmed gene expression results, being 79% of medium for LPDF, 101% for BLPDF, 51% for CPDF and 23% for IPDF. Amino acid-containing PDF (84%) incubated cells were as viable as BPDF (86%). ♦ Conclusion: In conclusion, PD solutions substantially differ with regard to their gene regulating profile and impact on vital functions of PMC, i.e. on cells known to be essential for peritoneal membrane homeostasis. PMID:25082841

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.

Chemical Composition and antiproliferative activity of essential oil from the leaves of a medicinal herb, Levisticum officinale, against UMSCC1 head and neck squamous carcinoma cells.

PubMed

Sertel, Serkan; Eichhorn, Tolga; Plinkert, Peter K; Efferth, Thomas

2011-01-01

Oral squamous cell carcinoma (OSCC) is a challenging disease with a high mortality rate. Natural products represent a valuable source for the development of novel anticancer drugs. We investigated the cytotoxic potential of essential oil from the leaves of a medicinal plant, Levisticum officinale (lovage) on head and neck squamous carcinoma cells (HNSCC). Cytotoxicity of lovage essential oil was investigated on the HNSCC cell line, UMSCC1. Additionally, we performed pharmacogenomics analyses. Lovage essential oil extract had an IC₅₀ value of 292.6 μg/ml. Genes involved in apoptosis, cancer, cellular growth and cell cycle regulation were the most prominently affected in microarray analyses. The three pathways to be most significantly regulated were extracellular signal-regulated kinase 5 (ERK5) signaling, integrin-linked kinase (ILK) signaling, virus entry via endocytic pathways and p53 signaling. Levisticum officinale essential oil inhibits human HNSCC cell growth.

Evaluation of novel trans-sulfonamide platinum complexes against tumor cell lines.

PubMed

Pérez, Carlos; Díaz-García, C Vanesa; Agudo-López, Alba; del Solar, Virginia; Cabrera, Silvia; Agulló-Ortuño, M Teresa; Navarro-Ranninger, Carmen; Alemán, José; López-Martín, José A

2014-04-09

Platinum-based drugs, mainly cisplatin, are employed for the treatment of solid malignancies. However, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. Here, the antitumor activity of different trans-sulfonamide platinum complexes in a panel of human cell lines is presented. The cytotoxicity profiles and cell cycle analyses of these platinum sulfonamide complexes were different from those of cisplatin. These studies showed that complex 2b with cyclohexyldiamine and dansyl moieties had the best antitumoral activities. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

GST-M1 is transcribed moreso than AKR7A2 in AFB₁-exposed human monocytes and lymphocytes.

PubMed

Bahari, Abbas; Mehrzad, Jalil; Mahmoudi, Mahmoud; Bassami, Mohammad Reza; Dehghani, Hesam

2015-01-01

Glutathione-S-transferases (GST) and aldo-keto reductases (AKR) are key aflatoxin (AF)-detoxifying enzymes. In this study, the expression of GST-M1, GST-T1, AKR-7A2, and AKR-7A3 genes in human monocytes and lymphocytes was analyzed after in vitro exposure to 10 or 100 ng AFB1/ml for 2 h. Unlike in pilot studies that showed that all four examined genes were present in HepG2 cells, in lymphocytes and monocytes, only GST-M1 and AKR-7A2 were detected. In fact, the induced expression of both GST-M1 and AKR-7A2 genes in human monocytes was moreso than that seen in AFB1-exposed lymphocytes. In addition, analyses of the effects of the exposures on cell cycle status were performed as, in cells lacking adequate detoxification capacities, it would be expected the cells would arrest at checkpoints in the cell cycle or progress to apoptotic/necrotic states. The results here indicated that only the high dose of AFB1 led to a change in cell cycle profiles and only in the monocytes (i.e. cells in S phase were significantly reduced). In general, the results here strongly suggest that human immune cell lineages appear to be able to increase their expression of AFB1-detoxifying enzymes (albeit to differing degrees) and, as a result, are able to counter potential toxicities from AFB1 and (likely) its metabolites.

Jasmonate Controls Leaf Growth by Repressing Cell Proliferation and the Onset of Endoreduplication while Maintaining a Potential Stand-By Mode1[W][OA

PubMed Central

Noir, Sandra; Bömer, Moritz; Takahashi, Naoki; Ishida, Takashi; Tsui, Tjir-Li; Balbi, Virginia; Shanahan, Hugh; Sugimoto, Keiko; Devoto, Alessandra

2013-01-01

Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coi1-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COI1-dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular “stand-by mode” by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COI1-dependent leaf growth inhibition. PMID:23439917

Investigation of long term storage effects on aerospace nickel-cadmium cell performance

NASA Technical Reports Server (NTRS)

Yi, T. Y.

1986-01-01

A study on evaluation of the long term storage effects on aerospace nickel-cadmium cells currently being performed at NASA/Goddard Space Flight Center (GSFC) is described. A number of cells of 6 Ah and 12 Ah capacities which were stored in shorted condition for 8 to 9 years at the GSFC were selected for this study. These cells will undergo electrical acceptance testing the the GSFC, and life cycling at the NASA Battery Test Facility at the Naval Weapons Facility at the Naval Weapons Support Center (NWSC) in Crane, Indiana; in addition, some cells from the study will undergo destructive analyses.

Investigation of long term storage effects on aerospace nickel-cadmium cell performance

NASA Astrophysics Data System (ADS)

Yi, T. Y.

1986-09-01

A study on evaluation of the long term storage effects on aerospace nickel-cadmium cells currently being performed at NASA/Goddard Space Flight Center (GSFC) is described. A number of cells of 6 Ah and 12 Ah capacities which were stored in shorted condition for 8 to 9 years at the GSFC were selected for this study. These cells will undergo electrical acceptance testing the the GSFC, and life cycling at the NASA Battery Test Facility at the Naval Weapons Facility at the Naval Weapons Support Center (NWSC) in Crane, Indiana; in addition, some cells from the study will undergo destructive analyses.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

PubMed

Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4 + T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4 + T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1 + CD4 + T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4 + T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation

PubMed Central

Celada, Lindsay J.; Rotsinger, Joseph E.; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = −0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression. PMID:27564547

Synthesis, characterization, DNA-binding study and anticancer properties of ternary metal(II) complexes of edda and an intercalating ligand.

PubMed

Ng, Chew Hee; Kong, King Chow; Von, Sze Tin; Balraj, Pauline; Jensen, Paul; Thirthagiri, Eswary; Hamada, Hirokazu; Chikira, Makoto

2008-01-28

A series of ternary metal(ii) complexes {M(phen)(edda); 1a (Cu), 1b (Co), 1c (Zn), 1d (Ni); H(2)edda = N,N(')-ethylenediaminediacetic acid} of N,N'-ethylene-bridged diglycine and 1,10-phenanthroline were synthesized and characterized by elemental analysis, FTIR, UV-visible spectroscopy and magnetic susceptibility measurement. The interaction of these complexes with DNA was investigated using CD and EPR spectroscopy. MTT assay results of 1a-1c , screened on MCF-7 cancer cell lines, show that synergy between the metal and ligands results in significant enhancement of their antiproliferative properties. Preliminary results from apoptosis and cell cycle analyses with flow cytometry are reported. seems to be able to induce cell cycle arrest at G(0)/G(1). The crystal structure of 1a is also included.

Molecular identification and transmission studies of X-cell parasites from Atlantic cod Gadus morhua (Gadiformes: Gadidae) and the northern black flounder Pseudopleuronectes obscurus (Pleuronectiformes: Pleuronectidae)

PubMed Central

2011-01-01

Background Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata. Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan. Results Phylogenetic analyses of small subunit ribosomal DNA (SSU rDNA) sequence data from Atlantic cod X-cells show that they are a related parasite that occupies a basal position to the clade containing other X-cell parasites. The X-cell parasite causing epidermal pseudotumours in P. obscurus is the same parasite that causes pseudotumours in H. dubius. Direct, fish to fish, transmission of the X-cell parasites used in this study, via oral feeding or injection, was not achieved. Non-amoeboid X-cells are contained within discrete sac-like structures that are loosely attached to epidermal pseudotumours in flatfish; these X-cells are able to tolerate exposure to seawater. A sensitive nested PCR assay was developed for the sub clinical detection of both parasites and to assist in future life cycle studies. PCR revealed that the parasite in P. obscurus was detectable in non-pseudotumourous areas of fish that had pseudotumours present in other areas of the body. Conclusions The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites. Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum. Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish. PMID:21299903

[Pancreatic acinar neoplasms : Comparative molecular characterization].

PubMed

Bergmann, F

2016-11-01

Pancreatic acinar cell carcinomas are biologically aggressive neoplasms for which treatment options are very limited. The molecular mechanisms of tumor initiation and progression are largely not understood and precursor lesions have not yet been identified. In this study, pancreatic acinar cell carcinomas were cytogenetically characterized as well as by molecular and immunohistochemical analyses. Corresponding investigations were carried out on pancreatic ductal adenocarcinomas and pancreatic neuroendocrine neoplasms augmented by functional analyses. We show that pancreatic acinar cell carcinomas display a microsatellite stable, chromosomal unstable genotype, characterized by recurrent chromosomal imbalances that clearly discriminate them from pancreatic ductal adenocarcinomas and neuroendocrine neoplasms. Based on findings obtained from comparative genomic hybridization, candidate genes could be identified, such as deleted in colorectal cancer (DCC) and c-MYC. Furthermore, several therapeutic targets were identified in acinar cell carcinomas and other pancreatic neoplasms, including epidermal growth factor receptor (EGFR), L1 cell adhesion molecule (L1CAM) and heat shock protein 90 (HSP90). Moreover, L1CAM was shown to play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma. Functional analyses in cell lines derived from pancreatic neuroendocrine neoplasms revealed promising anti-tumorigenic effects using EGFR and HSP90 inhibitors affecting the cell cycle and in the case of HSP90, regulating several other oncogenes. Finally, based on mutational analyses of mitochondrial DNA, molecular evidence is provided that acinar cell cystadenomas (or better cystic acinar transformation) represent non-clonal lesions, suggesting an inflammatory reactive non-neoplastic nature.

Alteronol induces cell cycle arrest and apoptosis via increased reactive oxygen species production in human breast cancer T47D cells.

PubMed

Ren, Boxue; Li, Defang; Si, Lingling; Ding, Yangfang; Han, Jichun; Chen, Xiaoyu; Zheng, Qiusheng

2018-04-01

Emerging evidence showed that alteronol has a potential antitumour effect in several tumour cells. However, the antitumour effect of alteronol on breast cancer has not been reported. This study investigated the mechanisms of alteronol-induced cell proliferation inhibition in human breast cancer T47D cells. After treatment with alteronol, T47D cell proliferation was examined by MTT assay. The cell cycle distribution, cell apoptosis, reactive oxygen species level and mitochondrial membrane potential were evaluated via flow cytometry. Next, the protein levels of cyclin B1, cdc2, p21, p-cyclin B1, p-cdc2, p53, Bax, Bcl-2 and cytochrome c were analysed using Western blot analysis. Meanwhile, the mRNA levels of cyclin B1, cdc2, p21 and p53 were examined by qRT-PCR. Our data showed that alteronol inhibited the proliferation of T47D cells via inducing G2-phase arrest and cell apoptosis. Compared with control group, alteronol significantly increased ROS level and triggered mitochondrial dysfunction in alteronol-treated T47D cells. Further studies showed that the mRNA and protein levels of cdc2 and cyclin B1 were downregulated, while the mRNA and protein levels of p21, p53, p-cyclin B1, p-cdc2 and cytochrome c were upregulated. In addition, the expression level of Bax was increased, and the expression level of Bcl-2 was decreased. Alteronol induced T47D cell cycle arrest and cell apoptosis through increasing ROS production and triggering mitochondrial dysfunction, and subsequently inhibiting T47D cell proliferation. © 2018 Royal Pharmaceutical Society.

Analysis of 12 AH aerospace nickel-cadmium cells from the design variable program

NASA Technical Reports Server (NTRS)

Vasanth, Kunigahalli L.; Morrow, George

1987-01-01

The Design Variable Program of NASA/GSFC provided a systematic approach to evaluate the performance of 12 Ampere-Hour Nickel-Cadmium cells of different designs. Design Variables tested in this program included teflonated negative plates, silver treated negative plates, lightly loaded negative plates, positive plates with no cadmium treatment, plate design of 1968 utilizing old and new processing techniques and electrochemically impregnated positive plates. These cells were life cycled in a Low-Earth Orbit (LEO) regime for 3 to 4 years. Representative cells taken from the Design Variable Program were examined via chemical, electrochemical and surface analyses. The results indicate the following: (1) positive swelling and carbonate content in the electrolyte increase as a function of number of cycles; (2) electrolyte distribution follows a general order NEG greater than POS greater than SEP; (3) control and No PQ groups outperformed the rest of the groups; and (4) the polyproylene group exhibited heavy cadmium migration and poor performance.

BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma.

PubMed

Sime, Wondossen; Niu, Qiankun; Abassi, Yasmin; Masoumi, Katarzyna Chmielarska; Zarrizi, Reihaneh; Køhler, Julie Bonne; Kjellström, Sven; Lasorsa, Vito Alessandro; Capasso, Mario; Fu, Haian; Massoumi, Ramin

2018-04-24

BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.

[Study on garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M].

PubMed

Wu, Fayin; Zhou, Hefeng; Fan, Zhiying; Zhu, Yawen; Li, Yongye; Yao, Yukun; Ran, Dan

2014-02-01

To observe the effect of garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M. Human salivary in adenoid cystic carcinoma cell line AC-M was cultured, divided into the experimental group (5-FU group, garlic oil group, garlic oil + 5-FU group) and the control group, to observe the growth activity of tumor cells by MTT methods; to analyse the changes of cell cycle and apoptosis rate by flow cytometry. MTT experiments showed that 5-FU, garlic oil, garlic oil and 5-FU on ACC-M cells have inhibition in different concentration, with the increase of concentration and action time of the rise; Cell cycle analysis showed significant changes in flow cytometry. With the increase of concentration and the acting time, the G0/G1, phase of the cell ratio increased, S had no significant change, but G2/M phase cells decreased. Apoptosis rate display showed garlic oil combined with 5-FU induced apoptosis of ACC-M cells was significantly stronger than single group. Garlic oil can effectively induce the apoptosis of adenoid cystic carcinoma cell line ACC-M. The effect of garlic oil combined with 5-FU on ACC-M cells was stronger than the garlic oil, 5-FU used alone.

Preclinical Evaluation of Vemurafenib as Therapy for BRAFV600E Mutated Sarcomas.

PubMed

Gouravan, Sarina; Meza-Zepeda, Leonardo A; Myklebost, Ola; Stratford, Eva W; Munthe, Else

2018-03-23

The BRAF V600E mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAF V600E mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarcoma cell lines confirming that the MAPK pathway is active in these cell lines, and that the pathway can be inhibited by vemurafenib, but also that these cells can proliferate despite this. These findings indicate that vemurafenib alone would not be an efficient therapy against BRAF V600E mutated sarcomas. However, further investigations of combination with other drugs are warranted.

Alcohol dehydrogenase and hydrogenase transcript fluctuations during a day-night cycle in Chlamydomonas reinhardtii: the role of anoxia.

PubMed

Whitney, Larisa Angela Swirsky; Loreti, Elena; Alpi, Amedeo; Perata, Pierdomenico

2011-04-01

• The unicellular green alga Chlamydomonas reinhardtii contains two iron (Fe)-hydrogenases which are responsible for hydrogen production under anoxia. In the present work the patterns of expression of alcohol dehydrogenase, a typical anaerobic gene in plants, of the hydrogenases genes (HYD1, HYD2) and of the genes responsible for their maturation (HYDEF, HYDG), were analysed. • The expression patterns were analysed by real-time reverse-transcription polymerase chain reaction in Chlamydomonas cultures during the day-night cycle, as well as in response to oxygen availability. • The results indicated that ADH1, HYD1, HYD2, HYDEF and HYDG were expressed following precise day-night fluctuations. ADH1 and HYD2 were modulated by the day-night cycle. Low oxygen plays an important role for the induction of HYD1, HYDEF and HYDG, while ADH1 and HYD2 expression was relatively insensitive to oxygen availability. • The regulation of the anaerobic gene expression in Chlamydomonas is only partly explained by responses to anoxia. The cell cycle and light-dark cycles are equally important elements in the regulatory network modulating the anaerobic response in Chlamydomonas. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

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.

Progesterone-induced miR-133a inhibits the proliferation of endometrial epithelial cells.

PubMed

Pan, J-L; Yuan, D-Z; Zhao, Y-B; Nie, L; Lei, Y; Liu, M; Long, Y; Zhang, J-H; Blok, L J; Burger, C W; Yue, L-M

2017-03-01

This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G 1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Sites and Regulation of Polyamine Catabolism in the Tobacco Plant. Correlations with Cell Division/Expansion, Cell Cycle Progression, and Vascular Development1

PubMed Central

Paschalidis, Konstantinos A.; Roubelakis-Angelakis, Kalliopi A.

2005-01-01

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development. PMID:16040649

Effects of maple (Acer) plant part extracts on proliferation, apoptosis and cell cycle arrest of human tumorigenic and non-tumorigenic colon cells.

PubMed

González-Sarrías, Antonio; Li, Liya; Seeram, Navindra P

2012-07-01

Phenolic-enriched extracts of maple sap and syrup, obtained from the sugar and red maple species (Acer saccharum Marsh, A. rubrum L., respectively), are reported to show anticancer effects. Despite traditional medicinal uses of various other parts of these plants by Native Americans, they have not been investigated for anticancer activity. Here leaves, stems/twigs, barks and sapwoods of both maple species were evaluated for antiproliferative effects against human colon tumorigenic (HCT-116, HT-29, Caco-2) and non-tumorigenic (CCD-18Co) cells. Extracts were standardized to total phenolic and ginnalin-A (isolated in our laboratory) levels. Overall, the extracts inhibited the growth of the colon cancer more than normal cells (over two-fold), their activities increased with their ginnalin-A levels, with red > sugar maple extracts. The red maple leaf extract, which contained the highest ginnalin-A content, was the most active extract (IC₅₀  = 35 and 16 µg/mL for extract and ginnalin-A, respectively). The extracts were not cytotoxic nor did they induce apoptosis of the colon cancer cells. However, cell cycle analyses revealed that the antiproliferative effects of the extracts were mediated through cell cycle arrest in the S-phase. The results from the current study suggest that these maple plant part extracts may have potential anticolon cancer effects. Copyright © 2011 John Wiley & Sons, Ltd.

Basic statistical analyses of candidate nickel-hydrogen cells for the Space Station Freedom

NASA Technical Reports Server (NTRS)

Maloney, Thomas M.; Frate, David T.

1993-01-01

Nickel-Hydrogen (Ni/H2) secondary batteries will be implemented as a power source for the Space Station Freedom as well as for other NASA missions. Consequently, characterization tests of Ni/H2 cells from Eagle-Picher, Whittaker-Yardney, and Hughes were completed at the NASA Lewis Research Center. Watt-hour efficiencies of each Ni/H2 cell were measured for regulated charge and discharge cycles as a function of temperature, charge rate, discharge rate, and state of charge. Temperatures ranged from -5 C to 30 C, charge rates ranged from C/10 to 1C, discharge rates ranged from C/10 to 2C, and states of charge ranged from 20 percent to 100 percent. Results from regression analyses and analyses of mean watt-hour efficiencies demonstrated that overall performance was best at temperatures between 10 C and 20 C while the discharge rate correlated most strongly with watt-hour efficiency. In general, the cell with back-to-back electrode arrangement, single stack, 26 percent KOH, and serrated zircar separator and the cell with a recirculating electrode arrangement, unit stack, 31 percent KOH, zircar separators performed best.

Biological effects of low-dose-rate irradiation of pancreatic carcinoma cells in vitro using 125I seeds

PubMed Central

Wang, Zhong-Min; Lu, Jian; Zhang, Li-Yun; Lin, Xiao-Zhu; Chen, Ke-Min; Chen, Zhi-Jin; Liu, Fen-Ju; Yan, Fu-Hua; Teng, Gao-Jun; Mao, Ai-Wu

2015-01-01

AIM: To determine the mechanism of the radiation-induced biological effects of 125I seeds on pancreatic carcinoma cells in vitro. METHODS: SW1990 and PANC-1 pancreatic cancer cell lines were cultured in DMEM in a suitable environment. Gray’s model of iodine-125 (125I) seed irradiation was used. In vitro, exponential phase SW1990, and PANC-1 cells were exposed to 0, 2, 4, 6, and 8 Gy using 125I radioactive seeds, with an initial dose rate of 12.13 cGy/h. A clonogenic survival experiment was performed to observe the ability of the cells to maintain their clonogenic capacity and to form colonies. Cell-cycle and apoptosis analyses were conducted to detect the apoptosis percentage in the SW1990 and PANC-1 cells. DNA synthesis was measured via a tritiated thymidine (3H-TdR) incorporation experiment. After continuous low-dose-rate irradiation with 125I radioactive seeds, the survival fractions at 2 Gy (SF2), percentage apoptosis, and cell cycle phases of the SW1990 and PANC-1 pancreatic cancer cell lines were calculated and compared. RESULTS: The survival fractions of the PANC-1 and SW1990 cells irradiated with 125I seeds decreased exponentially as the dose increased. No significant difference in SF2 was observed between SW1990 and PANC-1 cells (0.766 ± 0.063 vs 0.729 ± 0.045, P < 0.05). The 125I seeds induced a higher percentage of apoptosis than that observed in the control in both the SW1990 and PANC-1 cells. The rate of apoptosis increased with increasing radiation dosage. The percentage of apoptosis was slightly higher in the SW1990 cells than in the PANC-1 cells. Dose-dependent G2/M cell-cycle arrest was observed after 125I seed irradiation, with a peak value at 6 Gy. As the dose increased, the percentage of G2/M cell cycle arrest increased in both cell lines, whereas the rate of DNA incorporation decreased. In the 3H-TdR incorporation experiment, the dosimetry results of both the SW1990 and PANC-1 cells decreased as the radiation dose increased, with a minimum at 6 Gy. There were no significant differences in the dosimetry results of the two cell lines when they were exposed to the same dose of radiation. CONCLUSION: The pancreatic cancer cell-killing effects induced by 125I radioactive seeds mainly occurred via apoptosis and G2/M cell cycle arrest. PMID:25741139

Reduced Mitogenicity of Sera Following Weight Loss in Premenopausal Women

PubMed Central

Azrad, Maria; Chang, Pi-Ling; Gower, Barbara A.; Hunter, Gary R.; Nagy, Tim R.

2011-01-01

We investigated whether serum from normal weight women is less mitogenic and more apoptotic than sera from the same women in the overweight state. Sera from premenopausal women, age (mean ±SEE) 34.6±0.53 years, who were randomized to caloric restriction (CR) (n=13), CR + aerobic exercise (AE) (n=14) or CR + resistance training (RT) (n=20) were used to culture endometrial cancer cells. Phases of the cell cycle were determined, proliferating cell nuclear antigen (PCNA) positivity was used to assess proliferation and apoptosis was assessed by determining cleaved caspase-3 and poly-ADP-ribose polymerase (PARP). Analyses showed that overall, cells grown in sera from the weight-reduced state had significantly more cells in G0/G1 and significantly fewer cells in the S and G2/M phases of the cell cycle than cells grown in sera from the overweight state. PCNA staining confirmed that cells grown in sera from the weight-reduced state had fewer proliferating cells. Cleaved caspase-3 and PARP were not different in cells grown in sera from the weight-reduced state compared to the overweight state. We conclude that weight loss with or without exercise could lower the risk for cancer through changes in serum that result in reduced cellular mitogenicity. PMID:21774593

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

Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142

NASA Technical Reports Server (NTRS)

Schneegurt, M. A.; Sherman, D. M.; Nayar, S.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

1994-01-01

It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.

Ulk4 Regulates Neural Stem Cell Pool.

PubMed

Liu, Min; Guan, Zhenlong; Shen, Qin; Flinter, Frances; Domínguez, Laura; Ahn, Joo Wook; Collier, David A; O'Brien, Timothy; Shen, Sanbing

2016-09-01

The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4(tm1a/tm1a) mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4(tm1a/tm1a) mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016;34:2318-2331. © 2016 AlphaMed Press.

Impact of cycling at low temperatures on the safety behavior of 18650-type lithium ion cells: Combined study of mechanical and thermal abuse testing accompanied by post-mortem analysis

NASA Astrophysics Data System (ADS)

Friesen, Alex; Horsthemke, Fabian; Mönnighoff, Xaver; Brunklaus, Gunther; Krafft, Roman; Börner, Markus; Risthaus, Tim; Winter, Martin; Schappacher, Falko M.

2016-12-01

The impact of cycling at low temperatures on the thermal and mechanical abuse behavior of commercial 18650-type lithium ion cells was compared to fresh cells. Post-mortem analyses revealed a deposition of high surface area lithium (HSAL) metal on the graphite surface accompanied by severe electrolyte decomposition. Heat wait search (HWS) tests in an accelerating rate calorimeter (ARC) were performed to investigate the thermal abuse behavior of aged and fresh cells under quasi-adiabatic conditions, showing a strong shift of the onset temperature for exothermic reactions. HSAL deposition promotes the reduction of the carbonate based electrolyte due to the high reactivity of lithium metal with high surface area, leading to a thermally induced decomposition of the electrolyte to produce volatile gaseous products. Nail penetration tests showed a change in the thermal runaway (TR) behavior affected by the decomposition reaction. This study indicates a greater thermal hazard for LIB cells at higher SOC and experiencing aging at low temperature.

Discovery of Potent Antiproliferative Agents Targeting EGFR Tyrosine Kinase Based on the Pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine Scaffold.

PubMed

Aziz, Yasmine Mohamed Abdel; Said, Mohamed Mokhtar; El Shihawy, Hosam Ahmed; Tolba, Mai Fathy; Abouzid, Khaled Abouzid Mohamed

2015-01-01

A series of pyridothieno[3,2-d]pyrimidin-4-amines was designed and synthesized as congeners to the classical 4-anilinoquinazolines as ATP-competitive epidermal growth factor receptor (EGFR) inhibitors. Compound 5a exhibited the most potent and selective inhibitory activity against EGFR with an IC50 value of 36.7 nM. Moreover, compounds 4b and 5a showed remarkable cell growth inhibition against leukemia, central nervous system cancer, and non-small cell lung cancer cell lines that overexpress EGFR, with growth inhibition of 50% (GI50) values of around 10 nM in the full U.S. National Cancer Institute 60 cell panel assay. Cell cycle studies indicated that compounds 4b and 5a induced significant cell cycle arrest in the S-phase and G0/G1, respectively, in addition to boosting P27(kip) expression. Compound 5a did not alter the viability of placental trophoblasts, which reflects its safety for normal cells. The standard COMPARE analyses demonstrated considerable correlation levels between compounds 4b and 5a and erlotinib, with pyridinium chlorochromate (PCC) values of 0.707 and 0.727, respectively.

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

PubMed

Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

2015-12-15

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Prognostic value of cell cycle regulatory proteins in muscle-infiltrating bladder cancer.

PubMed

Galmozzi, Fabia; Rubagotti, Alessandra; Romagnoli, Andrea; Carmignani, Giorgio; Perdelli, Luisa; Gatteschi, Beatrice; Boccardo, Francesco

2006-12-01

The aims of this study were to investigate the expression levels of proteins involved in cell cycle regulation in specimens of bladder cancer and to correlate them with the clinicopathological characteristics, proliferative activity and survival. Eighty-two specimens obtained from patients affected by muscle-invasive bladder cancer were evaluated immunohistochemically for p53, p21 and cyclin D1 expression, as well as for the tumour proliferation index, Ki-67. The statistical analysis included Kaplan-Meier curves with log-rank test and Cox proportional hazards models. In univariate analyses, low Ki-67 proliferation index (P = 0.045) and negative p21 immunoreactivity (P = 0.04) were associated to patient's overall survival (OS), but in multivariate models p21 did not reach statistical significance. When the combinations of the variables were assessed in two separate multivariate models that included tumour stage, grading, lymph node status, vascular invasion and perineural invasion, the combined variables p21/Ki-67 or p21/cyclin D1 expression were independent predictors for OS; in particular, patients with positive p21/high Ki-67 (P = 0.015) or positive p21/negative cyclin D1 (P = 0.04) showed the worst survival outcome. Important alterations in the cell cycle regulatory pathways occur in muscle-invasive bladder cancer and the combined use of cell cycle regulators appears to provide significant prognostic information that could be used to select the patients most suitable for multimodal therapeutic approaches.

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells

PubMed Central

Janzer, Andreas; German, Natalie J.; Gonzalez-Herrera, Karina N.; Asara, John M.; Haigis, Marcia C.; Struhl, Kevin

2014-01-01

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation. PMID:25002509

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells.

PubMed

Janzer, Andreas; German, Natalie J; Gonzalez-Herrera, Karina N; Asara, John M; Haigis, Marcia C; Struhl, Kevin

2014-07-22

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.

Elucidating the functional role of endoreduplication in tomato fruit development

PubMed Central

Chevalier, Christian; Nafati, Mehdi; Mathieu-Rivet, Elodie; Bourdon, Matthieu; Frangne, Nathalie; Cheniclet, Catherine; Renaudin, Jean-Pierre; Gévaudant, Frédéric; Hernould, Michel

2011-01-01

Background Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination. Scope Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described. Conclusions The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits. PMID:21199834

Zn/gelled 6 M KOH/O 2 zinc-air battery

NASA Astrophysics Data System (ADS)

Mohamad, A. A.

The gel electrolyte for the zinc-air cell was prepared by mixing hydroponics gel with a 6 M potassium hydroxide aqueous solution. The self-discharge of cells was characterized by measuring the open-circuit voltage. The effect of a discharge rate of 50 mA constant current on cell voltage and plateau hour, as well as the voltage-current and current density-power density were measured and analysed. The electrode degradation after discharge cycling was characterized by structural and surface methods. The oxidation of the electrode surface further blocked the utilization of the Zn anode and was identified as a cause for the failure of the cell.

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

PubMed

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-10-03

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo . Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo . Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro . Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy

PubMed Central

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-01-01

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence. PMID:29108242

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

In vitro and in vivo anticancer effects of Lithospermum erythrorhizon extract on B16F10 murine melanoma.

PubMed

Rajasekar, Seetharaman; Park, Da Jung; Park, Cheol; Park, Sejin; Park, Young Hoon; Kim, Sun Tae; Choi, Yung Hyun; Choi, Young Whan

2012-11-21

Lithospermum erythrorhizon has long been used in traditional Asian medicine for the treatment of diseases including skin cancer. In this study, hexane extract from the roots of Lithospermum erythrorhizon (LEH) was chemically characterized and its anticancer activity was tested against the most aggressive form of skin cancer. The in vitro anticancer studies viz. cell growth, cell cycle and apoptosis, and the expression of tumor regulating proteins were analyzed against B16F10 melanoma cells. In addition, C57BL/6 mice models were used to evaluate the in vivo anticancer potential of LEH. Mice were intraperitoneally injected with LEH at doses of 0.1 and 10mg/kg every 3 days. The tumor inhibition ratio was determined after 21 days of treatment and the histopathological analyses of the tumor tissues were compared. Further, LEH was purified and its active compounds were structurally elucidated and identified by NMR spectra and quantified by HPLC analyses. LEH effectively inhibits the growth of melanoma cells with an IC(50) of 2.73μg/ml. Cell cycle analysis revealed that LEH increased the percentage of cells in sub-G1 phase by dose dependent manner. LEH exhibited down regulation of anti-apoptotic Bcl-2 family proteins and up regulation of apoptotic Bax protein expression. Importantly, LEH induced cleavage of poly (ADP-ribose) polymerase (PARP) and activated the caspase cascade (caspase 3) with this cleavage mediating the apoptosis of B16F10 cells. LEH treatment at a dose of 10mg/kg for 21 days in experimental mice implanted with tumors resulted in significant reduction of the tumor growth (43%) and weight (36%). Histopathology analysis of LEH treated tumor tissues showed evidence of increased necrotic cells in a concentration dependent manner. Meanwhile, five naphthoquinone compounds [Shikonin (1); Deoxyshikonin (2); β-Hydroxyisovalerylshikonin (3); Acetylshikonin (4) and Isobutyrylshikonin (5)] were purified from LEH and responsible for its anticancer activity. LEH induced apoptosis in B16F10 cells by activation of caspase 3 and inducing sub-G1 cell cycle arrest. LEH exhibited both in vitro and in vivo anticancer activity. Shikonin derivatives in the LEH are responsible for the anticancer activity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The cytotoxic activity of Ziziphus Jujube on cervical cancer cells: In Vitro study.

PubMed

Hoshyar, R; Jamali, S; Fereidouni, M; Abedini, M R

2015-12-30

Recently, there are tendency to use natural products such as Ziziphus Jujube (Jujube) as therapeutic agents for cancer. Understanding the molecular mechanisms of anti-cancer effects of Jujube may improve the current therapeutic strategies against cervical cancer. Our MTT data showed a significant dose- and time-dependent inhibition of OV-2008 cell proliferation following Jujube administration. Moreover, qRT-PCR analyses significantly revealed the suppression of cyclin D1 and the enhancement of P53, P21 and P27 expression in treated cells. These results suggest that the herb exerts a cytotoxic effect on cervical cancer cells through alternation of the expression of the genes that are involved in regulation of cell cycle.

Cadmium inhibits the protein degradation of Sml1 by inhibiting the phosphorylation of Sml1 in Saccharomyces cerevisiae

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

Baek, In-Joon; Kang, Hyun-Jun; Chang, Miwha

Highlights: Black-Right-Pointing-Pointer Cd inhibits Sml1-p formation. Black-Right-Pointing-Pointer Cd affects cell cycle. Black-Right-Pointing-Pointer Cd inhibits Sml1 ubiquitination. -- Abstract: Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels ofmore » SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.« less

Inhibition of ovarian cancer cell proliferation by Pien Tze Huang via the AKT-mTOR pathway

PubMed Central

HE, FAN; WU, HUI-NI; CAI, MU-YAN; LI, CHANG-PENG; ZHANG, XIN; WAN, QUAN; TANG, SHUANG-BO; CHENG, JIAN-DING

2014-01-01

Pien Tze Huang (PZH) is a well-known Chinese medicine that has been used as a therapeutic drug in the treatment of a number of diseases, such as hepatocellular carcinoma and colon cancer. However, few studies have analyzed the effects of PZH on ovarian cancer cell proliferation. In the present study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays, cell cycle and apoptosis rate analyses and western blotting were conducted to investigate the effects of PZH on the proliferation rate of ovarian cancer cells and its potential molecular pathway. The results showed that PZH inhibits the proliferation of the human ovarian cancer OVCAR-3 cell line by blocking the progression of the cell cycle from the G1 to S phase, however, PZH did not induce OVCAR-3 cell apoptosis. Increased PZH concentration may downregulate the expression of AKT, phosphorylated (p)-AKT, mammalian target of rapamycin (mTOR) and p-mTOR proteins in the OVCAR-3 cell line. In addition, it was observed that PZH may suppress the protein expression of cyclin-dependent kinase (CDK)4 and CDK6. Overall, the results of the present study indicated that PZH may inhibit ovarian cancer cell proliferation by modulating the activity of the AKT-mTOR pathway. PMID:24932287

Megakaryocyte polyploidization is associated with decreased expression of polo-like kinase (PLK).

PubMed

Yagi, M; Roth, G J

2006-09-01

During differentiation, megakaryocytes (MK), the bone marrow precursors of circulating blood platelets, undergo polyploidization, repeated rounds of DNA replication without cell division. Mature normal MK may contain a DNA content of up to 128N, in contrast to normal diploid (2N) cells. The extent of polyploidy may influence the number of platelets produced by the MK. Therefore, understanding the molecular mechanisms regulating polyploidization could identify events involved in controlling both cell division and thrombopoiesis. We investigated the expression of several proteins involved in mitosis in cultured mouse MK, and tested the effect of expression on polyploidization. Western blot and immunofluorescent analyses were used to assess expression of cell cycle proteins in cultured MK. Populations of polyploidizing MK were separated on the basis of DNA content by flow cytometry. The gene encoding mouse polo-like kinase 1 (PLK-1) was introduced into MK by retroviral transduction, and its effects measured by flow cytometry. Polyploid mouse MK expressed lower levels of two proteins, p55CDC and PLK-1, whose activity is necessary for cell cycle progression and completion of mitosis. Comparison of sorted 2N/4N and polyploid MK indicated that PLK-1 expression was absent in polyploid MK, while expression of other cell cycle proteins was similar in both populations. Forced expression of PLK-1 during MK differentiation was associated with decreased polyploidization. These experiments suggest that PLK-1 is an important regulator of polyploidization in differentiating MK.

Clock genes and their genomic distributions in three species of salmonid fishes: Associations with genes regulating sexual maturation and cell cycling

PubMed Central

2010-01-01

Background Clock family genes encode transcription factors that regulate clock-controlled genes and thus regulate many physiological mechanisms/processes in a circadian fashion. Clock1 duplicates and copies of Clock3 and NPAS2-like genes were partially characterized (genomic sequencing) and mapped using family-based indels/SNPs in rainbow trout (RT)(Oncorhynchus mykiss), Arctic charr (AC)(Salvelinus alpinus), and Atlantic salmon (AS)(Salmo salar) mapping panels. Results Clock1 duplicates mapped to linkage groups RT-8/-24, AC-16/-13 and AS-2/-18. Clock3/NPAS2-like genes mapped to RT-9/-20, AC-20/-43, and AS-5. Most of these linkage group regions containing the Clock gene duplicates were derived from the most recent 4R whole genome duplication event specific to the salmonids. These linkage groups contain quantitative trait loci (QTL) for life history and growth traits (i.e., reproduction and cell cycling). Comparative synteny analyses with other model teleost species reveal a high degree of conservation for genes in these chromosomal regions suggesting that functionally related or co-regulated genes are clustered in syntenic blocks. For example, anti-müllerian hormone (amh), regulating sexual maturation, and ornithine decarboxylase antizymes (oaz1 and oaz2), regulating cell cycling, are contained within these syntenic blocks. Conclusions Synteny analyses indicate that regions homologous to major life-history QTL regions in salmonids contain many candidate genes that are likely to influence reproduction and cell cycling. The order of these genes is highly conserved across the vertebrate species examined, and as such, these genes may make up a functional cluster of genes that are likely co-regulated. CLOCK, as a transcription factor, is found within this block and therefore has the potential to cis-regulate the processes influenced by these genes. Additionally, clock-controlled genes (CCGs) are located in other life-history QTL regions within salmonids suggesting that at least in part, trans-regulation of these QTL regions may also occur via Clock expression. PMID:20670436

Long non‑coding RNA AK001796 contributes to cisplatin resistance of non‑small cell lung cancer.

PubMed

Liu, Bin; Pan, Chun-Feng; Ma, Teng; Wang, Jun; Yao, Guo-Liang; Wei, Ke; Chen, Yi-Jiang

2017-10-01

Cisplatin (DDP)‑based chemotherapy is the most widely used therapy for non‑small cell lung cancer (NSCLC). However, the existence of chemoresistance has become a major limitation in its efficacy. Long non‑coding RNAs (lncRNAs) have been shown to be involved in chemotherapy drug resistance. The aim of the present study was to investigate the biological role of lncRNA AK001796 in cisplatin‑resistant NSCLC A549/DDP cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to monitor the differences in the expression of AK001796 in cisplatin-resistant (A549/DDP) cells and parental A549 cells. Cellular sensitivity to cisplatin and cell viability were examined using an MTT assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. The expression levels of cell cycle proteins cyclin C (CCNC), baculoviral IAP repeat containing 5 (BIRC5), cyclin‑dependent kinase 1 (CDK1) and G2 and S phase‑expressed 1 (GTSE1) were assessed using RT‑qPCR and western blot analyses. It was found that the expression of AK001796 was increased in A549/DDP cells, compared with that in A549 cells. The knockdown of AK001796 by small interfering RNA reduced cellular cisplatin resistance and cell viability, and resulted in cell‑cycle arrest, with a marked increase in the proportion of A549/DDP cells in the G0/G1 phase. By contrast, the knockdown of AK001796 increased the number of apoptotic cancer cells during cisplatin treatment. It was also shown that the knockdown of AK001796 positively induced the expression of cell apoptosis‑associated factors, CCNC and BIRC5, and suppressed the expression of cell cycle‑associated factors, CDK1 and GTSE5. Taken together, these findings indicated that lncRNA AK001796 increased the resistance of NSCLC cells to cisplatin through regulating cell apoptosis and cell proliferation, and thus provides an attractive therapeutic target for NSCLC.

Antitumor action of 3-bromopyruvate implicates reorganized tumor growth regulatory components of tumor milieu, cell cycle arrest and induction of mitochondria-dependent tumor cell death.

PubMed

Yadav, Saveg; Kujur, Praveen Kumar; Pandey, Shrish Kumar; Goel, Yugal; Maurya, Babu Nandan; Verma, Ashish; Kumar, Ajay; Singh, Rana Pratap; Singh, Sukh Mahendra

2018-01-15

Evidences demonstrate that metabolic inhibitor 3-bromopyruvate (3-BP) exerts a potent antitumor action against a wide range of malignancies. However, the effect of 3-BP on progression of the tumors of thymic origin remains unexplored. Although, constituents of tumor microenvironment (TME) plays a pivotal role in regulation of tumor progression, it remains unclear if 3-BP can alter the composition of the crucial tumor growth regulatory components of the external surrounding of tumor cells. Thus, the present investigation attempts to understand the effect of 3-BP administration to a host bearing a progressively growing tumor of thymic origin on tumor growth regulatory soluble, cellular and biophysical components of tumor milieu vis-à-vis understanding its association with tumor progression, accompanying cell cycle events and mode of cell death. Further, the expression of cell survival regulatory molecules and hemodynamic characteristics of the tumor milieu were analysed to decipher mechanisms underlying the antitumor action of 3-BP. Administration of 3-BP to tumor-bearing hosts retarded tumor progression accompanied by induction of tumor cell death, cell cycle arrest, declined metabolism, inhibited mitochondrial membrane potential, elevated release of cytochrome c and altered hemodynamics. Moreover, 3-BP reconstituted the external milieu, in concurrence with deregulated glucose and pH homeostasis and increased tumor infiltration by NK cells, macrophages, and T lymphocytes. Further, 3-BP administration altered the expression of key regulatory molecules involved in glucose uptake, intracellular pH and tumor cell survival. The outcomes of this study will help in optimizing the therapeutic application of 3-BP by targeting crucial tumor growth regulatory components of tumor milieu. Copyright © 2017 Elsevier Inc. All rights reserved.

Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

PubMed Central

Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; Patel, Neil K; Lu, Hua; Zeng, Shelya X; Wang, Guangdi; Zhang, Changde; You, Zongbing

2014-01-01

Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA’s effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, also named cIAP1), leading to activation of caspases 7 and 3 and turning on the downstream apoptotic events. MAA-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and CDK2 expression at the late time. MAA up-regulated p21 expression through inhibition of HDAC activities, independently of p53/p63/p73. These findings demonstrate that MAA suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which suggests that MAA could be used as a potential therapeutic drug for prostate cancer. PMID:25606576

Inhibition of Midkine Suppresses Prostate Cancer CD133+ Stem Cell Growth and Migration.

PubMed

Erdogan, Suat; Doganlar, Zeynep B; Doganlar, Oguzhan; Turkekul, Kader; Serttas, Riza

2017-09-01

Midkine (MDK) is a tumor-promoting factor that is often overexpressed in various human carcinomas, and the role of MDK has not yet been fully investigated in prostate cancer stem cells. Prostate cancer CD133 + stem cells (PCSCs) were isolated from human castration-resistant PC3 cells. PCSCs were treated with different concentrations of MDK inhibitor, iMDK, for 24-72 hours. The IC 50 values were determined by the MTT test. Endogenous MDK messenger RNA expression was knocked down by small interfering RNA. Quantitative reverse transcription polymerase chain reaction, Western blot analyses and image-based cytometry were used to investigate apoptosis and cell cycle progression as well as their underlying molecular mechanisms. Cell migration was evaluated by the wound healing test. iMDK caused dose- and time-dependent inhibition of PCSC survival. Similar growth inhibition was also obtained by small interfering RNA-mediated knockdown of endogenous MDK expression. iMDK was shown to preferentially induce cell cycle arrest at the S and G2/M phases. Suppressed PCSC growth was also accompanied by increases in p53 and the cell cycle inhibitor p21 genes. Combinatorial treatment of iMDK with docetaxel significantly inhibited cell proliferation versus either of the agents used alone. Inhibition of MDK expression strongly suppressed the migration of PCSCs compared to untreated and docetaxel-treated cells. iMDK and the knockdown of MDK decreased p-Akt and significantly upregulated the expression of PI3K/phosphatase/tensin homolog. Our data indicate that MDK plays a crucial role in controlling PCSC proliferation and migration. Therefore, suppression of endogenous expression of MDK would, in combination with traditional chemotherapy drugs, be a potential treatment for PCSCs. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

Oligonucleotide microarray analysis of apoptosis induced by 15-methoxypinusolidic acid in microglial BV2 cells

PubMed Central

Choi, Y; Lim, SY; Jeong, HS; Koo, KA; Sung, SH; Kim, YC

2009-01-01

Background and purpose: We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia. Experimental approach: Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry. Key results: A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45α, gadd45γ and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells. Conclusion and implications: 15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA. PMID:19466985

Correlation of the expression of CD32 and CD180 receptors on CLL cells and MEC1 cell line.

PubMed

Tsertsvadze, T; Mitskevich, N; Ghirdaladze, D; Porakishvili, N

2015-03-01

Chronic Lymphocytic Leukemia (CLL) presents with clonal expansion and accumulation of CD5+CD19+CD23+ cells in peripheral lymphoid organs and tissues and in bone marrow. CLL is supposedly driven by exogenous and/or endogenous (auto)antigen(s) and there is increasing evidence that CLL cells receive microenvironmental signals which support their growth, survival and expansion in vivo. We have previously shown that powerful signals are received by CLL cells through CD180 orphan toll-like receptor. Additional accessory signals could be generated through FcγRII (CD32), since both are expressed on CLL cells as well as on control B cells. Here we studied correlation of the expression of CD32 and CD180 on CLL cells as well as on MEC1 cell line. Peripheral blood mononuclear cells (PBMC) from CLL patients and age-matched healthy volunteers were separated, stained with appropriate antibodies to CD19, CD32 and CD180 and analysed by flow cytometry. CD32 and CD180 expression on MEC1 cells was studied at different time-points. The data was statistically analysed using the Mann-Whitney non-parametrical test. Our data indicates that expression of CD32 is significantly increased on CLL cells compared to control B cells as well as in long-term MEC1 cell culture. In contrast, CD180 expression on MEC1 cells significantly decreased throughout 0-96h of MEC1 cell culture. We have recently shown that CD180 ligation can redirect sIgM-mediated signaling from pro-survival to pro-apoptotic. This data indicates that a drop in the expression of CD180 on cycling CLL cells might lead to a weakening of this effect and enhance further survival and expansion of CLL cells in proliferative centres of lymphoid tissues. Since MEC1 cells are derived from a CLL patient with mutated IGVH genes (M-CLL) negative correlation between CD180 and CD32 expression on cycling MEC1 cells could be limited to M-CLL.

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

PubMed Central

Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609

Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer.

PubMed

Madhu Krishna, B; Chaudhary, Sanjib; Mishra, Dipti Ranjan; Naik, Sanoj K; Suklabaidya, S; Adhya, A K; Mishra, Sandip K

2018-05-30

Breast cancer (BC) is highly heterogeneous with ~ 60-70% of estrogen receptor positive BC patient's response to anti-hormone therapy. Estrogen receptors (ERs) play an important role in breast cancer progression and treatment. Estrogen related receptors (ERRs) are a group of nuclear receptors which belong to orphan nuclear receptors, which have sequence homology with ERs and share target genes. Here, we investigated the possible role and clinicopathological importance of ERRβ in breast cancer. Estrogen related receptor β (ERRβ) expression was examined using tissue microarray slides (TMA) of Breast Carcinoma patients with adjacent normal by immunohistochemistry and in breast cancer cell lines. In order to investigate whether ERRβ is a direct target of ERα, we investigated the expression of ERRβ in short hairpin ribonucleic acid knockdown of ERα breast cancer cells by western blot, qRT-PCR and RT-PCR. We further confirmed the binding of ERα by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), Re-ChIP and luciferase assays. Fluorescence-activated cell sorting analysis (FACS) was performed to elucidate the role of ERRβ in cell cycle regulation. A Kaplan-Meier Survival analysis of GEO dataset was performed to correlate the expression of ERRβ with survival in breast cancer patients. Tissue microarray (TMA) analysis showed that ERRβ is significantly down-regulated in breast carcinoma tissue samples compared to adjacent normal. ER + ve breast tumors and cell lines showed a significant expression of ERRβ compared to ER-ve tumors and cell lines. Estrogen treatment significantly induced the expression of ERRβ and it was ERα dependent. Mechanistic analyses indicate that ERα directly targets ERRβ through estrogen response element and ERRβ also mediates cell cycle regulation through p18, p21 cip and cyclin D1 in breast cancer cells. Our results also showed the up-regulation of ERRβ promoter activity in ectopically co-expressed ERα and ERRβ breast cancer cell lines. Fluorescence-activated cell sorting analysis (FACS) showed increased G0/G1 phase cell population in ERRβ overexpressed MCF7 cells. Furthermore, ERRβ expression was inversely correlated with overall survival in breast cancer. Collectively our results suggest cell cycle and tumor suppressor role of ERRβ in breast cancer cells which provide a potential avenue to target ERRβ signaling pathway in breast cancer. Our results indicate that ERRβ is a negative regulator of cell cycle and a possible tumor suppressor in breast cancer. ERRβ could be therapeutic target for the treatment of breast cancer.

Senescence-associated reprogramming promotes cancer stemness.

PubMed

Milanovic, Maja; Fan, Dorothy N Y; Belenki, Dimitri; Däbritz, J Henry M; Zhao, Zhen; Yu, Yong; Dörr, Jan R; Dimitrova, Lora; Lenze, Dido; Monteiro Barbosa, Ines A; Mendoza-Parra, Marco A; Kanashova, Tamara; Metzner, Marlen; Pardon, Katharina; Reimann, Maurice; Trumpp, Andreas; Dörken, Bernd; Zuber, Johannes; Gronemeyer, Hinrich; Hummel, Michael; Dittmar, Gunnar; Lee, Soyoung; Schmitt, Clemens A

2018-01-04

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16 INK4a , p21 CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

Anaplastic Thyroid Carcinoma: A ceRNA Analysis Pointed to a Crosstalk between SOX2, TP53, and microRNA Biogenesis

PubMed Central

Carina, Valeria; Tomasello, Laura; Pitrone, Maria; Baiamonte, Concetta; Amato, Marco Calogero

2015-01-01

It has been suggested that cancer stem cells (CSC) may play a central role in oncogenesis, especially in undifferentiated tumours. Anaplastic thyroid carcinoma (ATC) has characteristics suggestive of a tumour enriched in CSC. Previous studies suggested that the stem cell factor SOX2 has a preeminent hierarchical role in determining the characteristics of stem cells in SW1736 ATC cell line. In detail, silencing SOX2 in SW1736 is able to suppress the expression of the stem markers analysed, strongly sensitizing the line to treatment with chemotherapeutic agents. Therefore, in order to further investigate the role of SOX2 in ATC, a competing endogenous RNA (ceRNA) analysis was conducted in order to isolate new functional partners of SOX2. Among the interactors, of particular interest are genes involved in the biogenesis of miRNAs (DICER1, RNASEN, and EIF2C2), in the control cell cycle (TP53, CCND1), and in mitochondrial activity (COX8A). The data suggest that stemness, microRNA biogenesis and functions, p53 regulatory network, cyclin D1, and cell cycle control, together with mitochondrial activity, might be coregulated. PMID:25705224

Molecular biology of bladder cancer.

PubMed

Martin-Doyle, William; Kwiatkowski, David J

2015-04-01

Classic as well as more recent large-scale genomic analyses have uncovered multiple genes and pathways important for bladder cancer development. Genes involved in cell-cycle control, chromatin regulation, and receptor tyrosine and PI3 kinase-mammalian target of rapamycin signaling pathways are commonly mutated in muscle-invasive bladder cancer. Expression-based analyses have identified distinct types of bladder cancer that are similar to subsets of breast cancer, and have prognostic and therapeutic significance. These observations are leading to novel therapeutic approaches in bladder cancer, providing optimism for therapeutic progress. Copyright © 2015 Elsevier Inc. All rights reserved.

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

CMC-544 (inotuzumab ozogamicin) shows less effect on multidrug resistant cells: analyses in cell lines and cells from patients with B-cell chronic lymphocytic leukaemia and lymphoma.

PubMed

Takeshita, Akihiro; Shinjo, Kaori; Yamakage, Nozomi; Ono, Takaaki; Hirano, Isao; Matsui, Hirotaka; Shigeno, Kazuyuki; Nakamura, Satoki; Tobita, Tadasu; Maekawa, Masato; Ohnishi, Kazunori; Sugimoto, Yoshikazu; Kiyoi, Hitoshi; Naoe, Tomoki; Ohno, Ryuzo

2009-06-01

The effect of CMC-544, a calicheamicin-conjugated anti-CD22 monoclonal antibody, was analysed in relation to CD22 and P-glycoprotein (P-gp) in B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B-cell CLL or NHL were also used. The effect of CMC-544 was analysed by viable cell count, morphology, annexin-V staining, and cell cycle distribution. A dose-dependent, selective cytotoxic effect of CMC-544 was observed in cell lines that expressed CD22. CMC-544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC-544 in P-gp-expressing sublines. In clinical samples, the cytotoxic effect of CMC-544 was inversely related to the amount of P-gp (P = 0.003), and to intracellular rhodamine-123 accumulation (P < 0.001). On the other hand, the effect positively correlated with the amount of CD22 (P = 0.010). The effect of CMC-544 depends on the levels of CD22 and P-gp. Our findings will help to predict the clinical effectiveness of this drug on these B-cell malignancies, suggesting a beneficial effect with combined use of CMC-544 and MDR modifiers.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome.

PubMed

Waugh, Stacey; Kashon, Michael L; Li, Shengqiao; Miller, Gerome R; Johnson, Claud; Krajnak, Kristine

2016-04-01

The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612

Conditional ablation of the Notch2 receptor in the ocular lens

PubMed Central

Saravanamuthu, Senthil S.; Le, Tien T.; Gao, Chun Y.; Cojocaru, Radu I.; Pandiyan, Pushpa; Liu, Chunqiao; Zhang, Jun; Zelenka, Peggy S.; Brown, Nadean L.

2011-01-01

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. PMID:22173065

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells.

PubMed

Al-Qatati, Abeer; Aliwaini, Saeb

2017-12-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

PubMed Central

Al-Qatati, Abeer; Aliwaini, Saeb

2017-01-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma. PMID:29344241

Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

PubMed Central

Szafran, Adam T.; Szwarc, Maria; Marcelli, Marco; Mancini, Michael A.

2008-01-01

Background Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. Conclusions/Significance HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations. PMID:18978937

Picomonas judraskeda Gen. Et Sp. Nov.: The First Identified Member of the Picozoa Phylum Nov., a Widespread Group of Picoeukaryotes, Formerly Known as ‘Picobiliphytes’

PubMed Central

Seenivasan, Ramkumar; Sausen, Nicole; Medlin, Linda K.; Melkonian, Michael

2013-01-01

In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe Picomonas judraskeda gen. et sp. nov., from marine coastal surface waters, which has a ‘picobiliphyte’ 18S rDNA signature. Using vital mitochondrial staining and cell sorting by flow cytometry, a single cell-derived culture was established. The cells are biflagellate, 2.5–3.8×2–2.5 µm in size, lack plastids and display a novel stereotypic cycle of cell motility (described as the “jump, drag, and skedaddle”-cycle). They consist of two hemispherical parts separated by a deep cleft, an anterior part that contains all major cell organelles including the flagellar apparatus, and a posterior part housing vacuoles/vesicles and the feeding apparatus, both parts separated by a large vacuolar cisterna. From serial section analyses of cells, fixed at putative stages of the feeding cycle, it is concluded that cells are not bacterivorous, but feed on small marine colloids of less than 150 nm diameter by fluid-phase, bulk flow endocytosis. Based on the novel features of cell motility, ultrastructure and feeding, and their isolated phylogenetic position, we establish a new phylum, Picozoa, for Picomonas judraskeda, representing an apparently widespread and ecologically important group of heterotrophic picoeukaryotes, formerly known as ‘picobiliphytes’. PMID:23555709

Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation.

PubMed

Zeng, Huawei; Claycombe, Kate J; Reindl, Katie M

2015-10-01

Consumption of a high-fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk, while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer-preventive effects. To distinguish these opposing effects of DCA and butyrate (two major metabolites in colon lumen), we examined the effects of physiologically relevant doses of butyrate (0.5-2 mmol/l) and DCA (0.05-0.3 mmol/l) on colon cell proliferation. We hypothesize that butyrate and DCA each modulates the cell cycle and apoptosis via common and distinct cellular signaling targets. In this study, we demonstrated that both butyrate and DCA inhibited cell proliferation by up to 89% and 92% and increased cell apoptosis rate by up to 3.1- and 4.5-fold, respectively. Cell cycle analyses revealed that butyrate led to an increase in G1 and G2 fractions with a concomitant drop in the S-phase fraction, but DCA induced an increase in only G1 fraction with a concomitant drop in the S-phase fraction when compared with the untreated cells. The examination of early cellular signaling revealed that DCA but not butyrate increased intracellular reactive oxygen species, genomic DNA breakage, the activation of ERK1/2, caspase-3 and PARP. In contrast, DCA decreased activated Rb protein level, and butyrate but not DCA increased p21 expression. Collectively, although both butyrate and DCA inhibit colonic cell proliferation, butyrate increases tumor suppressor gene expression, whereas DCA decreases tumor suppressor activation in cell cycle and apoptosis pathways. Published by Elsevier Inc.

Influence of salinomycin treatment on division and movement of individual cancer cells cultured in normoxia or hypoxia evaluated with time-lapse digital holographic microscopy

PubMed Central

Kamlund, Sofia; Strand, Daniel; Janicke, Birgit; Alm, Kersti; Oredsson, Stina

2017-01-01

ABSTRACT Most studies on new cancer drugs are based on population-derived data, where the absence of response of a small population may pass unnoticed. Thus, individual longitudinal tracking of cells is important for the future development of efficient cancer treatments. We have used digital holographic microscopy to track individual JIMT-1 human breast cancer cells and L929 mouse fibroblast cultivated in normoxia or hypoxia. In addition, JIMT-1 cells were treated with salinomycin, a cancer stem cell targeting compound. Three-day time-lapse movies were captured and individual cells were analysed with respect to cell division (cell cycle length) and cell movement. Comparing population-doubling time derived from population-based growth curves and individual cell cycle time data from time-lapse movies show that the former hide a sub-population of dividing cells. Salinomycin treatment increased the motility of cells, however, this motility did not result in an increased distant migration i.e. the cells increased their local movement. MCF-7 breast cancer cells showed similar motility behaviour as salinomycin-treated JIMT-1 cells. We suggest that combining features, such as motility and migration, can be used to distinguish cancer cells with mesenchymal (JIMT-1) and epithelial (MCF-7) features. The data clearly emphasize the importance of longitudinal cell tracking to understand the biology of individual cells under different conditions. PMID:28933990

MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome

PubMed Central

Wang, Kunning; Liang, Qiaoyi; Li, Xiaoxing; Tsoi, Ho; Zhang, Jingwan; Wang, Hua; Go, Minnie Y Y; Chiu, Philip W Y; Ng, Enders K W; Sung, Joseph J Y; Yu, Jun

2016-01-01

Background Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. Methods MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. Results MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1–S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan–Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer. Conclusions MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer. PMID:26206665

Forces Generated by Cell Intercalation Tow Epidermal Sheets in Mammalian Tissue Morphogenesis

PubMed Central

Heller, Evan; Kumar, K. Vijay; Grill, Stephan W.; Fuchs, Elaine

2014-01-01

Summary While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting and cell cycle inhibitors reveal that closure does not require overlying periderm, proliferation or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound-repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA and α5β1-fibronectin-mediated migration, and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue. PMID:24697897

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

NASA Astrophysics Data System (ADS)

Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

2016-05-01

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

Cell cycle perturbations and genotoxic effects in human primary fibroblasts induced by low-energy protons and X/gamma-rays.

PubMed

Antoccia, Antonio; Sgura, Antonella; Berardinelli, Francesco; Cavinato, Maria; Cherubini, Roberto; Gerardi, Silvia; Tanzarella, Caterina

2009-09-01

The effect of graded doses of high-linear energy transfer (LET) low-energy protons to induce cycle perturbations and genotoxic damage was investigated in normal human fibroblasts. Furthermore, such effects were compared with those produced by low-LET radiations. HFFF2, human primary fibroblasts were exposed to either protons (LET = 28.5 keV/microm) or X/gamma-rays, and endpoints related to cell cycle kinetics and DNA damage analysed. Following both type of irradiations, unsynchronized cells suffered an inhibition to entry into S-phase for doses of 1-4 Gy and remained arrested in the G(1)-phase for several days. The levels of induction of regulator proteins, such as TP53 and CDKN1A showed a clear LET-dependence. DSB induction and repair as measured by scoring for gamma-H2AX foci indicated that protons, with respect to X-rays, yielded a lower number of DSBs per Gy, which showed a slower kinetics of disappearance. Such result was in agreement with the extent of MN induction in binucleated cells after X-irradiation. No significant differences between the two types of radiations were observed with the clonogenic assay, resulting anyway the slope of gamma-ray curve higher than that the proton one. In conclusion, in normal human primary fibroblasts cell cycle arrest at the G(1)/S transition can be triggered shortly after irradiation and maintained for several hours post-irradiation of both protons and X-rays. DNA damage produced by protons appears less amenable to be repaired and could be transformed in cytogenetic damage in the form of MN.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

PubMed

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

PubMed

Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

2010-04-01

Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

A western-style diet, with and without chronic androgen treatment, alters the number, structure and function of small antral follicles in ovaries of young adult monkeys

PubMed Central

Bishop, Cecily V.; Xu, Fuhua; Xu, Jing; Ting, Alison Y.; Galbreath, Etienne; McGee, Whitney K.; Zelinski, Mary B.; Hennebold, Jon D.; Cameron, Judy L.; Stouffer, Richard L.

2015-01-01

Objective To examine the small antral follicle (SAF) cohort in ovaries of adult rhesus monkeys following consumption of a western-style diet (WSD), with or without chronically elevated androgen levels since before puberty. Design Cholesterol or testosterone (T; n=6/group) implants were placed subcutaneously in female rhesus macaques beginning at 1 yr of age (pre-pubertal), with addition of a WSD (high fat/fructose) at 5.5 yrs (menarche ~2.6 yrs). Ovaries were collected at 7 yrs of age. One ovary/female was embedded in paraffin for morphological and immunohistochemical analyses. The SAFs (<2.5mm) were dissected from the other ovary obtained at/near menses in a subgroup of females (n=3/group), and processed for microarray analyses of the SAF transcriptome. Ovaries of adult monkeys consuming a standard macaque diet (low in fats and sugars) were obtained at similar stages of the menstrual cycle and used as controls for all analyses. Setting National primate research center Animals Adult, female rhesus monkeys (Macaca mulatta) Interventions None Main outcome measures Histological analyses, SAF counts and morphology, protein localization and abundance in SAFs, transcriptome in SAFs (mRNAs) Results Compared to controls, consumption of a WSD, with and without T treatment, increased the numbers of SAFs per ovary, due to the presence of more atretic follicles. Numbers of granulosa cells expressing cellular proliferation markers (pRb and pH3) was greater in healthy SAFs, while numbers of cells expressing the cell cycle inhibitor (p21) was higher in atretic SAFs. Intense CYP17A1 staining was observed in the theca cells of SAFs from WSD+/− T groups, compared to controls. Microarray analyses of the transcriptome in SAFs isolated from WSD and WSD+T treated females and controls consuming a standard diet, identified 1944 genes whose mRNA levels changed ≥2-fold among the three groups. Further analyses identified several gene pathways altered by WSD and/or WSD+T associated with steroid, carbohydrate and lipid metabolism, plus ovarian processes. Alterations in levels of several SAF mRNAs are similar to those observed in follicular cells from women with polycystic ovary syndrome (PCOS). Conclusion These data indicate that consumption of a WSD high in fats and sugars in the presence and absence of chronically elevated T alters the structure and function of SAFs within primate ovaries. PMID:26718060

A life cycle assessment of distributed energy production from organic waste: Two case studies in Europe.

PubMed

Evangelisti, Sara; Clift, Roland; Tagliaferri, Carla; Lettieri, Paola

2017-06-01

By means of the life cycle assessment methodology, the purpose of this study is to assess the environmental impact when biomethane from organic waste produced at residential level is used to supply energy to a group of dwellings in the distributed generation paradigm. Three different Combined Heat and Power systems, such as fuel cells, Stirling engine and micro gas turbine, installed at household level are assessed in two different settings: one in Northern Europe (UK) and one in Southern Europe (Italy). Different operating strategies are investigated for each technology. Moreover, marginal electricity production technologies are analysed to assess their influence on the results. This study has demonstrated that the type of bio-methane fed micro-CHP technology employed has a significantly different environmental impact: fuel cells are the most environmentally friendly solution in every category analysed; Stirling engines, although can supply heat to the largest number of dwellings are the least environmentally friendly technology. However, key factors investigated in the model presented in this paper influence the decision making on the type of technology adopted and the operating strategy to be implemented. Copyright © 2017 Elsevier Ltd. All rights reserved.

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions.

PubMed

Lee, Yong-Uk; Son, Miseol; Kim, Jiyoung; Shim, Yhong-Hee; Kawasaki, Ichiro

2016-01-01

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions

PubMed Central

Lee, Yong-Uk; Son, Miseol; Kim, Jiyoung; Shim, Yhong-Hee; Kawasaki, Ichiro

2016-01-01

ABSTRACT Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine. PMID:27104746

Ethylene carbonate-free fluoroethylene carbonate-based electrolyte works better for freestanding Si-based composite paper anodes for Li-ion batteries

NASA Astrophysics Data System (ADS)

Yao, K.; Zheng, J. P.; Liang, R.

2018-03-01

Fluoroethylene carbonate (FEC)-based electrolytes using FEC as the co-solvent (50 wt%) are investigated and compared with the electrolyte using FEC as the additive (10 wt%) for freestanding Si-carbon nanotubes (CNTs) composite paper anodes for Li-ion batteries. The ethylene carbonate (EC)-free FEC-based electrolyte is found to achieve higher specific capacity and better capacity retention in terms of long-term cycling. After 500 cycles, the capacity retention of the cell using diethyl carbonate (DEC)-FEC (1:1 w/w) is increased by 88% and 60% compared to the cells using EC-DEC-FEC (45:45:10 w/w/w) and EC-FEC (1:1 w/w), respectively. Through SEM-EDX and XPS analyses, a possible reaction route of formation of fluorinated semicarbonates and polyolefins from FEC is proposed. The inferior cell performance related to the EC-containing electrolytes is likely due to the formation of more polyolefins, which do not favor Li ion migration.

Coatomer subunit beta 2 (COPB2), identified by label-free quantitative proteomics, regulates cell proliferation and apoptosis in human prostate carcinoma cells.

PubMed

Mi, Yuanyuan; Sun, Chuanyu; Wei, Bingbing; Sun, Feiyu; Guo, Yijun; Hu, Qingfeng; Ding, Weihong; Zhu, Lijie; Xia, Guowei

2018-01-01

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G 1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA. Copyright © 2017 Elsevier Inc. All rights reserved.

Spinal Cord Injury Causes Brain Inflammation Associated with Cognitive and Affective Changes: Role of Cell Cycle Pathways

PubMed Central

Zhao, Zaorui; Sabirzhanov, Boris; Stoica, Bogdan A.; Kumar, Alok; Luo, Tao; Skovira, Jacob; Faden, Alan I.

2014-01-01

Experimental spinal cord injury (SCI) causes chronic neuropathic pain associated with inflammatory changes in thalamic pain regulatory sites. Our recent studies examining chronic pain mechanisms after rodent SCI showed chronic inflammatory changes not only in thalamus, but also in other regions including hippocampus and cerebral cortex. Because changes appeared similar to those in our rodent TBI models that are associated with neurodegeneration and neurobehavioral dysfunction, we examined effects of mouse SCI on cognition, depressive-like behavior, and brain inflammation. SCI caused spatial and retention memory impairment and depressive-like behavior, as evidenced by poor performance in the Morris water maze, Y-maze, novel objective recognition, step-down passive avoidance, tail suspension, and sucrose preference tests. SCI caused chronic microglial activation in the hippocampus and cerebral cortex, where microglia with hypertrophic morphologies and M1 phenotype predominated. Stereological analyses showed significant neuronal loss in the hippocampus at 12 weeks but not 8 d after injury. Increased cell-cycle-related gene (cyclins A1, A2, D1, E2F1, and PCNA) and protein (cyclin D1 and CDK4) expression were found chronically in hippocampus and cerebral cortex. Systemic administration of the selective cyclin-dependent kinase inhibitor CR8 after SCI significantly reduced cell cycle gene and protein expression, microglial activation and neurodegeneration in the brain, cognitive decline, and depression. These studies indicate that SCI can initiate a chronic brain neurodegenerative response, likely related to delayed, sustained induction of M1-type microglia and related cell cycle activation, which result in cognitive deficits and physiological depression. PMID:25122899

Regulatory network analysis of Epstein-Barr virus identifies functional modules and hub genes involved in infectious mononucleosis.

PubMed

Poorebrahim, Mansour; Salarian, Ali; Najafi, Saeideh; Abazari, Mohammad Foad; Aleagha, Maryam Nouri; Dadras, Mohammad Nasr; Jazayeri, Seyed Mohammad; Ataei, Atousa; Poortahmasebi, Vahdat

2017-05-01

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis (IM) and establishes lifetime infection associated with a variety of cancers and autoimmune diseases. The aim of this study was to develop an integrative gene regulatory network (GRN) approach and overlying gene expression data to identify the representative subnetworks for IM and EBV latent infection (LI). After identifying differentially expressed genes (DEGs) in both IM and LI gene expression profiles, functional annotations were applied using gene ontology (GO) and BiNGO tools, and construction of GRNs, topological analysis and identification of modules were carried out using several plugins of Cytoscape. In parallel, a human-EBV GRN was generated using the Hu-Vir database for further analyses. Our analysis revealed that the majority of DEGs in both IM and LI were involved in cell-cycle and DNA repair processes. However, these genes showed a significant negative correlation in the IM and LI states. Furthermore, cyclin-dependent kinase 2 (CDK2) - a hub gene with the highest centrality score - appeared to be the key player in cell cycle regulation in IM disease. The most significant functional modules in the IM and LI states were involved in the regulation of the cell cycle and apoptosis, respectively. Human-EBV network analysis revealed several direct targets of EBV proteins during IM disease. Our study provides an important first report on the response to IM/LI EBV infection in humans. An important aspect of our data was the upregulation of genes associated with cell cycle progression and proliferation.

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

PubMed Central

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

2017-01-01

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

The response of single human cells to zero gravity

NASA Technical Reports Server (NTRS)

Montgomery, P. O., Jr.; Cook, J. E.; Reynolds, R. C.; Paul, J. S.; Hayflick, L.; Stock, D.; Schulz, W. W.; Kimzey, S. L.; Thirolf, R. G.; Rogers, T.

1974-01-01

The SO15 experiment was designed to extend observations of the effects of zero-gravity to living human cells during and subsequent to a 59-day flight on Skylab 3. A strain of diploid human embryonic lung cells, WI-38, was chosen for this purpose. The studies were concerned with observations designed to detect the effects of zero-gravity on cell growth rates and on cell structure as observed by light microscopy, transmission and scanning electron microscopy and histochemistry. Studies of the effects of zero-gravity on the cell function and the cell cycle were performed by time lapse motion picture photography and microspectrophotometry. Subsequent study of the returned living cells included karotyping, G- and C-banding, and analyses of the culture media used. Some of the living cells returned were banked by deep freeze techniques for possible future experiments.

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.

The importance of ion fluxes for cancer proliferation and metastasis: A thermodynamic analysis.

PubMed

Lucia, Umberto; Deisboeck, Thomas S

2018-05-14

Following a thermodynamic approach, we develop a new theoretical analysis of ion transfer across cell membranes. Supported also by experimental data from the literature, we highlight that ion channels determine the typical features of cancer cells, i.e. independence from growth-regulatory signals, avoidance of apoptosis, indefinite proliferative potential, and the capability of inducing angiogenesis. Specifically, we analyse how ion transport, with particular regards to Ca 2+ fluxes, modulates cancer cell proliferation, and regulates cell cycle checkpoints. Finally, our analysis also suggests that in malignant tumours aerobic glycolysis is the more efficient metabolic process when taking the required solvent capacity into account. Copyright © 2018 Elsevier Ltd. All rights reserved.

New sesquiterpene lactones from Ambrosia cumanensis Kunth.

PubMed

Jimenez-Usuga, Nora Del Socorro; Malafronte, Nicola; Cotugno, Roberta; De Leo, Marinella; Osorio, Edison; De Tommasi, Nunziatina

2016-09-01

Eleven sesquiterpene lactones, including three new natural products (1-3), were isolated from the n-butanolic extract of Ambrosia cumanensis Kunth. aerial parts. The structure of all isolated compounds was elucidated by 1D- and 2D-NMR, and MS analyses. All compounds were tested for their antiproliferative activity on HeLa, Jurkat, and U937 cell lines. Compound 3, 2,3-dehydropsilostachyn C, showed cytotoxic activity with different potency in all cell lines. By means of flow cytometric studies, compound 3 was demonstrated to induce in Jurkat cells a G2/M cell cycle block, while in U937 elicited both cytostatic and cytotoxic responses. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve

PubMed Central

dos Santos, Cyndia Mara Bezerra; Ludwig, Adriana; Kessler, Rafael Luis; Rampazzo, Rita de Cássia Pontello; Inoue, Alexandre Haruo; Krieger, Marco Aurélio; Pavoni, Daniela Parada; Probst, Christian Macagnan

2018-01-01

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins. PMID:29668769

Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer.

PubMed

Shao, Yaping; Ye, Guozhu; Ren, Shancheng; Piao, Hai-Long; Zhao, Xinjie; Lu, Xin; Wang, Fubo; Ma, Wang; Li, Jia; Yin, Peiyuan; Xia, Tian; Xu, Chuanliang; Yu, Jane J; Sun, Yinghao; Xu, Guowang

2018-07-15

Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene-metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography-mass spectrometry (GC-MS)-based metabolomics and RNA-seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network-based analyses to gain a comprehensive and in-depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies. © 2018 UICC.

Landscape and flux reveal a new global view and physical quantification of mammalian cell cycle

PubMed Central

Li, Chunhe; Wang, Jin

2014-01-01

Cell cycles, essential for biological function, have been investigated extensively. However, enabling a global understanding and defining a physical quantification of the stability and function of the cell cycle remains challenging. Based upon a mammalian cell cycle gene network, we uncovered the underlying Mexican hat landscape of the cell cycle. We found the emergence of three local basins of attraction and two major potential barriers along the cell cycle trajectory. The three local basins of attraction characterize the G1, S/G2, and M phases. The barriers characterize the G1 and S/G2 checkpoints, respectively, of the cell cycle, thus providing an explanation of the checkpoint mechanism for the cell cycle from the physical perspective. We found that the progression of a cell cycle is determined by two driving forces: curl flux for acceleration and potential barriers for deceleration along the cycle path. Therefore, the cell cycle can be promoted (suppressed), either by enhancing (suppressing) the flux (representing the energy input) or by lowering (increasing) the barrier along the cell cycle path. We found that both the entropy production rate and energy per cell cycle increase as the growth factor increases. This reflects that cell growth and division are driven by energy or nutrition supply. More energy input increases flux and decreases barrier along the cell cycle path, leading to faster oscillations. We also identified certain key genes and regulations for stability and progression of the cell cycle. Some of these findings were evidenced from experiments whereas others lead to predictions and potential anticancer strategies. PMID:25228772

Identification of Cell Cycle-Regulated Genes by Convolutional Neural Network.

PubMed

Liu, Chenglin; Cui, Peng; Huang, Tao

2017-01-01

The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype are analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight to the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

PubMed

Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

1984-11-01

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts

PubMed Central

Nath, Rahul; Mant, Christine A; Kell, Barbara; Cason, John; Bible, Jon M

2006-01-01

Background Human papillomavirus type 16 (HPV-16) E5 protein co-operates with epidermal growth factor to stimulate mitogenesis of murine fibroblasts. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-16 E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-growth of murine fibroblasts. Results We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-16 abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-16 E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-growth; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-growth and repression of p21 expression, despite high transcription levels. Conclusion The effects of HPV-16 E5 variants upon mitosis help to explain why Leu44Val65 HPV-16 E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK. PMID:16899131

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.

The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

PubMed Central

Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173

Indirect-fired gas turbine dual fuel cell power cycle

DOEpatents

Micheli, Paul L.; Williams, Mark C.; Sudhoff, Frederick A.

1996-01-01

A fuel cell and gas turbine combined cycle system which includes dual fuel cell cycles combined with a gas turbine cycle wherein a solid oxide fuel cell cycle operated at a pressure of between 6 to 15 atms tops the turbine cycle and is used to produce CO.sub.2 for a molten carbonate fuel cell cycle which bottoms the turbine and is operated at essentially atmospheric pressure. A high pressure combustor is used to combust the excess fuel from the topping fuel cell cycle to further heat the pressurized gas driving the turbine. A low pressure combustor is used to combust the excess fuel from the bottoming fuel cell to reheat the gas stream passing out of the turbine which is used to preheat the pressurized air stream entering the topping fuel cell before passing into the bottoming fuel cell cathode. The CO.sub.2 generated in the solid oxide fuel cell cycle cascades through the system to the molten carbonate fuel cell cycle cathode.

The global regulatory architecture of transcription during the Caulobacter cell cycle.

PubMed

Zhou, Bo; Schrader, Jared M; Kalogeraki, Virginia S; Abeliuk, Eduardo; Dinh, Cong B; Pham, James Q; Cui, Zhongying Z; Dill, David L; McAdams, Harley H; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

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.

Pathogenesis of Gastric Cancer: Genetics and Molecular Classification.

PubMed

Figueiredo, Ceu; Camargo, M C; Leite, Marina; Fuentes-Pananá, Ezequiel M; Rabkin, Charles S; Machado, José C

Gastric cancer is the fifth most incident and the third most common cause of cancer-related death in the world. Infection with Helicobacter pylori is the major risk factor for this disease. Gastric cancer is the final outcome of a cascade of events that takes decades to occur and results from the accumulation of multiple genetic and epigenetic alterations. These changes are crucial for tumor cells to expedite and sustain the array of pathways involved in the cancer development, such as cell cycle, DNA repair, metabolism, cell-to-cell and cell-to-matrix interactions, apoptosis, angiogenesis, and immune surveillance. Comprehensive molecular analyses of gastric cancer have disclosed the complex heterogeneity of this disease. In particular, these analyses have confirmed that Epstein-Barr virus (EBV)-positive gastric cancer is a distinct entity. The identification of gastric cancer subtypes characterized by recognizable molecular profiles may pave the way for a more personalized clinical management and to the identification of novel therapeutic targets and biomarkers for screening, prognosis, prediction of response to treatment, and monitoring of gastric cancer progression.

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

PubMed

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells

PubMed Central

Zhang, Yiqiang; Zhong, Jiang F; Qiu, Hongyu; Robb MacLellan, W.; Marbán, Eduardo; Wang, Charles

2015-01-01

It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. PMID:26657817

S100A11 promotes human pancreatic cancer PANC-1 cell proliferation and is involved in the PI3K/AKT signaling pathway.

PubMed

Xiao, Mingbing; Li, Tao; Ji, Yifei; Jiang, Feng; Ni, Wenkai; Zhu, Jing; Bao, Baijun; Lu, Cuihua; Ni, Runzhou

2018-01-01

S100A11, a member of S100 calcium-binding protein family, is associated with the numerous processes of tumorigenesis and metastasis. In the present study, the role of S100A11, and its possible underlying mechanisms in cell proliferation, apoptosis and cell cycle distribution in human pancreatic cancer were explored. Immunohistochemical analyses of S100A11 and phosphorylated (p)-AKT serine/threonine kinase (AKT) were performed in 30 resected specimens from patients with pancreatic cancer. PANC-1 cells were transfected with pcDNA3.1-S100A11 or treated with 50 µmol/l LY294002 for 48 h. Cell proliferation was determined using a cell counting kit-8 assay, whereas apoptosis and cell cycle distribution were determined by flow cytometry analysis. The mRNA and protein levels of S100A11, and AKT were determined using semi quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. Pearson correlation analysis revealed that the expression levels of S100A11 and p-AKT were positively correlated (r, 0.802; P<0.05). Compared with the control group, S100A11 overexpression significantly promoted PANC-1 cell proliferation and reduced the percentage of early apoptotic cells. Flow cytometric analysis indicated that the proportion of PANC-1 cells in the S phase was significantly elevated and cell percentage in the G0/G1 phase declined in response to S100A11 overexpression (all P<0.05). S100A11 overexpression also significantly increased AKT mRNA and p-AKT protein expression levels (both P<0.05). The phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, significantly inhibited PANC-1 cell proliferation, promoted apoptosis and caused G1/S phase arrest in PANC-1 cells (all P<0.05). These findings together suggest that S100A11 promotes the viability and proliferation of human pancreatic cancer PANC-1 cells through the upregulation of the PI3K/AKT signaling pathway. Thus, S100A11 may be considered as a novel drug target for targeted therapy of pancreatic cancer.

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

PubMed Central

Johard, Helena; Mahdessian, Diana; Fedr, Radek; Marks, Carolyn; Medalová, Jiřina; Souček, Karel; Lundberg, Emma; Linnarsson, Sten; Bryja, Vítězslav; Sekyrova, Petra; Altun, Mikael; Andäng, Michael

2017-01-01

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. PMID:29228002

Cell life and death in the anterior pituitary gland: role of oestrogens.

PubMed

Seilicovich, A

2010-07-01

Apoptotic processes play an important role in the maintenance of cell numbers in the anterior pituitary gland during physiological endocrine events. In this review, we summarise the regulation of apoptosis of anterior pituitary cells, particularly lactotrophs, somatotrophs and gonadotrophs, and analyse the possible mechanisms involved in oestrogen-induced apoptosis in anterior pituitary cells. Oestrogens exert apoptotic actions in several cell types and act as modulators of pituitary cell renewal, sensitising cells to both mitogenic and apoptotic signals. Local synthesis of growth factors and cytokines induced by oestradiol as well as changes in phenotypic features that enhance the responsiveness of anterior pituitary cells to pro-apoptotic factors may account for cyclical apoptotic activity in anterior pituitary cells during the oestrous cycle. Considering that tissue homeostasis results from a balance between cell proliferation and death and that mechanisms involved in apoptosis are tightly regulated, defects in cell death processes could have a considerable physiopathological impact.

Attentional modulation of cell-class specific gamma-band synchronization in awake monkey area V4

PubMed Central

Vinck, Martin; Womelsdorf, Thilo; Buffalo, Elizabeth A.; Desimone, Robert; Fries, Pascal

2013-01-01

Summary Selective visual attention is subserved by selective neuronal synchronization, entailing precise orchestration among excitatory and inhibitory cells. We tentatively identified these as broad (BS) and narrow spiking (NS) cells and analyzed their synchronization to the local field potential in two macaque monkeys performing a selective visual attention task. Across cells, gamma phases scattered widely but were unaffected by stimulation or attention. During stimulation, NS cells lagged BS cells on average by ~60° and gamma synchronized twice as strongly. Attention enhanced and reduced the gamma locking of strongly and weakly activated cells, respectively. During a pre-stimulus attentional cue period, BS cells showed weak gamma synchronization, while NS cells gamma synchronized as strongly as with visual stimulation. These analyses reveal the cell-type specific dynamics of the gamma cycle in macaque visual cortex and suggest that attention affects neurons differentially depending on cell type and activation level. PMID:24267656

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cell - Update II

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1992-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV) nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles, compared to 3500 cycles for cells containing 31 percent KOH. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2X normal rate). The depth-of-discharge was 80 percent. Six 48-Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16,000 cycles during the continuing test.

Day/night regulation of aquaporins during the CAM cycle in Mesembryanthemum crystallinum.

PubMed

Vera-Estrella, Rosario; Barkla, Bronwyn J; Amezcua-Romero, Julio C; Pantoja, Omar

2012-03-01

Mesembryanthemum crystallinum exhibits induction of Crassulacean acid metabolism (CAM) after a threshold stage of development, by exposure to long days with high light intensities or by water and salt stress. During the CAM cycle, fluctuations in carbon partitioning within the cell lead to transient drops in osmotic potential, which are likely stabilized/balanced by passive movement of water via aquaporins (AQPs). Protoplast swelling assays were used to detect changes in water permeability during the day/night cycle of CAM. To assess the role of AQPs during the same period, we followed transcript accumulation and protein abundance of four plasma membrane intrinsic proteins (PIPs) and one tonoplast intrinsic protein (TIP). CAM plants showed a persistent rhythm of specific AQP protein abundance changes throughout the day/night cycle, including changes in amount of McPIP2;1, McTIP1;2, McPIP1;4 and McPIP1;5, while the abundance of McPIP1;2 was unchanged. These protein changes did not appear to be coordinated with transcript levels for any of the AQPs analysed; however, they did occur in parrallel to alterations in water permeability, as well as variations in cell osmolarity, pinitol, glucose, fructose and phosphoenolpyruvate carboxylase (PEPc) levels measured throughout the day/night CAM cycle. Results suggest a role for AQPs in maintaining water balance during CAM and highlight the complexity of protein expression during the CAM cycle. © 2011 Blackwell Publishing Ltd.

The cell cycle.

PubMed

Singh, N; Lim, R B; Sawyer, M A

2000-07-01

The cell cycle and the cell cycle control system are the engines that drive life. They allow for the processes of cell renewal and the growth of organisms, under controlled conditions. The control system is essential for the monitoring of normal cell growth and replication of genetic material and to ensure that normal, functional daughter cells are produced at completion of each cell cycle. Although certain clinical applications exist which take advantage of the events of the cell cycle, our understanding of its mechanisms and how to manipulate them is infantile. The next decades will continue to see the effort of many researchers focused upon unlocking the mysteries of the cell cycle and the cell cycle control system.

Pathological implications of cell cycle re-entry in Alzheimer disease.

PubMed

Bonda, David J; Lee, Hyun-pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

2010-06-29

The complex neurodegeneration underlying Alzheimer disease (AD), although incompletely understood, is characterised by an aberrant re-entry into the cell cycle in neurons. Pathological evidence, in the form of cell cycle markers and regulatory proteins, suggests that cell cycle re-entry is an early event in AD, which precedes the formation of amyloid-beta plaques and neurofibrillary tangles (NFTs). Although the exact mechanisms that induce and mediate these cell cycle events in AD are not clear, significant advances have been made in further understanding the pathological role of cell cycle re-entry in AD. Importantly, recent studies indicate that cell cycle re-entry is not a consequence, but rather a cause, of neurodegeneration, suggesting that targeting of cell cycle re-entry may provide an opportunity for therapeutic intervention. Moreover, multiple inducers of cell cycle re-entry and their interactions in AD have been proposed. Here, we review the most recent advances in understanding the pathological implications of cell cycle re-entry in AD.

Synergistic effect of aluminum and ionizing radiation upon ultrastructure, oxidative stress and apoptotic alterations in Paneth cells of rat intestine.

PubMed

Eltahawy, N A; Elsonbaty, S M; Abunour, S; Zahran, W E

2017-03-01

Environmental and occupational exposure to aluminum along with ionizing radiation results in serious health problems. This study was planned to investigate the impact of oxidative stress provoked by exposure to ionizing radiation with aluminum administration upon cellular ultra structure and apoptotic changes in Paneth cells of rat small intestine . Animals received daily aluminum chloride by gastric gavage at a dose 0.5 mg/Kg BW for 4 weeks. Whole body gamma irradiation was applied at a dose 2 Gy/week up to 8 Gy. Ileum malondialdehyde, advanced oxidative protein products, protein carbonyl and tumor necrosis factor-alpha were assessed as biomarkers of lipid peroxidation, protein oxidation and inflammation respectively along with superoxide dismutase, catalase, and glutathione peroxidase activities as enzymatic antioxidants. Moreover, analyses of cell cycle division and apoptotic changes were evaluated by flow cytometry. Intestinal cellular ultra structure was investigated using transmission electron microscope.Oxidative and inflammatory stresses assessment in the ileum of rats revealed that aluminum and ionizing radiation exposures exhibited a significant effect upon the increase in oxidative stress biomarkers along with the inflammatory marker tumor necrosis factor-α accompanied by a significant decreases in the antioxidant enzyme activities. Flow cytometric analyses showed significant alterations in the percentage of cells during cell cycle division phases along with significant increase in apoptotic cells. Ultra structurally, intestinal cellular alterations with marked injury in Paneth cells at the sites of bacterial translocation in the crypt of lumens were recorded. The results of this study have clearly showed that aluminum and ionizing radiation exposures induced apoptosis with oxidative and inflammatory disturbance in the Paneth cells of rat intestine, which appeared to play a major role in the pathogenesis of cellular damage. Furthermore, the interaction of these two intestinal toxic routes was found to be synergistic.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

PubMed

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

PubMed Central

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R.

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase. PMID:19729042

Chloride-Reinforced Carbon Nanofiber Host as Effective Polysulfide Traps in Lithium-Sulfur Batteries.

PubMed

Fan, Lei; Zhuang, Houlong L; Zhang, Kaihang; Cooper, Valentino R; Li, Qi; Lu, Yingying

2016-12-01

Lithium-sulfur (Li-S) battery is one of the most promising alternatives for the current state-of-the-art lithium-ion batteries due to its high theoretical energy density and low production cost from the use of sulfur. However, the commercialization of Li-S batteries has been so far limited to the cyclability and the retention of active sulfur materials. Using co-electrospinning and physical vapor deposition procedures, we created a class of chloride-carbon nanofiber composites, and studied their effectiveness on polysulfides sequestration. By trapping sulfur reduction products in the modified cathode through both chemical and physical confinements, these chloride-coated cathodes are shown to remarkably suppress the polysulfide dissolution and shuttling between lithium and sulfur electrodes. From adsorption experiments and theoretical calculations, it is shown that not only the sulfide-adsorption effect but also the diffusivity in the vicinity of these chlorides materials plays an important role on the reversibility of sulfur-based cathode upon repeated cycles. Balancing the adsorption and diffusion effects of these nonconductive materials could lead to the enhanced cycling performance of an Li-S cell. Electrochemical analyses over hundreds of cycles indicate that cells containing indium chloride-modified carbon nanofiber outperform cells with other halogenated salts, delivering an average specific capacity of above 1200 mAh g -1 at 0.2 C.

7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide.

PubMed

Patridge, Eric V; Eriksson, Emma S E; Penketh, Philip G; Baumann, Raymond P; Zhu, Rui; Shyam, Krishnamurthy; Eriksson, Leif A; Sartorelli, Alan C

2012-10-01

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC(50) = 12.1 μM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide

PubMed Central

Eriksson, Emma S. E.; Penketh, Philip G.; Baumann, Raymond P.; Zhu, Rui; Shyam, Krishnamurthy; Eriksson, Leif A.; Sartorelli, Alan C.

2013-01-01

Here, we report on 7-nitro-4-(phenylthio) benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein–protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity. PMID:22669514

Toxoplasma gondii tissue cyst purification using Percoll gradients

PubMed Central

Watts, Elizabeth A.; Dhara, Animesh; Sinai, Anthony P.

2017-01-01

The protozoan parasite Toxoplasma gondii is capable of infecting all warm blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the CNS and muscle during the asexual cycle within all chronically infected warm-blooded hosts. These tissue cysts are populated with slow growing bradyzoites which have been until recently thought to be dormant entities in the context of immune sufficiency. Reactivation to active growth during immune suppression is of critical clinical importance. Yet we know little about tissue cysts or the bradyzoites they house as the diversity of tissue cysts cannot be replicated in cell culture systems. Our optimization of tissue cyst purification from the brains of infected mice using Percoll gradients provides an efficient means to recover in vivo derived tissue cysts that can be applied to imaging, cell-biologic, biochemical, transcriptomic and proteomic analyses. PMID:28510363

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma

PubMed Central

Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B.; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M.; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E.; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H.; Deubzer, Hedwig E.

2016-01-01

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma. PMID:27572323

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma.

PubMed

Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H; Deubzer, Hedwig E

2016-10-11

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

PubMed

Bridger, J M; Kill, I R; Lichter, P

1998-01-01

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Genomic profiling of bovine corpus luteum maturation

PubMed Central

Wigoda, Noa; Ben-Dor, Shifra; Orr, Irit; Meidan, Rina

2018-01-01

To unveil novel global changes associated with corpus luteum (CL) maturation, we analyzed transcriptome data for the bovine CL on days 4 and 11, representing the developing vs. mature gland. Our analyses revealed 681 differentially expressed genes (363 and 318 on day 4 and 11, respectively), with ≥2 fold change and FDR of <5%. Different gene ontology (GO) categories were represented prominently in transcriptome data at these stages (e.g. days 4: cell cycle, chromosome, DNA metabolic process and replication and on day 11: immune response; lipid metabolic process and complement activation). Based on bioinformatic analyses, select genes expression in day 4 and 11 CL was validated with quantitative real-time PCR. Cell specific expression was also determined in enriched luteal endothelial and steroidogenic cells. Genes related to the angiogenic process such as NOS3, which maintains dilated vessels and MMP9, matrix degrading enzyme, were higher on day 4. Importantly, our data suggests day 11 CL acquire mechanisms to prevent blood vessel sprouting and promote their maturation by expressing NOTCH4 and JAG1, greatly enriched in luteal endothelial cells. Another endothelial specific gene, CD300LG, was identified here in the CL for the first time. CD300LG is an adhesion molecule enabling lymphocyte migration, its higher levels at mid cycle are expected to support the transmigration of immune cells into the CL at this stage. Together with steroidogenic genes, most of the genes regulating de-novo cholesterol biosynthetic pathway (e.g HMGCS, HMGCR) and cholesterol uptake from plasma (LDLR, APOD and APOE) were upregulated in the mature CL. These findings provide new insight of the processes involved in CL maturation including blood vessel growth and stabilization, leucocyte transmigration as well as progesterone synthesis as the CL matures. PMID:29590145

10 CFR 436.24 - Uncertainty analyses.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Procedures for Life Cycle Cost Analyses § 436.24 Uncertainty analyses. If particular items of cost data or... impact of uncertainty on the calculation of life cycle cost effectiveness or the assignment of rank order... and probabilistic analysis. If additional analysis casts substantial doubt on the life cycle cost...

10 CFR 436.24 - Uncertainty analyses.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Procedures for Life Cycle Cost Analyses § 436.24 Uncertainty analyses. If particular items of cost data or... impact of uncertainty on the calculation of life cycle cost effectiveness or the assignment of rank order... and probabilistic analysis. If additional analysis casts substantial doubt on the life cycle cost...

10 CFR 436.24 - Uncertainty analyses.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Procedures for Life Cycle Cost Analyses § 436.24 Uncertainty analyses. If particular items of cost data or... impact of uncertainty on the calculation of life cycle cost effectiveness or the assignment of rank order... and probabilistic analysis. If additional analysis casts substantial doubt on the life cycle cost...

10 CFR 436.24 - Uncertainty analyses.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Procedures for Life Cycle Cost Analyses § 436.24 Uncertainty analyses. If particular items of cost data or... impact of uncertainty on the calculation of life cycle cost effectiveness or the assignment of rank order... and probabilistic analysis. If additional analysis casts substantial doubt on the life cycle cost...

Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

PubMed

Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd; Juhl, Morten; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

2017-01-01

Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Inhibition of mTOR's Catalytic Site by PKI-587 Is a Promising Therapeutic Option for Gastroenteropancreatic Neuroendocrine Tumor Disease

PubMed Central

Freitag, Helma; Christen, Friederike; Lewens, Florentine; Grass, Irina; Briest, Franziska; Iwaszkiewicz, Sara; Siegmund, Britta; Grabowski, Patricia

2017-01-01

Background The characteristic clinical heterogeneity and mostly slow-growing behavior of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) cause problems in finding appropriate treatments. Thus, the current therapy options are not satisfactory. PKI-587 is a highly potent, novel dual inhibitor of PI3K and mTORC1/C2. Aim We assessed the effects of PKI-587 in different GEP-NEN tumor models, including the poorly differentiated cell line LCC-18, and compared them with those of the established mTORC1 inhibitor everolimus. Methods We treated BON, QGP-1, KRJ-I, and LCC-18 cell lines with increasing concentrations of the inhibitor PKI-587, and compared the results with those of everolimus and DMSO. We assessed the impact of the treatments on viability (WST-1 assay), on apoptotic processes (caspase 3/7 assay, JC-1), and on cell cycle regulation (flow cytometry). We determined alterations in signaling mediators by phosphor-specific Western blot analysis and conducted multiplexed gene expression analysis (nCounter® technology). Results In all cell lines, PKI-587 dose-dependently inhibited proliferation, whereas everolimus was less effective. Treatment with PKI-587 led to cell cycle arrest and induction of apoptosis and successfully suppressed activity of the direct mTORC1 target 4E-BP1, a crucial factor for tumor genesis only partially inhibited by everolimus. Gene expression analyses revealed relevant changes of RAS, MAPK, STAT, and PI3K pathway genes after treatment. Treatment-dependent and cell line-characteristic effects on AKT/Rb/E2F signaling regarding cell cycle control and apoptosis are extensively discussed in this paper. Conclusion PI3K/mTOR dual targeting is a promising new therapeutic approach in neuroendocrine tumor disease that should be evaluated in further clinical trials. PMID:27513674

Overcoming the response plateau in multiple myeloma: a novel bortezomib-based strategy for secondary induction and high-yield CD34+ stem cell mobilization.

PubMed

Niesvizky, Ruben; Mark, Tomer M; Ward, Maureen; Jayabalan, David S; Pearse, Roger N; Manco, Megan; Stern, Jessica; Christos, Paul J; Mathews, Lena; Shore, Tsiporah B; Zafar, Faiza; Pekle, Karen; Xiang, Zhaoying; Ely, Scott; Skerret, Donna; Chen-Kiang, Selina; Coleman, Morton; Lane, Maureen E

2013-03-15

This phase II study evaluated bortezomib-based secondary induction and stem cell mobilization in 38 transplant-eligible patients with myeloma who had an incomplete and stalled response to, or had relapsed after, previous immunomodulatory drug-based induction. Patients received up to six 21-day cycles of bortezomib plus dexamethasone, with added liposomal doxorubicin for patients not achieving partial response or better by cycle 2 or very good partial response or better (≥VGPR) by cycle 4 (DoVeD), followed by bortezomib, high-dose cyclophosphamide, and filgrastim mobilization. Gene expression/signaling pathway analyses were conducted in purified CD34+ cells after bortezomib-based mobilization and compared against patients who received only filgrastim ± cyclophosphamide. Plasma samples were similarly analyzed for quantification of associated protein markers. The response rate to DoVeD relative to the pre-DoVeD baseline was 61%, including 39% ≥ VGPR. Deeper responses were achieved in 10 of 27 patients who received bortezomib-based mobilization; postmobilization response rate was 96%, including 48% ≥ VGPR, relative to the pre-DoVeD baseline. Median CD34+ cell yield was 23.2 × 10(6) cells/kg (median of 1 apheresis session). After a median follow-up of 46.6 months, median progression-free survival was 47.1 months from DoVeD initiation; 5-year overall survival rate was 76.4%. Grade ≥ 3 adverse events included thrombocytopenia (13%), hand-foot syndrome (11%), peripheral neuropathy (8%), and neutropenia (5%). Bortezomib-based mobilization was associated with modulated expression of genes involved in stem cell migration. Bortezomib-based secondary induction and mobilization could represent an alternative strategy for elimination of tumor burden in immunomodulatory drug-resistant patients that does not impact stem cell yield.

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

Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei

Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs havemore » been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.« less

Transcription and methylation analyses of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

PubMed Central

Gaillard, Coline; Tokuyasu, Taku A.; Rosen, Galit; Sotzen, Jason; Vitaliano-Prunier, Adeline; Roy, Ritu; Passegué, Emmanuelle; de Thé, Hugues; Figueroa, Maria E.; Kogan, Scott C.

2015-01-01

Acute promyelocytic leukemia is an aggressive malignancy characterized by the accumulation of promyelocytes in the bone marrow. PML/RARA is the primary abnormality implicated in this pathology, but the mechanisms by which this chimeric fusion protein initiates disease are incompletely understood. Identifying PML/RARA targets in vivo is critical for comprehending the road to pathogenesis. Utilizing a novel sorting strategy, we isolated highly purified promyelocyte populations from normal and young preleukemic animals, carried out microarray and methylation profiling analyses, and compared the results from the two groups of animals. Surprisingly, in the absence of secondary lesions, PML/RARA had an overall limited impact on both the transcriptome and methylome. Of interest, we did identify down-regulation of secondary and tertiary granule genes as the first step engaging the myeloid maturation block. Although initially not sufficient to arrest terminal granulopoiesis in vivo, such alterations set the stage for the later, complete differentiation block seen in leukemia. Further, gene set enrichment analysis revealed that PML/RARA promyelocytes exhibit a subtle increase in expression of cell cycle genes, and we show that this leads to both increased proliferation of these cells and expansion of the promyelocyte compartment. Importantly, this proliferation signature was absent from the poorly leukemogenic p50/RARA fusion model, implying a critical role for PML in the altered cell-cycle kinetics and ability to initiate leukemia. Thus, our findings challenge the predominant model in the field and we propose that PML/RARA initiates leukemia by subtly shifting cell fate decisions within the promyelocyte compartment. PMID:26088929

Metabolic Profiling in Association with Vascular Endothelial Cell Dysfunction Following Non-Toxic Cadmium Exposure

PubMed Central

Li, Xiaofei; Nong, Qingjiao; Mao, Baoyu; Pan, Xue

2017-01-01

This study aimed to determine the metabolic profile of non-toxic cadmium (Cd)-induced dysfunctional endothelial cells using human umbilical vein endothelial cells (HUVECs). HUVECs (n = 6 per group) were treated with 0, 1, 5, or 10 μM cadmium chloride (CdCl2) for 48 h. Cell phenotypes, including nitric oxide (NO) production, the inflammatory response, and oxidative stress, were evaluated in Cd-exposed and control HUVECs. Cd-exposed and control HUVECs were analysed using gas chromatography time-of-flight/mass spectrometry. Compared to control HUVECs, Cd-exposed HUVECs were dysfunctional, exhibiting decreased NO production, a proinflammatory state, and non-significant oxidative stress. Further metabolic profiling revealed 24 significantly-altered metabolites in the dysfunctional endothelial cells. The significantly-altered metabolites were involved in the impaired tricarboxylic acid (TCA) cycle, activated pyruvate metabolism, up-regulated glucogenic amino acid metabolism, and increased pyrimidine metabolism. The current metabolic findings further suggest that the metabolic changes linked to TCA cycle dysfunction, glycosylation of the hexosamine biosynthesis pathway (HBP), and compensatory responses to genomic instability and energy deficiency may be generally associated with dysfunctional phenotypes, characterized by decreased NO production, a proinflammatory state, and non-significant oxidative stress, in endothelial cells following non-toxic Cd exposure. PMID:28872622

Antitumor Activity of Ethanolic Extract of Dendrobium formosum in T-Cell Lymphoma: An In Vitro and In Vivo Study

PubMed Central

Prasad, Ritika; Koch, Biplob

2014-01-01

Dendrobium, a genus of orchid, was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. The study was aimed to evaluate the anticancer property of the ethanolic extract of Dendrobium formosum on Dalton's lymphoma. In vitro cytotoxicity was determined by MTT assay, apoptosis was determined by fluorescence microscopy, and cell cycle progression was analysed using flow cytometry; in vivo antitumor activity was performed in Dalton's lymphoma bearing mice. The IC50 value of ethanolic extract was obtained at 350 μg/mL in Dalton's lymphoma cells. Fluorescence microscopy analysis showed significant increase in apoptotic cell death in dose- and time-dependent manner which was further confirmed through the resulting DNA fragmentation. Further, flow cytometry analysis showed that the ethanolic extract arrests the cells in G2/M phase of the cell cycle. The in vivo anticancer activity study illustrates significant increase in the survival time of Dalton's lymphoma bearing mice on treatment with ethanolic extract when compared to control. These results substantiate the antitumor properties of ethanolic extract of Dendrobium formosum and suggest an alternative in treatment of cancer. Further studies are required regarding the isolation and characterization of bioactive components along with the analysis of molecular mechanism involved. PMID:24959588

Metformin directly acts on mitochondria to alter cellular bioenergetics

PubMed Central

2014-01-01

Background Metformin is widely used in the treatment of diabetes, and there is interest in ‘repurposing’ the drug for cancer prevention or treatment. However, the mechanism underlying the metabolic effects of metformin remains poorly understood. Methods We performed respirometry and stable isotope tracer analyses on cells and isolated mitochondria to investigate the impact of metformin on mitochondrial functions. Results We show that metformin decreases mitochondrial respiration, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, cells treated with metformin become energetically inefficient, and display increased aerobic glycolysis and reduced glucose metabolism through the citric acid cycle. Conflicting prior studies proposed mitochondrial complex I or various cytosolic targets for metformin action, but we show that the compound limits respiration and citric acid cycle activity in isolated mitochondria, indicating that at least for these effects, the mitochondrion is the primary target. Finally, we demonstrate that cancer cells exposed to metformin display a greater compensatory increase in aerobic glycolysis than nontransformed cells, highlighting their metabolic vulnerability. Prevention of this compensatory metabolic event in cancer cells significantly impairs survival. Conclusions Together, these results demonstrate that metformin directly acts on mitochondria to limit respiration and that the sensitivity of cells to metformin is dependent on their ability to cope with energetic stress. PMID:25184038

CD9 monoclonal antibody-conjugated PEGylated liposomes for targeted delivery of rapamycin in the treatment of cellular senescence

NASA Astrophysics Data System (ADS)

Thuy Nguyen, Hanh; Thapa, Raj Kumar; Shin, Beom Soo; Jeong, Jee-Heon; Kim, Jae-Ryong; Yong, Chul Soon; Kim, Jong Oh

2017-03-01

Premature cellular senescence refers to the state of irreversible cell cycle arrest due to DNA damage or other stresses. In this study, CD9 monoclonal antibody (CD9mAb) was successfully conjugated to the surface of PEGylated liposomes for targeted delivery of rapamycin (LR-CD9mAb) to overcome senescence of CD9 receptor-overexpressing cells. LR-CD9mAb has a small particle size (143.3 ± 2.4 nm), narrow size distribution (polydispersity index: 0.220 ± 0.036), and negative zeta potential (-14.6 ± 1.2 mV). The uptake of CD9-targeted liposomes by premature senescent human dermal fibroblasts (HDFs) was higher than that by young HDFs, as displayed by confocal microscopic images. The senescence might not be reversed by treatment with rapamycin; however, the drug promoted cell proliferation and reduced the number of cells that expressed the senescence-associated-β-galactosidase (SA-β-gal). These effects were further confirmed by cell viability, cell cycle, and Western blotting analyses. Moreover, CD9-targeted liposomes showed better anti-senescence activity, in comparison with free rapamycin or the conventional liposomal formulation, suggesting the potential application of this system in further in vivo studies.

The metastasis suppressor SOX11 is an independent prognostic factor for improved survival in gastric cancer

PubMed Central

QU, YING; ZHOU, CHENFEI; ZHANG, JIANIAN; CAI, QU; LI, JIANFANG; DU, TAO; ZHU, ZHENGGANG; CUI, XIAOJIANG; LIU, BINGYA

2014-01-01

SOX11 is involved in gastrulation and in malignant diseases. The aim of this study was to investigate the role of SOX11 in gastric cancer and its expression pattern and clinical significance. SOX11 overexpression cell model was used to examine in vitro and in vivo the role of SOX11 in cell growth and metastasis. Cell cycle analysis and Annexin V/PI double staining were used to investigate the effect of SOX11 on cell cycle progression and apoptosis. The expression of SOX11 in human gastric cancer was examined by immunohistochemistry. The correlation of SOX11 expression with clinicopathological characteristics and survival of patients was analyzed by Pearson’s χ2 and Kaplan-Meier analyses, respectively. Cox’s proportional hazard model was employed in multivariate analysis. SOX11 overexpression did not inhibit cell growth but strongly suppressed cell migration/invasion in vitro and in vivo. We found a significant correlation between high SOX11 protein levels and Lauren’s classification (intestinal type), differentiation status (high and medium), and early TNM stage. SOX11 is an independent prognostic factor for improved survival in gastric cancer patients. SOX11 was a potential tumor-suppressor and an independent positive prognostic factor in gastric cancer patients with less advanced clinicopathological features. PMID:24604109

Berberine displays antitumor activity in esophageal cancer cells in vitro.

PubMed

Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

2017-04-14

To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G 2 /M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

Berberine displays antitumor activity in esophageal cancer cells in vitro

PubMed Central

Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

2017-01-01

AIM To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients. PMID:28465635

Quantitative imaging with Fucci and mathematics to uncover temporal dynamics of cell cycle progression.

PubMed

Saitou, Takashi; Imamura, Takeshi

2016-01-01

Cell cycle progression is strictly coordinated to ensure proper tissue growth, development, and regeneration of multicellular organisms. Spatiotemporal visualization of cell cycle phases directly helps us to obtain a deeper understanding of controlled, multicellular, cell cycle progression. The fluorescent ubiquitination-based cell cycle indicator (Fucci) system allows us to monitor, in living cells, the G1 and the S/G2/M phases of the cell cycle in red and green fluorescent colors, respectively. Since the discovery of Fucci technology, it has found numerous applications in the characterization of the timing of cell cycle phase transitions under diverse conditions and various biological processes. However, due to the complexity of cell cycle dynamics, understanding of specific patterns of cell cycle progression is still far from complete. In order to tackle this issue, quantitative approaches combined with mathematical modeling seem to be essential. Here, we review several studies that attempted to integrate Fucci technology and mathematical models to obtain quantitative information regarding cell cycle regulatory patterns. Focusing on the technological development of utilizing mathematics to retrieve meaningful information from the Fucci producing data, we discuss how the combined methods advance a quantitative understanding of cell cycle regulation. © 2015 Japanese Society of Developmental Biologists.

Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.

PubMed

Coskun, Devrim; Britto, Dev T; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J

2013-12-01

Futile transmembrane NH3/NH4(+) cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4(+) toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4(+)) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope (13)N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4(+) ion. Influx of (13)NH3/(13)NH4(+), which exceeded 200 µmol g(-1) h(-1), was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4(+)), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g(-1) h(-1)). Efflux of (13)NH3/(13)NH4(+) responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.

Cell Cycle Control in the Early Embryonic Development of Aquatic Animal Species

PubMed Central

Siefert, Joseph C.; Clowdus, Emily A.; Sansam, Christopher L.

2016-01-01

The cell cycle is integrated with many aspects of embryonic development. Not only is proper control over the pace of cell proliferation important, but also the timing of cell cycle progression is coordinated with transcription, cell migration, and cell differentiation. Due to the ease with which the embryos of aquatic organisms can be observed and manipulated, they have been a popular choice for embryologists throughout history. In the cell cycle field, aquatic organisms have been extremely important because they have played a major role in the discovery and analysis of key regulators of the cell cycle. In particular, the frog Xenopus laevis has been instrumental for understanding how the basic embryonic cell cycle is regulated. More recently, the zebrafish has been used to understand how the cell cycle is remodeled during vertebrate development and how it is regulated during morphogenesis. This review describes how some of the unique strengths of aquatic species have been leveraged for cell cycle research and suggests how species such as Xenopus and zebrafish will continue to reveal the roles of the cell cycle in human biology and disease. PMID:26475527

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

PubMed

Soares, Júlia Ribeiro; José Tenório de Melo, Edésio; da Cunha, Maura; Fernandes, Kátia Valevski Sales; Taveira, Gabriel Bonan; da Silva Pereira, Lidia; Pimenta, Samy; Trindade, Fernanda Gomes; Regente, Mariana; Pinedo, Marcela; de la Canal, Laura; Gomes, Valdirene Moreira; de Oliveira Carvalho, André

2017-01-01

Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef 1 -Saccharomyces cerevisiae interaction. ApDef 1 -S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. ApDef 1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef 1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef 1 induced death. ApDef 1 -S. cerevisiae interaction resulted in membrane permeabilization, H 2 O 2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. ApDef 1 -S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef 1 -S. cerevisiae interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

Curcumin induces apoptosis in pancreatic cancer cells through the induction of forkhead box O1 and inhibition of the PI3K/Akt pathway.

PubMed

Zhao, Zhiming; Li, Chenggang; Xi, Hao; Gao, Yuanxing; Xu, Dabin

2015-10-01

Previous population investigations have suggested that the application of curcumin may be associated with decreased incidence and improved prognosis in certain types of cancer. Forkhead box O1 (FOXO1) has been implicated in the regulation of several biological processes, including stress resistance, metabolism, DNA repair, cell cycle and apoptosis. The aims of the present study were to investigate the effects and molecular mechanisms of curcumin on the induction of anti‑proliferation, cell cycle arrest and apoptosis, by FOXO1, in pancreatic cancer cells. The MTT assay and ELISA‑Brdu assay were used to assess cell proliferation. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to detect the expression of PCNA, Ki‑67, B‑cell lymphoma‑2 (Bcl‑2), B‑cell‑associated X protein (Bax), cyclin D1, p21, p27 and FOXO1. Cell apoptosis was detected using a Cell Death ELISA detection kit. A Caspase‑3/9 Fluorescent Assay kit was used to detect caspase activity. The findings revealed that curcumin significantly decreased cell proliferation, which was associated with increased expression of the p21/CIP1 and p27/KIP1 cyclin‑dependent kinase inhibitors, and inhibited expression of cyclin D1. In addition, curcumin induced apoptosis by decreasing the Bcl‑2/Bax protein ratio and increasing caspase‑9/3 activation in the pancreatic cancer cells. Using siRNA against FOXO1, and Akt inhibitor and activator, the present study confirmed that curcumin induced the expression of FOXO1 by inhibition of phosphoinositide 3‑kinase/Akt signaling, leading to cell cycle arrest and apoptosis. In conclusion, these findings offer support for a mechanism that may underlie the anti‑neoplastic effects of curcumin and justify further investigation to examine the potential roles for activators of FOXO1 in the prevention and treatment of pancreatic cancer.

New in vitro insights on a cell death pathway induced by magnolol and honokiol in aristolochic acid tubulotoxicity.

PubMed

Bunel, Valérian; Antoine, Marie-Hélène; Stévigny, Caroline; Nortier, Joëlle; Duez, Pierre

2016-01-01

Aristolochic acids (AA) are nephrotoxic agents found in Aristolochia species whose consumption leads to the onset of a progressive tubulointerstitial fibrosis. This AA-nephropathy was first reported during the Belgian outbreak of the 1990's in which more than a hundred patients consumed slimming pills containing an Aristolochia species and Magnolia officinalis. The patients developed an end-stage kidney disease requiring dialysis or transplantation. Magnolol and honokiol are bioactive compounds from M. officinalis known for their potent antioxidant activity. As they can alleviate oxidative stress, we investigated their respective effects on AA-mediated tubulotoxicity using HK-2 cells. Magnolol and honokiol were able to reduce the oxidative stress associated with AA-treatment. Cytotoxicity alleviation was further investigated and overall cell viability measurements unexpectedly revealed that both compounds worsened the survival of AA-treated cells. Flow cytometry analyses of annexin V/PI stained cells indicated that the lignans efficiently prevented AA-induced apoptosis; but favored necrosis. Microscopy observations highlighted extensive vacuolization; other types of cell death, including autophagy, paraptosis or accelerated senescence were excluded. Ki-67 index and cell cycle analysis indicated that both magnolol and honokiol inhibited proliferation by blocking the cell cycle at the G1 phase; they also prevented the AA-induced G2/M arrest. Copyright © 2015 Elsevier Ltd. All rights reserved.

Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells.

PubMed

Zeng, Huawei; Botnen, James H; Briske-Anderson, Mary

2010-01-01

The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer nutrients. Methylselenol has been hypothesized to be a critical Se metabolite for anticancer activity in vivo. In this study, we demonstrated that both DCA (75-300 micromol/l) and submicromolar methylselenol inhibited colon cancer cell proliferation by up to 64% and 63%, respectively. In addition, DCA and methylselenol each increased colon cancer cell apoptosis rate by up to twofold. Cell cycle analyses revealed that DCA induced an increase in only the G1 fraction with a concomitant drop in G2 and S-phase; in contrast, methylselenol led to an increase in the G1 and G2 fractions with a concomitant drop only in the S-phase. Although both DCA and methylselenol significantly promoted apoptosis and inhibited cell growth, examination of mitogen-activated protein kinase (MAPK) pathway activation showed that DCA, but not methylselenol, induced SAPK/JNK1/2, p38 MAPK, ERK1/2 activation. Thus, our data provide, for the first time, the molecular basis for opposite effects of methylselenol and DCA on colon tumorigenesis.

Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause.

PubMed

Shimizu, Yuta; Mukai, Ayumu; Goto, Shin G

2018-04-01

Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modelling cell cycle synchronisation in networks of coupled radial glial cells.

PubMed

Barrack, Duncan S; Thul, Rüdiger; Owen, Markus R

2015-07-21

Radial glial cells play a crucial role in the embryonic mammalian brain. Their proliferation is thought to be controlled, in part, by ATP mediated calcium signals. It has been hypothesised that these signals act to locally synchronise cell cycles, so that clusters of cells proliferate together, shedding daughter cells in uniform sheets. In this paper we investigate this cell cycle synchronisation by taking an ordinary differential equation model that couples the dynamics of intracellular calcium and the cell cycle and extend it to populations of cells coupled via extracellular ATP signals. Through bifurcation analysis we show that although ATP mediated calcium release can lead to cell cycle synchronisation, a number of other asynchronous oscillatory solutions including torus solutions dominate the parameter space and cell cycle synchronisation is far from guaranteed. Despite this, numerical results indicate that the transient and not the asymptotic behaviour of the system is important in accounting for cell cycle synchronisation. In particular, quiescent cells can be entrained on to the cell cycle via ATP mediated calcium signals initiated by a driving cell and crucially will cycle in near synchrony with the driving cell for the duration of neurogenesis. This behaviour is highly sensitive to the timing of ATP release, with release at the G1/S phase transition of the cell cycle far more likely to lead to near synchrony than release during mid G1 phase. This result, which suggests that ATP release timing is critical to radial glia cell cycle synchronisation, may help us to understand normal and pathological brain development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Unraveling Interfaces between Energy Metabolism and Cell Cycle in Plants.

PubMed

Siqueira, João Antonio; Hardoim, Pablo; Ferreira, Paulo C G; Nunes-Nesi, Adriano; Hemerly, Adriana S

2018-06-19

Oscillation in energy levels is widely variable in dividing and differentiated cells. To synchronize cell proliferation and energy fluctuations, cell cycle-related proteins have been implicated in the regulation of mitochondrial energy-generating pathways in yeasts and animals. Plants have chloroplasts and mitochondria, coordinating the cell energy flow. Recent findings suggest an integrated regulation of these organelles and the nuclear cell cycle. Furthermore, reports indicate a set of interactions between the cell cycle and energy metabolism, coordinating the turnover of proteins in plants. Here, we discuss how cell cycle-related proteins directly interact with energy metabolism-related proteins to modulate energy homeostasis and cell cycle progression. We provide interfaces between cell cycle and energy metabolism-related proteins that could be explored to maximize plant yield. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Cell cycle-related metabolism and mitochondrial dynamics in a replication-competent pancreatic beta-cell line.

PubMed

Montemurro, Chiara; Vadrevu, Suryakiran; Gurlo, Tatyana; Butler, Alexandra E; Vongbunyong, Kenny E; Petcherski, Anton; Shirihai, Orian S; Satin, Leslie S; Braas, Daniel; Butler, Peter C; Tudzarova, Slavica

2017-01-01

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca 2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca 2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca 2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca 2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.

Solar power satellites - Heat engine or solar cells

NASA Technical Reports Server (NTRS)

Oman, H.; Gregory, D. L.

1978-01-01

A solar power satellite is the energy-converting element of a system that can deliver some 10 GW of power to utilities on the earth's surface. We evaluated heat engines and solar cells for converting sunshine to electric power at the satellite. A potassium Rankine cycle was the best of the heat engines, and 50 microns thick single-crystal silicon cells were the best of the photovoltaic converters. Neither solar cells nor heat engines had a clear advantage when all factors were considered. The potassium-turbine power plant, however, was more difficult to assemble and required a more expensive orbital assembly base. We therefore based our cost analyses on solar-cell energy conversion, concluding that satellite-generated power could be delivered to utilities for around 4 to 5 cents a kWh.

A Short-Term Advantage for Syngamy in the Origin of Eukaryotic Sex: Effects of Cell Fusion on Cell Cycle Duration and Other Effects Related to the Duration of the Cell Cycle-Relationship between Cell Growth Curve and the Optimal Size of the Species, and Circadian Cell Cycle in Photosynthetic Unicellular Organisms.

PubMed

Mancebo Quintana, J M; Mancebo Quintana, S

2012-01-01

The origin of sex is becoming a vexatious issue for Evolutionary Biology. Numerous hypotheses have been proposed, based on the genetic effects of sex, on trophic effects or on the formation of cysts and syncytia. Our approach addresses the change in cell cycle duration which would cause cell fusion. Several results are obtained through graphical and mathematical analysis and computer simulations. (1) In poor environments, cell fusion would be an advantageous strategy, as fusion between cells of different size shortens the cycle of the smaller cell (relative to the asexual cycle), and the majority of mergers would occur between cells of different sizes. (2) The easiest-to-evolve regulation of cell proliferation (sexual/asexual) would be by modifying the checkpoints of the cell cycle. (3) A regulation of this kind would have required the existence of the G2 phase, and sex could thus be the cause of the appearance of this phase. Regarding cell cycle, (4) the exponential curve is the only cell growth curve that has no effect on the optimal cell size in unicellular species; (5) the existence of a plateau with no growth at the end of the cell cycle explains the circadian cell cycle observed in unicellular algae.

Transcription Factor Binding Profiles Reveal Cyclic Expression of Human Protein-coding Genes and Non-coding RNAs

PubMed Central

Cheng, Chao; Ung, Matthew; Grant, Gavin D.; Whitfield, Michael L.

2013-01-01

Cell cycle is a complex and highly supervised process that must proceed with regulatory precision to achieve successful cellular division. Despite the wide application, microarray time course experiments have several limitations in identifying cell cycle genes. We thus propose a computational model to predict human cell cycle genes based on transcription factor (TF) binding and regulatory motif information in their promoters. We utilize ENCODE ChIP-seq data and motif information as predictors to discriminate cell cycle against non-cell cycle genes. Our results show that both the trans- TF features and the cis- motif features are predictive of cell cycle genes, and a combination of the two types of features can further improve prediction accuracy. We apply our model to a complete list of GENCODE promoters to predict novel cell cycle driving promoters for both protein-coding genes and non-coding RNAs such as lincRNAs. We find that a similar percentage of lincRNAs are cell cycle regulated as protein-coding genes, suggesting the importance of non-coding RNAs in cell cycle division. The model we propose here provides not only a practical tool for identifying novel cell cycle genes with high accuracy, but also new insights on cell cycle regulation by TFs and cis-regulatory elements. PMID:23874175

Cycle life test and failure model of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Smithrick, J. J.

1983-01-01

Six ampere hour individual pressure vessel nickel hydrogen cells were charge/discharge cycled to failure. Failure as used here is defined to occur when the end of discharge voltage degraded to 0.9 volts. They were cycled under a low earth orbit cycle regime to a deep depth of discharge (80 percent of rated ampere hour capacity). Both cell designs were fabricated by the same manufacturer and represent current state of the art. A failure model was advanced which suggests both cell designs have inadequate volume tolerance characteristics. The limited existing data base at a deep depth of discharge (DOD) was expanded. Two cells of each design were cycled. One COMSAT cell failed at cycle 1712 and the other failed at cycle 1875. For the Air Force/Hughes cells, one cell failed at cycle 2250 and the other failed at cycle 2638. All cells, of both designs, failed due to low end of discharge voltage (0.9 volts). No cell failed due to electrical shorts. After cell failure, three different reconditioning tests (deep discharge, physical reorientation, and open circuit voltage stand) were conducted on all cells of each design. A fourth reconditioning test (electrolyte addition) was conducted on one cell of each design. In addition post cycle cell teardown and failure analysis were performed on the one cell of each design which did not have electrolyte added after failure.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

DOE PAGES

Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; ...

2014-10-10

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow onmore » urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.« less

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

Vissers, Daniel R.; Isheim, Dieter; Zhan, Chun

Lithium-ion batteries utilizing 5 V spinel material, LixMn1.5Ni0.5O4 have received considerable interest in recent years for their ability to deliver high energy and power densities. In this paper, we report an atomic scale analysis of the surface layer of a core–shell 5 V spinel structure where a small amount of the manganese lattice sites have been substituted with cobalt in the shell to reach a stoichiometry of LixMn1.18Ni0.55Co0.27O4. Our analyses include electrochemical analysis, atom probe tomography (APT) analysis, kinetic analysis of the interfacial reactions, and high resolution scanning transmission electron microscopy (HR-TEM) analysis. The APT analysis is performed on themore » material before and after long-term cycling at room temperature to provide insights into the atomic scale phenomena within the surface layer of the electrode material. Our APT data reveals a 25–30 nano-meter (nm) region which forms after cycling. From our analyses, we believe that the outer few nanometers of this region stabilizes the 5 V spinel within the chemical environment of the lithium-ion cell such that its structure is not compromised and thereby enables this material to cycle without significant capacity fading.« less

Cell Cycle Control by PTEN.

PubMed

Brandmaier, Andrew; Hou, Sheng-Qi; Shen, Wen H

2017-07-21

Continuous and error-free chromosome inheritance through the cell cycle is essential for genomic stability and tumor suppression. However, accumulation of aberrant genetic materials often causes the cell cycle to go awry, leading to malignant transformation. In response to genotoxic stress, cells employ diverse adaptive mechanisms to halt or exit the cell cycle temporarily or permanently. The intrinsic machinery of cycling, resting, and exiting shapes the cellular response to extrinsic stimuli, whereas prevalent disruption of the cell cycle machinery in tumor cells often confers resistance to anticancer therapy. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a guardian of the genome that is frequently mutated or deleted in human cancer. Moreover, it is increasingly evident that PTEN deficiency disrupts the fundamental processes of genetic transmission. Cells lacking PTEN exhibit cell cycle deregulation and cell fate reprogramming. Here, we review the role of PTEN in regulating the key processes in and out of cell cycle to optimize genomic integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrochemical activity of Fe-MIL-100 as a positive electrode for Na-ion batteries

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

Sava Gallis, Dorina F.; Pratt III, Harry D.; Anderson, Travis M.

2016-01-01

Here we investigate the electrochemical activity of metal-organic frameworks (MOFs) as positive electrodes for Na-ion batteries in coin cell configurations. The performance of Fe-MIL-100 material is highly dependent on the choice of sodium salt source, and electrolyte system. The overall capacity fades over many cycles, however the high Coulombic efficiency is maintained. This can be correlated with inaccessibility of active sites for Na intercalation, due to the increase of extra carbonaceous material inside the pores. High resolution synchrotron powder X-ray and pair distribution function analyses of the as-made and cycled electrodes reveal the structure maintains the long-range order with progressivemore » cycling. This finding suggests that careful consideration of all variables in battery components, and especially electrolyte selection can lead to greatly improved performances.« less

A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity

PubMed Central

Lasker, Keren; Ahrens, Daniel G.; Eckart, Michael R.

2017-01-01

ABSTRACT Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment. PMID:28246363

Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

PubMed

Nordin, Noraziah; Majid, Nazia Abdul; Hashim, Najihah Mohd; Rahman, Mashitoh Abd; Hassan, Zalila; Ali, Hapipah Mohd

2015-01-01

Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.

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.

Live-Cell Imaging of Filoviruses.

PubMed

Schudt, Gordian; Dolnik, Olga; Becker, Stephan

2017-01-01

Observation of molecular processes inside living cells is fundamental to a deeper understanding of virus-host interactions in filoviral-infected cells. These observations can provide spatiotemporal insights into protein synthesis, protein-protein interaction dynamics, and transport processes of these highly pathogenic viruses. Thus, live-cell imaging provides the possibility for antiviral screening in real time and gives mechanistic insights into understanding filovirus assembly steps that are dependent on cellular factors, which then represent potential targets against this highly fatal disease. Here we describe analysis of living filovirus-infected cells under maximum biosafety (i.e., BSL4) conditions using plasmid-driven expression of fluorescently labeled viral and cellular proteins and/or viral genome-encoded expression of fluorescently labeled proteins. Such multiple-color and multidimensional time-lapse live-cell imaging analyses are a powerful method to gain a better understanding of the filovirus infection cycle.

NASA Lewis advanced IPV nickel-hydrogen technology

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Britton, Doris L.

1993-01-01

Individual pressure vessel (IPV) nickel-hydrogen technology was advanced at NASA Lewis and under Lewis contracts. Some of the advancements are as follows: to use 26 percent potassium hydroxide electrolyte to improve cycle life and performance, to modify the state of the art cell design to eliminate identified failure modes and further improve cycle life, and to develop a lightweight nickel electrode to reduce battery mass, hence reduce launch and/or increase satellite payload. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen battery cells was reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 accelerated LEO cycles at 80 percent DOD compared to 3,500 cycles for cells containing 31 percent KOH. Results of the boiler plate cell tests have been validated at NWSC, Crane, Indiana. Forty-eight ampere-hour flight cells containing 26 and 31 percent KOH have undergone real time LEO cycle life testing at an 80 percent DOD, 10 C. The three cells containing 26 percent KOH failed on the average at cycle 19,500. The three cells containing 31 percent KOH failed on the average at cycle 6,400. Validation testing of NASA Lewis 125 Ah advanced design IPV nickel-hydrogen flight cells is also being conducted at NWSC, Crane, Indiana under a NASA Lewis contract. This consists of characterization, storage, and cycle life testing. There was no capacity degradation after 52 days of storage with the cells in the discharged state, on open circuit, 0 C, and a hydrogen pressure of 14.5 psia. The catalyzed wall wick cells have been cycled for over 22,694 cycles with no cell failures in the continuing test. All three of the non-catalyzed wall wick cells failed (cycles 9,588; 13,900; and 20,575). Cycle life test results of the Fibrex nickel electrode has demonstrated the feasibility of an improved nickel electrode giving a higher specific energy nickel-hydrogen cell. A nickel-hydrogen boiler plate cell using an 80 mil thick, 90 percent porous Fibrex nickel electrode has been cycled for 10,000 cycles at 40 percent DOD.

Effect of cycling on the lithium/electrolyte interface in organic electrolytes

NASA Technical Reports Server (NTRS)

Surampudi, S.; Shen, D. H.; Huang, C.-K.; Narayanan, S. R.; Attia, A.; Halpert, G.; Peled, E.

1993-01-01

Nondestructive methods such as ac impedance spectroscopy and microcalorimetry are used to study the effect of cell cycling on the lithium/electrolyte interface. The reactivity of both uncycled and cycled lithium towards various electrolytes is examined by measuring the heat evolved from the cells under open-circuit conditions at 25 C by microcalorimetry. Cycled cells at the end of charge/discharge exhibited considerably higher heat output compared with the uncycled cells. After 30 d of storage, the heat output of the cycled cells is similar to that of the uncycled cells. The cell internal resistance increases with cycling, and this is attributed to the degradation of the electrolyte with cycling.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome

PubMed Central

Waugh, Stacey; Kashon, Michael L.; Li, Shengqiao; Miller, Gerome R.; Johnson, Claud; Krajnak, Kristine

2016-01-01

Objective The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. Methods Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s2, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. Results Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. Conclusion Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division. PMID:27058473

Proteomic studies in zebrafish liver cells exposed to the brominated flame retardants HBCD and TBBPA.

PubMed

Kling, Peter; Förlin, Lars

2009-10-01

Proteomic effect screening in zebrafish liver cells was performed to generate hypotheses regarding single and mixed exposure to the BFRs HBCD and TBBPA. Responses at sublethal exposure were analysed by two-dimensional gel electrophoresis followed by MALDI-TOF and FT-ICR protein identification. Mixing of HBCD and TBBPA at sublethal doses of individual substances seemed to increase toxicity. Proteomic analyses revealed distinct exposure-specific and overlapping responses suggesting novel mechanisms with regard to HBCD and TBBPA exposure. While distinct HBCD responses were related to decreased protein metabolism, TBBPA revealed effects related to protein folding and NADPH production. Overlapping responses suggest increased gluconeogenesis (GAPDH and aldolase) while distinct mixture effects suggest a pronounced NADPH production and changes in proteins related to cell cycle control (prohibitin and crk-like oncogene). We conclude that mixtures containing HBCD and TBBPA may result in unexpected effects highlighting proteomics as a sensitive tool for detecting and hypothesis generation of mixture effects.

Antileukaemic effect of PI3K-mTOR inhibitors in acute myeloid leukaemia-gene expression profiles reveal CDC25B expression as determinate of pharmacological effect.

PubMed

Reikvam, Håkon; Tamburini, Jerome; Skrede, Silje; Holdhus, Rita; Poulain, Laury; Ersvaer, Elisabeth; Hatfield, Kimberley J; Bruserud, Øystein

2014-01-01

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC-0941, 3-methyladenine) were studied, and we investigated cytokine-dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K-mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K-mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K-Akt-mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K-Akt-mTOR pathway and mediate rapamycin resistance in experimental models. © 2013 John Wiley & Sons Ltd.

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

PubMed Central

Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

2016-01-01

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3–4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting. PMID:27189321

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.

PubMed

Ahn, Eunyong; Kumar, Praveen; Mukha, Dzmitry; Tzur, Amit; Shlomi, Tomer

2017-11-06

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Playing with the cell cycle to build the spinal cord.

PubMed

Molina, Angie; Pituello, Fabienne

2017-12-01

A fundamental issue in nervous system development and homeostasis is to understand the mechanisms governing the balance between the maintenance of proliferating progenitors versus their differentiation into post-mitotic neurons. Accumulating data suggest that the cell cycle and core regulators of the cell cycle machinery play a major role in regulating this fine balance. Here, we focus on the interplay between the cell cycle and cellular and molecular events governing spinal cord development. We describe the existing links between the cell cycle and interkinetic nuclear migration (INM). We show how the different morphogens patterning the neural tube also regulate the cell cycle machinery to coordinate proliferation and patterning. We give examples of how cell cycle core regulators regulate transcriptionally, or post-transcriptionally, genes involved in controlling the maintenance versus the differentiation of neural progenitors. Finally, we describe the changes in cell cycle kinetics occurring during neural tube patterning and at the time of neuronal differentiation, and we discuss future research directions to better understand the role of the cell cycle in cell fate decisions. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model

PubMed Central

Sokhi, Upneet K.; Bacolod, Manny D.; Dasgupta, Santanu; Emdad, Luni; Das, Swadesh K.; Dumur, Catherine I.; Miles, Michael F.; Sarkar, Devanand; Fisher, Paul B.

2013-01-01

Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes. PMID:24143183

Genetic bases of fungal white rot wood decay predicted by phylogenomic analysis of correlated gene-phenotype evolution

Treesearch

László G. Nagy; Robert Riley; Philip J. Bergmann; Krisztina Krizsán; Francis M. Martin; Igor V. Grigoriev; Dan Cullen; David S. Hibbett

2016-01-01

Fungal decomposition of plant cell walls (PCW) is a complex process that has diverse industrial applications and huge impacts on the carbon cycle. White rot (WR) is a powerful mode of PCW decay in which lignin and carbohydrates are both degraded. Mechanistic studies of decay coupled with comparative genomic analyses have provided clues to the enzymatic components of WR...

Integrative Genomic Analyses Yields Cell Cycle Regulatory Programs with Prognostic Value

PubMed Central

Cheng, Chao; Lou, Shaoke; Andrews, Erik H.; Ung, Matthew H.; Varn, Frederick S.

2016-01-01

Liposarcoma is the second most common form of sarcoma, which has been categorized into four molecular subtypes, which are associated with differential prognosis of patients. However, the transcriptional regulatory programs associated with distinct histological and molecular subtypes of liposarcoma have not been investigated. This study uses integrative analyses to systematically define the transcriptional regulatory programs associated with liposarcoma. Likewise, computational methods are used to identify regulatory programs associated with different liposarcoma subtypes as well as programs that are predictive of prognosis. Further analysis of curated gene sets was used to identify prognostic gene signatures. The integration of data from a variety sources including gene expression profiles, transcription factor (TF) binding data from ChIP-seq experiments, curated gene sets, and clinical information of patients indicated discrete regulatory programs (e.g., controlled by E2F1 and E2F4) with significantly different regulatory activity in one or multiple subtypes of liposarcoma with respect to normal adipose tissue. These programs were also shown to be prognostic, wherein liposarcoma patients with higher E2F4 or E2F1 activity associated with unfavorable prognosis. A total of 259 gene sets were significantly associated with patient survival in liposarcoma, among which >50% are involved in cell cycle and proliferation. PMID:26856934

[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

PubMed Central

Park, Yon Jung; Wen, Jing; Bang, Seungmin; Park, Seung Woo

2006-01-01

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild-type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53-expressing cells by inducing temporal growth arrest. PMID:17066513

Interferon-inducible effector mechanisms in cell-autonomous immunity

PubMed Central

MacMicking, John D.

2014-01-01

Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells — a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world. PMID:22531325

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.

Expression of the nifH Gene of a Herbaspirillum Endophyte in Wild Rice Species: Daily Rhythm during the Light-Dark Cycle

PubMed Central

You, Mu; Nishiguchi, Tomohiro; Saito, Asami; Isawa, Tsuyoshi; Mitsui, Hisayuki; Minamisawa, Kiwamu

2005-01-01

The expression of nitrogenase genes of Herbaspirillum sp. B501 associated in shoot (leaf and stem) of wild rice, Oryza officinalis, was studied by means of reverse transcription-PCR (RT-PCR) targeted at the nifH gene. RT-PCR analyses indicate that nifH transcript was detected exclusively from nitrogen-fixing cells of gfp-tagged strain B501gfp1 in both free-living and endophytic states by using a constitutive gfp gene transcript as a positive control. Transcription of nifH and nitrogen fixation in free-living cells were induced maximally at a 2% O2 concentration and repressed in free air (21% O2). nifH transcription was monitored in the endophytic cells by using total RNA extracted from B501gfp1-inoculated wild rice plants during daily light-dark cycles. The level of nifH transcription in planta varied dramatically, with a maximum during the light period. Moreover, the light radiation enhanced nifH expression even in free-living cells grown in culture. These results suggest that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment. PMID:16332801

Cell cycle nucleic acids, polypeptides and uses thereof

DOEpatents

Gordon-Kamm, William J [Urbandale, IA; Lowe, Keith S [Johnston, IA; Larkins, Brian A [Tucson, AZ; Dilkes, Brian R [Tucson, AZ; Sun, Yuejin [Westfield, IN

2007-08-14

The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.

Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification.

PubMed

Zerjatke, Thomas; Gak, Igor A; Kirova, Dilyana; Fuhrmann, Markus; Daniel, Katrin; Gonciarz, Magdalena; Müller, Doris; Glauche, Ingmar; Mansfeld, Jörg

2017-05-30

Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations. Copyright © 2017 The Author(s). Published by Elsevier Inc. 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.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells. An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent potassium hydroxide (KOH) electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen cells is reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells - An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent potassium hydroxide (KOH) electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen cells is reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH.

Effect of LEO cycling on 125 Ah advanced design IPV nickel-hydrogen flight cells - An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent potassium hydroxide (KOH) electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen cells is reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH.

Exploring the Underlying Mechanisms of the Xenopus laevis Embryonic Cell Cycle.

PubMed

Zhang, Kun; Wang, Jin

2018-05-31

The cell cycle is an indispensable process in proliferation and development. Despite significant efforts, global quantification and physical understanding are still challenging. In this study, we explored the mechanisms of the Xenopus laevis embryonic cell cycle by quantifying the underlying landscape and flux. We uncovered the Mexican hat landscape of the Xenopus laevis embryonic cell cycle with several local basins and barriers on the oscillation path. The local basins characterize the different phases of the Xenopus laevis embryonic cell cycle, and the local barriers represent the checkpoints. The checkpoint mechanism of the cell cycle is revealed by the landscape basins and barriers. While landscape shape determines the stabilities of the states on the oscillation path, the curl flux force determines the stability of the cell cycle flow. Replication is fundamental for biology of living cells. We quantify the input energy (through the entropy production) as the thermodynamic requirement for initiation and sustainability of single cell life (cell cycle). Furthermore, we also quantify curl flux originated from the input energy as the dynamical requirement for the emergence of a new stable phase (cell cycle). This can provide a new quantitative insight for the origin of single cell life. In fact, the curl flux originated from the energy input or nutrition supply determines the speed and guarantees the progression of the cell cycle. The speed of the cell cycle is a hallmark of cancer. We characterized the quality of the cell cycle by the coherence time and found it is supported by the flux and energy cost. We are also able to quantify the degree of time irreversibility by the cross correlation function forward and backward in time from the stochastic traces in the simulation or experiments, providing a way for the quantification of the time irreversibility and the flux. Through global sensitivity analysis upon landscape and flux, we can identify the key elements for controlling the cell cycle speed. This can help to design an effective strategy for drug discovery against cancer.

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

PubMed Central

Arzumanyan, Alla; Kulathinal, Rob J.; Blain, Stacy W.; Holcombe, Randall F.; Mahajna, Jamal; Marino, Maria; Martinez-Chantar, Maria L.; Nawroth, Roman; Sanchez-Garcia, Isidro; Sharma, Dipali; Saxena, Neeraj K.; Singh, Neetu; Vlachostergios, Panagiotis J.; Guo, Shanchun; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Niccolai, Elena; Amin, Amr; Ashraf, S. Salman; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Aquilano, Katia; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Nowsheen, Somaira

2016-01-01

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). This data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression. PMID:25892662

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells-update 2

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in low earth orbit (LEO) cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40 000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. This test was conducted at Hughes Aircraft Company under a NASA Lewis contract. The purpose was to investigate the effect of KOH concentration on cycle life. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The boiler plate test results are in the process of being validated using flight hardware and real time LEO test at the Naval Weapons Support Center (NWSC), Crane, Indiana under a NASA Lewis Contract. Six 48 Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16600 cycles during the continuing test.

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.

Presence of nanosilica (E551) in commercial food products: TNF-mediated oxidative stress and altered cell cycle progression in human lung fibroblast cells.

PubMed

Athinarayanan, Jegan; Periasamy, Vaiyapuri Subbarayan; Alsaif, Mohammed A; Al-Warthan, Abdulrahman A; Alshatwi, Ali A

2014-04-01

Silica (E551) is commonly used as an anti-caking agent in food products. The morphology and the dimension of the added silica particles are not, however, usually stated on the food product label. The food industry has adapted nanotechnology using engineered nanoparticles to improve the quality of their products. However, there has been increased debate regarding the health and safety concerns related to the use of engineered nanoparticles in consumer products. In this study, we investigated the morphology and dimensions of silica (E551) particles in food. The silica content of commercial food products was determined using inductively coupled plasma optical emission spectrometry. The result indicates that 2.74-14. 45 μg/g silica was found in commercial food products; however, the daily dietary intake in increase causes adverse effects on human health. E551 was isolated from food products and the morphology, particle size, crystalline nature, and purity of the silica particles were analyzed using XRD, FTIR, TEM, EDX and DLS. The results of these analyses confirmed the presence of spherical silica nanoparticles (of amorphous nature) in food, approximately 10-50 nm in size. The effects of E551 on human lung fibroblast cell viability, intracellular ROS levels, cell cycle phase, and the expression levels of metabolic stress-responsive genes (CAT, GSTA4, TNF, CYP1A, POR, SOD1, GSTM3, GPX1, and GSR1) were studied. The results suggest that E551 induces a dose-dependent cytotoxicity and changes in ROS levels and alters the gene expression and cell cycle. Treatment with a high concentration of E551 caused significant cytotoxic effects on WI-38 cells. These findings have implications for the use of these nanoparticles in the food industry.

The adverse effects of high fat induced obesity on female reproductive cycle and hormones

NASA Astrophysics Data System (ADS)

Donthireddy, Laxminarasimha Reddy

The prevalence of obesity, an established risk and progression factor for abnormal reproductive cycle and tissue damage in female mice. It leads to earlier puberty, menarche in young females and infertility. There are extensive range of consequences of obesity which includes type-2 diabetes, cardiovascular disease and insulin resistance. Obesity is the interaction between dietary intake, genes, life style and environment. The interplay of hormones estrogen, insulin, and leptin is well known on energy homeostasis and reproduction. The aim of this study is to determine the effect of high fat induced obesity on reproductive cycles and its hormonal abnormalities on mice model. Two week, 3 month and 8 month long normal (WT) and very high fat diet (VHFD) diet course is followed. When mice are fed with very high fat diet, there is a drastic increase in weight within the first week later. There was a significant (p<0.001) increase in leptin levels in 6 month VHFD treated animals. 2 week, 3 month and 6 month time interval pap smear test results showed number of cells, length of estrous cycle and phases of the estrous cycle changes with VHFD mice(n=30) compared to normal diet mice(n=10). These results also indicate that the changes in the reproductive cycles in VHFD treated female mice could be due to the changes in hormones. Histo-pathological analyses of kidney, ovary, liver, pancreas, heart and lungs showed remarkable changes in some tissue on exposure to very high fat. Highly deposited fat packets observed surrounding the hepatocytes and nerve cells.

Fluctuations of the transcription factor ATML1 generate the pattern of giant cells in the Arabidopsis sepal

PubMed Central

Meyer, Heather M; Teles, José; Formosa-Jordan, Pau; Refahi, Yassin; San-Bento, Rita; Ingram, Gwyneth; Jönsson, Henrik; Locke, James C W; Roeder, Adrienne H K

2017-01-01

Multicellular development produces patterns of specialized cell types. Yet, it is often unclear how individual cells within a field of identical cells initiate the patterning process. Using live imaging, quantitative image analyses and modeling, we show that during Arabidopsis thaliana sepal development, fluctuations in the concentration of the transcription factor ATML1 pattern a field of identical epidermal cells to differentiate into giant cells interspersed between smaller cells. We find that ATML1 is expressed in all epidermal cells. However, its level fluctuates in each of these cells. If ATML1 levels surpass a threshold during the G2 phase of the cell cycle, the cell will likely enter a state of endoreduplication and become giant. Otherwise, the cell divides. Our results demonstrate a fluctuation-driven patterning mechanism for how cell fate decisions can be initiated through a random yet tightly regulated process. DOI: http://dx.doi.org/10.7554/eLife.19131.001 PMID:28145865

The cell cycle of early mammalian embryos: lessons from genetic mouse models.

PubMed

Artus, Jérôme; Babinet, Charles; Cohen-Tannoudji, Michel

2006-03-01

Genes coding for cell cycle components predicted to be essential for its regulation have been shown to be dispensable in mice, at the whole organism level. Such studies have highlighted the extraordinary plasticity of the embryonic cell cycle and suggest that many aspects of in vivo cell cycle regulation remain to be discovered. Here, we discuss the particularities of the mouse early embryonic cell cycle and review the mutations that result in cell cycle defects during mouse early embryogenesis, including deficiencies for genes of the cyclin family (cyclin A2 and B1), genes involved in cell cycle checkpoints (Mad2, Bub3, Chk1, Atr), genes involved in ubiquitin and ubiquitin-like pathways (Uba3, Ubc9, Cul1, Cul3, Apc2, Apc10, Csn2) as well as genes the function of which had not been previously ascribed to cell cycle regulation (Cdc2P1, E4F and Omcg1).

Building Maintenance and Repair Data for Life-Cycle Cost Analyses: Electrical Systems.

DTIC Science & Technology

1991-05-01

Repair Data for Life-Cycle Cost Analyses: Electrical Systems by Edgar S. Neely Robert D. Neathammer James R. Stirn Robert P. Winkler This research...systems have been developed to assist planners in preparing DD Form 1391 documentation, designers in life-cycle cost component selection, and maintainers...Maintenance and Repair Data for Life-Cycle Cost Analyses: RDTE dated 1980 Electrical Systems REIMB 1984 - 1989 6. AUTH4OR(S) Edgar S. Neely, Robert D

[Flowcytometry DNA analysis of oral and maxillofacial non-Hodgkin's lymphoma].

PubMed

Ma, Li; He, Zhixiu; Wu, Lanyan; Cai, Yixin; Huang, Hechang; Lei, Song

2002-06-01

The purpose of this study was to investigate the relationship between the results of flowcytometry analyses of different clinical stage, location, pathologic grade and cell origin of oral and maxillofacial non-Hodgkin's lymphoma (NHL), and the diagnostic value of flowcytometry analysis in lymphoma. This study analyzed 50 oral and maxillofacial NHL cases and 10 reactive lymph nodes (formalin fixed and paraffin embedded) by flowcytometry (FCM). Reactive lymph nodes were all diploid. The diploid rate of NHL was 54%, and aneuploidy rate was 46%. There was statistically significant difference between reactive lymph nodes and NHL in the DNA ploidy status and cell cycle data (SPF, CV, S + G2/M, DI). The S phase fraction (SPF) and S + G2/M had close relationship with the grade of NHL. SPF value and DNA ploidy status had no obvious relationship with the prognosis. The results suggested that the FCM had diagnostic value in NHL, especially when the morphological diagnosis was difficult. Although the cell cycle data had no prognostic value, SPF and SPF + G2/M can show the proliferative status of NHL, which can help clinical doctor select therapeutic method.

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

PubMed

Yau, Richard G; Doerner, Kerstin; Castellanos, Erick R; Haakonsen, Diane L; Werner, Achim; Wang, Nan; Yang, X William; Martinez-Martin, Nadia; Matsumoto, Marissa L; Dixit, Vishva M; Rape, Michael

2017-11-02

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information. Copyright © 2017 Elsevier Inc. All rights reserved.

Model-Based Analysis of Cell Cycle Responses to Dynamically Changing Environments

PubMed Central

Seaton, Daniel D; Krishnan, J

2016-01-01

Cell cycle progression is carefully coordinated with a cell’s intra- and extracellular environment. While some pathways have been identified that communicate information from the environment to the cell cycle, a systematic understanding of how this information is dynamically processed is lacking. We address this by performing dynamic sensitivity analysis of three mathematical models of the cell cycle in Saccharomyces cerevisiae. We demonstrate that these models make broadly consistent qualitative predictions about cell cycle progression under dynamically changing conditions. For example, it is shown that the models predict anticorrelated changes in cell size and cell cycle duration under different environments independently of the growth rate. This prediction is validated by comparison to available literature data. Other consistent patterns emerge, such as widespread nonmonotonic changes in cell size down generations in response to parameter changes. We extend our analysis by investigating glucose signalling to the cell cycle, showing that known regulation of Cln3 translation and Cln1,2 transcription by glucose is sufficient to explain the experimentally observed changes in cell cycle dynamics at different glucose concentrations. Together, these results provide a framework for understanding the complex responses the cell cycle is capable of producing in response to dynamic environments. PMID:26741131

A dual-color marker system for in vivo visualization of cell cycle progression in Arabidopsis.

PubMed

Yin, Ke; Ueda, Minako; Takagi, Hitomi; Kajihara, Takehiro; Sugamata Aki, Shiori; Nobusawa, Takashi; Umeda-Hara, Chikage; Umeda, Masaaki

2014-11-01

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M-specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S-phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy-terminal region is responsible for proteasome-mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S-specific promoter of a histone 3.1-type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M-specific CYCB1-GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time-lapse imaging of cell cycle progression. The resultant dual-color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

A Short-Term Advantage for Syngamy in the Origin of Eukaryotic Sex: Effects of Cell Fusion on Cell Cycle Duration and Other Effects Related to the Duration of the Cell Cycle—Relationship between Cell Growth Curve and the Optimal Size of the Species, and Circadian Cell Cycle in Photosynthetic Unicellular Organisms

PubMed Central

Mancebo Quintana, J. M.; Mancebo Quintana, S.

2012-01-01

The origin of sex is becoming a vexatious issue for Evolutionary Biology. Numerous hypotheses have been proposed, based on the genetic effects of sex, on trophic effects or on the formation of cysts and syncytia. Our approach addresses the change in cell cycle duration which would cause cell fusion. Several results are obtained through graphical and mathematical analysis and computer simulations. (1) In poor environments, cell fusion would be an advantageous strategy, as fusion between cells of different size shortens the cycle of the smaller cell (relative to the asexual cycle), and the majority of mergers would occur between cells of different sizes. (2) The easiest-to-evolve regulation of cell proliferation (sexual/asexual) would be by modifying the checkpoints of the cell cycle. (3) A regulation of this kind would have required the existence of the G2 phase, and sex could thus be the cause of the appearance of this phase. Regarding cell cycle, (4) the exponential curve is the only cell growth curve that has no effect on the optimal cell size in unicellular species; (5) the existence of a plateau with no growth at the end of the cell cycle explains the circadian cell cycle observed in unicellular algae. PMID:22666626

Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and novel therapeutic target in acute myeloid leukemia

PubMed Central

Porter, Christopher C.; Kim, Jihye; Fosmire, Susan; Gearheart, Christy M.; van Linden, Annemie; Baturin, Dmitry; Zaberezhnyy, Vadym; Patel, Purvi R.; Gao, Dexiang; Tan, Aik Choon; DeGregori, James

2011-01-01

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide shRNA screens to identify proteins that mediate AML cell fate after cytarabine exposure, gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context, and examination of existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. PMID:22289989

Cell Cycle Regulation of Stem Cells by MicroRNAs.

PubMed

Mens, Michelle M J; Ghanbari, Mohsen

2018-06-01

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

A Module Experimental Process System Development Unit (MEPSDU)

NASA Technical Reports Server (NTRS)

1981-01-01

Subsequent to the design review, a series of tests was conducted on simulated modules to demonstrate that all environmental specifications (wind loading, hailstone impact, thermal cycling, and humidity cycling) are satisfied by the design. All tests, except hailstone impact, were successfully completed. The assembly sequence was simplified by virtue of eliminating the frame components and assembly steps. Performance was improved by reducing the module edge border required to accommodate the frame of the preliminary design module. An ultrasonic rolling spot bonding technique was selected for use in the machine to perform the aluminum interconnect to cell metallization electrical joints required in the MEPSDU module configuration. This selection was based on extensive experimental tests and economic analyses.

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

Scratch2 prevents cell cycle re-entry by repressing miR-25 in postmitotic primary neurons.

PubMed

Rodríguez-Aznar, Eva; Barrallo-Gimeno, Alejandro; Nieto, M Angela

2013-03-20

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis

PubMed Central

2012-01-01

Background Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. Methods siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. Results The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. Conclusions In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines. PMID:22257483

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis.

PubMed

Kapp, Friedrich G; Sommer, Anette; Kiefer, Thomas; Dölken, Gottfried; Haendler, Bernard

2012-01-18

Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines.

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

PubMed Central

Żabka, Aneta; Polit, Justyna Teresa; Maszewski, Janusz

2012-01-01

Background and Aims Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. Methods Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H2O2 production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). Key Results Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H2O2, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. Conclusions The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus. PMID:23087128

An extensive program of periodic alternative splicing linked to cell cycle progression

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Weatheritt, Robert; Wang, Yang; Blencowe, Benjamin J; Wang, Zefeng

2016-01-01

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control. DOI: http://dx.doi.org/10.7554/eLife.10288.001 PMID:27015110

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo.

PubMed

Zeng, Yuanyuan; Zhu, Jianjie; Shen, Dan; Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-An

2017-05-09

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3'-untranslated region (3'-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3'-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment.

MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo

PubMed Central

Qin, Hualong; Lei, Zhe; Li, Wei; Liu, Zeyi; Huang, Jian-an

2017-01-01

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3′-untranslated region (3′-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3′-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment. PMID:28199217

Metabolomic analysis of the green microalga Chlamydomonas reinhardtii cultivated under day/night conditions.

PubMed

Willamme, Rémi; Alsafra, Zouheir; Arumugam, Rameshkumar; Eppe, Gauthier; Remacle, Françoise; Levine, R D; Remacle, Claire

2015-12-10

Biomass composition of Chlamydomonas reinhardtii was studied during two consecutive cycles of 12h light/12h dark. As in our experimental conditions the two synchronized divisions were separated by 20h, it was possible to show that accumulation of dry weight, proteins, chlorophyll and fatty acids mainly depends on cell division, whereas starch accumulation depends on a circadian rhythm as reported previously. Our metabolomics analyses also revealed that accumulation of five (Ser, Val, Leu, Ile and Thr) of the nine free amino acids detected displayed rhythmicity, depending on cell division while Glu was 20-50 times more abundant than the other ones probably because this free amino acid serves not only for protein synthesis but also for biosynthesis of nitrogen compounds. In addition, we performed a thermodynamic-motivated theoretical approach known as 'surprisal analysis'. The results from this analysis showed that cells were close to a steady state all along the 48h of the experiment. In addition, calculation of free energy of cellular metabolites showed that the transition point, i.e. the state which immediately precedes cell division, corresponds to the most unstable stage of the cell cycle and that division is identified as the greatest drop in the free energy of metabolites. Copyright © 2015 Elsevier B.V. All rights reserved.

Prevalence of Circulating Tumor Cells After Adjuvant Chemotherapy With or Without Anthracyclines in Patients With HER2-negative, Hormone Receptor-positive Early Breast Cancer.

PubMed

Schramm, Amelie; Schochter, Fabienne; Friedl, Thomas W P; de Gregorio, Nikolaus; Andergassen, Ulrich; Alunni-Fabbroni, Marianna; Trapp, Elisabeth; Jaeger, Bernadette; Heinrich, Georg; Camara, Oumar; Decker, Thomas; Ober, Angelika; Mahner, Sven; Fehm, Tanja N; Pantel, Klaus; Fasching, Peter A; Schneeweiss, Andreas; Janni, Wolfgang; Rack, Brigitte K

2017-07-01

Use of anthracycline-based chemotherapy in patients with early breast cancer (EBC) has been well-established but is often associated with cardiotoxicity. Based on data suggesting a limited benefit of anthracyclines in human epidermal growth factor receptor 2 (HER2)-negative patients, the Simultaneous Study of Docetaxel Based Anthracycline Free Adjuvant Treatment Evaluation, as well as Life Style Intervention Strategies (SUCCESS) C study randomized patients to either anthracycline-containing or anthracycline-free chemotherapy. Given the proven prognostic value of circulating tumor cells (CTCs) in EBC, we compared the prevalence of CTCs after chemotherapy between both treatment arms for a preliminary efficacy assessment. The SUCCESS C trial (NCT00847444) is an open-label, phase III study randomizing 3547 patients with HER2-negative EBC to either 3 cycles of epirubicin, 5-fluorouracil, and cyclophosphamide followed by 3 cycles of docetaxel (FEC-DOC) or 6 cycles of docetaxel and cyclophosphamide (DOC-C). CTC status was prospectively evaluated in hormone receptor-positive patients at the time of last chemotherapy cycle using the US Food and Drug Administration-approved CellSearch System (Janssen Diagnostics). Data on CTC status were available for 1766 patients. Overall, CTCs were found in 221 (12.5%) patients. Univariate analyses revealed that presence of CTCs at time of last chemotherapy cycle was not significantly associated with tumor or patient characteristics (all P > .1). There was no significant difference with respect to presence of CTCs between patients randomized to FEC-DOC or DOC-C (11.5% vs. 13.6%; P = .18). The comparable prevalence of CTCs at the time of last chemotherapy cycle may indicate that anthracycline-free chemotherapy is equally effective to anthracycline-containing chemotherapy in HER2-negative, hormone receptor-positive EBC. However, efficacy data from the final survival analysis of SUCCESS C have to be awaited to confirm these preliminary findings. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of growth of tetraploid nuclei in roots of Vicia faba.

PubMed

Bansal, J; Davidson, D

1978-03-01

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.

Flow cytometry analysis of cell cycle and specific cell synchronization with butyrate

USDA-ARS?s Scientific Manuscript database

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. The possibility of using butyrate-blocked cells to obtain synchronized cells was explored and the properties of butyrate-induced cell ...

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

A breakthrough in low earth orbit (LEO) cycle life of individual pressure vessel (IPV) nickel hydrogen battery cells was reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. The effect of KOH concentration on cycle life was studied. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min charge (2 x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The next step is to validate these results using flight hardware and a real time LEO test. NASA Lewis has a contract with the Naval Weapons Support Center (NWSC), Crane, Indiana, to validate the boiler plate test results. Six 48 A-hr Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells) and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The cells were cycled for over 8000 cycles in the continuing test. There were no failures for the cells containing 26 percent KOH. There was two failures, however, for the cells containing 31 percent KOH.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

A breakthrough in the low-earth-orbit (LEO) cycle life of individual pressure vessel (IPV) nickel hydrogen battery cells is reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. The effect of KOH concentration on cycle life was studied. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min charge (2 x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The next step is to validate these results using flight hardware and real time LEO test. NASA Lewis has a contract with the Naval Weapons Support Center (NWSC), Crane, Indiana to validate the boiler plate test results. Six 48 A-hr Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells) and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The cells were cycled for over 8000 cycles in the continuing test. There were no failures for the cells containing 26 percent KOH. There were two failures, however, for the cells containing 31 percent KOH.

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

PubMed Central

Cooper, James Ross; Abdullatif, Muhammad Bilal; Burnett, Edward C.; Kempsell, Karen E.; Conforti, Franco; Tolley, Howard; Collins, Jane E.; Davies, Donna E.

2016-01-01

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. PMID:27792742

Elevated lung cancer risk is associated with deficiencies in cell cycle checkpoints: Genotype and phenotype analyses from a case-control study

PubMed Central

Zheng, Yun-Ling; Kosti, Ourania; Loffredo, Christopher; Bowman, Elise; Mechanic, Leah; Perlmutter, Donna; Jones, Raymond; Shields, Peter G.; Harris, Curtis

2010-01-01

Cell cycle checkpoints play critical roles in the maintenance of genomic integrity and inactivation of checkpoint genes, and are frequently perturbed in most cancers. In a case-control study of 299 non-small cell lung cancer cases and 550 controls in Maryland, we investigated the association between γ-radiation-induced G2/M arrest in cultured blood lymphocytes and lung cancer risk, and examined genotype-phenotype correlations between genetic polymorphisms of 20 genes involving in DNA repair and cell cycle control and γ-radiation-induced G2/M arrest. The study was specifically designed to examine race and gender differences in risk factors. Our data indicated that a less efficient DNA damage-induced G2/M checkpoint was associated with an increased risk of lung cancer in African American women with an adjusted odds ratio (OR) of 2.63 (95% CI = 1.01 – 7.26); there were no statistically significant associations for Caucasians, or African American men. When the African American women were categorized into quartiles, a significant reverse trend of decreased G2/M checkpoint function and increased lung cancer risk was present, with lowest-vs-highest quartile OR of 13.72 (95% CI = 2.30 – 81.92, Ptrend < 0.01). Genotype-phenotype correlation analysis indicated that polymorphisms in ATM, CDC25C, CDKN1A, BRCA2, ERCC6, TP53, and TP53BP1 genes were significantly associated with the γ-radiation-induced G2/M arrest phenotype. This study provides evidence that a less efficient G2/M checkpoint is significantly associated with lung cancer risk in African American women. The data also suggested that the function of G2/M checkpoint is modulated by genetic polymorphisms in genes involved in DNA repair and cell cycle control. PMID:19626602

A new cell culture model to genetically dissect the complete human papillomavirus life cycle.

PubMed

Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Myers, Julia E; Keiffer, Timothy R; DiGiuseppe, Stephen; Polk, Paula; Bodily, Jason M; Scott, Rona S; Sapp, Martin

2018-03-01

Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of γH2AX- and NBS1-positive repair foci.

PubMed

Suchánková, Jana; Kozubek, Stanislav; Legartová, Soňa; Sehnalová, Petra; Küntziger, Thomas; Bártová, Eva

2015-12-01

The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most striking protein present not only at DNA lesions, but also spreading out in their vicinity. Our conclusions highlight the significant role of the spatiotemporal dynamics of DNA repair-related proteins and their specific assembly/disassembly at DNA lesions, which can be cell type- and cell cycle dependent. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

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.

Advanced vehicle systems assessment. Volume 3: Systems assessment

NASA Technical Reports Server (NTRS)

Hardy, K.

1985-01-01

The systems analyses integrate the advanced component and vehicle characteristics into conceptual vehicles with identical performance (for a given application) and evaluates the vehicles in typical use patterns. Initial and life-cycle costs are estimated and compared to conventional reference vehicles with comparable technological advances, assuming the vehicles will be in competition in the early 1990s. Electric vans, commuter vehicles, and full-size vehicles, in addition to electric/heat-engine hybrid and fuel-cell powered vehicles, are addressed in terms of performance and economics. System and subsystem recommendations for vans and two-passenger commuter vehicles are based on the economic analyses in this volume.

Restrictions in Cell Cycle Progression of Adult Vestibular Supporting Cells in Response to Ectopic Cyclin D1 Expression

PubMed Central

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H.; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells. PMID:22073316

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

PubMed

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.

Slow-cycling stem cells in hydra contribute to head regeneration

PubMed Central

Govindasamy, Niraimathi; Murthy, Supriya; Ghanekar, Yashoda

2014-01-01

ABSTRACT Adult stem cells face the challenge of maintaining tissue homeostasis by self-renewal while maintaining their proliferation potential over the lifetime of an organism. Continuous proliferation can cause genotoxic/metabolic stress that can compromise the genomic integrity of stem cells. To prevent stem cell exhaustion, highly proliferative adult tissues maintain a pool of quiescent stem cells that divide only in response to injury and thus remain protected from genotoxic stress. Hydra is a remarkable organism with highly proliferative stem cells and ability to regenerate at whole animal level. Intriguingly, hydra does not display consequences of high proliferation, such as senescence or tumour formation. In this study, we investigate if hydra harbours a pool of slow-cycling stem cells that could help prevent undesirable consequences of continuous proliferation. Hydra were pulsed with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) and then chased in the absence of EdU to monitor the presence of EdU-retaining cells. A significant number of undifferentiated cells of all three lineages in hydra retained EdU for about 8–10 cell cycles, indicating that these cells did not enter cell cycle. These label-retaining cells were resistant to hydroxyurea treatment and were predominantly in the G2 phase of cell cycle. Most significantly, similar to mammalian quiescent stem cells, these cells rapidly entered cell division during head regeneration. This study shows for the first time that, contrary to current beliefs, cells in hydra display heterogeneity in their cell cycle potential and the slow-cycling cells in this population enter cell cycle during head regeneration. These results suggest an early evolution of slow-cycling stem cells in multicellular animals. PMID:25432513

Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans

PubMed Central

Sierra, Crystal S.; Haase, Steven B.

2016-01-01

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation. PMID:27918582

Cytotoxic effect of Reseda lutea L.: A case of forgotten remedy.

PubMed

Radulović, Niko S; Zlatković, Dragan B; Ilić-Tomić, Tatjana; Senerović, Lidija; Nikodinovic-Runic, Jasmina

2014-04-11

Reseda lutea L. (Resedaceae) or Wild Mignonette is a widely distributed plant species. Pliny the Elder (AD 23-AD 79), a Roman scholar and naturalist, reported the use of R. lutea for reducing tumors in his Historia naturalis. Accounts of the beneficial effects of R. lutea in tumor treatment could also be found in the works of later authors, such as Étienne François Geoffroy (1672-1731) and Samuel Frederick Gray (1766-1828). However, to date no in vivo or in vitro evidence exists in support of the alleged tumor healing properties of R. lutea. The composition of autolysates obtained from different organs (root, flower and fruit) of R. lutea was investigated by GC and GC-MS analyses and IR, 1D and 2D NMR spectroscopy. These analyses led to the discovery of a new compound isolated in pure form from the flower autolysate. Autolysates and their major constituents were submitted to MTT-dye reduction cytotoxic assay on human A375 (melanoma) and MRC5 (fibroblast) cell lines. Mechanism of the cytotoxic effects was studied by cell cycle analysis and Annexin V assay. Benzyl isothiocyanate and 2-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate were identified as the major constituents of the root and flower autolysates, respectively (the later represents a new natural product). These compounds showed significant antiproliferative effects against both cell lines, which could also explain the observed high cytotoxic activity of the tested autolysates. Cell cycle analysis revealed apoptosis as the probable mechanism of cell death. Tumor healing properties attributed to R. lutea in the pre-modern texts were substantiated by the herein obtained results. Two isothiocyanates were found to be the major carriers of the observed activity. Although there was a relatively low differential effect of the plant metabolites on transformed and non-transformed cell lines, one can argue that the noted strong cytotoxicity provides first evidence that could explain the long forgotten use of this particular species. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome.

PubMed

Wang, Kunning; Liang, Qiaoyi; Li, Xiaoxing; Tsoi, Ho; Zhang, Jingwan; Wang, Hua; Go, Minnie Y Y; Chiu, Philip W Y; Ng, Enders K W; Sung, Joseph J Y; Yu, Jun

2016-10-01

Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1-S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan-Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer. MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

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

PubMed

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

2016-07-02

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

Clinical value of circulating endothelial cell levels in metastatic colorectal cancer patients treated with first-line chemotherapy and bevacizumab.

PubMed

Malka, D; Boige, V; Jacques, N; Vimond, N; Adenis, A; Boucher, E; Pierga, J Y; Conroy, T; Chauffert, B; François, E; Guichard, P; Galais, M P; Cvitkovic, F; Ducreux, M; Farace, F

2012-04-01

We investigated whether circulating endothelial cells (CECs) predict clinical outcome of first-line chemotherapy and bevacizumab in metastatic colorectal cancer (mCRC) patients. In a substudy of the randomized phase II FNCLCC ACCORD 13/0503 trial, CECs (CD45- CD31+ CD146+ 7-amino-actinomycin- cells) were enumerated in 99 patients by four-color flow cytometry at baseline and after one cycle of treatment. We correlated CEC levels with objective response rate (ORR), 6-month progression-free survival (PFS) rate (primary end point of the trial), PFS, and overall survival (OS). Multivariate analyses of potential prognostic factors, including CEC counts and Köhne score, were carried out. By multivariate analysis, high baseline CEC levels were the only independent prognostic factor for 6-month PFS rate (P < 0.01) and were independently associated with worse PFS (P = 0.02). High CEC levels after one cycle were the only independent prognostic factor for ORR (P = 0.03). High CEC levels at both time points independently predicted worse ORR (P = 0.025), 6-month PFS rate (P = 0.007), and PFS (P = 0.02). Köhne score was the only variable associated with OS. CEC levels at baseline and after one treatment cycle may independently predict ORR and PFS in mCRC patients starting first-line bevacizumab and chemotherapy.

An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

PubMed Central

Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

2012-01-01

During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

Ecotoxicological assessment of cobalt using Hydra model: ROS, oxidative stress, DNA damage, cell cycle arrest, and apoptosis as mechanisms of toxicity.

PubMed

Zeeshan, Mohammed; Murugadas, Anbazhagan; Ghaskadbi, Surendra; Ramaswamy, Babu Rajendran; Akbarsha, Mohammad Abdulkader

2017-05-01

The mechanisms underlying cobalt toxicity in aquatic species in general and cnidarians in particular remain poorly understood. Herein we investigated cobalt toxicity in a Hydra model from morphological, histological, developmental, and molecular biological perspectives. Hydra, exposed to cobalt (0-60 mg/L), were altered in morphology, histology, and regeneration. Exposure to standardized sublethal doses of cobalt impaired feeding by affecting nematocytes, which in turn affected reproduction. At the cellular level, excessive ROS generation, as the principal mechanism of action, primarily occurred in the lysosomes, which was accompanied by the upregulation of expression of the antioxidant genes SOD, GST, GPx, and G6PD. The number of Hsp70 and FoxO transcripts also increased. Interestingly, the upregulations were higher in the 24-h than in the 48-h time-point group, indicating that ROS overwhelmed the cellular defense mechanisms at the latter time-point. Comet assay revealed DNA damage. Cell cycle analysis indicated the induction of apoptosis accompanied or not by cell cycle arrest. Immunoblot analyses revealed that cobalt treatment triggered mitochondria-mediated apoptosis as inferred from the modulation of the key proteins Bax, Bcl-2, and caspase-3. From this data, we suggest the use of Hydra as a model organism for the risk assessment of heavy metal pollution in aquatic ecosystems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line

PubMed Central

Jiang, Linjia; Romero-Carvajal, Andres; Haug, Jeff S.; Seidel, Christopher W.; Piotrowski, Tatjana

2014-01-01

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/β-catenin signaling following hair cell death. We propose that Wnt/β-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals. PMID:24706903

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

Differentiation of endosperm transfer cells of barley: a comprehensive analysis at the micro-scale.

PubMed

Thiel, Johannes; Riewe, David; Rutten, Twan; Melzer, Michael; Friedel, Swetlana; Bollenbeck, Felix; Weschke, Winfriede; Weber, Hans

2012-08-01

Barley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection-based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange-like secondary-wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene-signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4-hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up-regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress-related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

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.

Expression and Function of the Progesterone Receptor in Human Prostate Stroma Provide Novel Insights to Cell Proliferation Control

PubMed Central

Yu, Yue; Liu, Liangliang; Xie, Ning; Xue, Hui; Fazli, Ladan; Buttyan, Ralph; Wang, Yuzhuo; Gleave, Martin

2013-01-01

Context: Like other tissues, the prostate is an admixture of many different cell types that can be segregated into components of the epithelium or stroma. Reciprocal interactions between these 2 types of cells are critical for maintaining prostate homeostasis, whereas aberrant stromal cell proliferation can disrupt this balance and result in diseases such as benign prostatic hyperplasia. Although the androgen and estrogen receptors are relatively well studied for their functions in controlling stromal cell proliferation and differentiation, the role of the progesterone receptor (PR) remains unclear. Objective: The aim of the study was to investigate the expression and function of the PR in the prostate. Design and Setting: Human prostate biopsies, renal capsule xenografts, and prostate stromal cells were used. Immunohistochemistry, Western blotting, real-time quantitative PCR, cell proliferation, flow cytometry, and gene microarray analyses were performed. Results: Two PR isoforms, PRA and PRB, are expressed in prostate stromal fibroblasts and smooth muscle cells, but not in epithelial cells. Both PR isoforms suppress prostate stromal cell proliferation through inhibition of the expression of cyclinA, cyclinB, and cdc25c, thus delaying cell cycling through S and M phases. Gene microarray analyses further demonstrated that PRA and PRB regulated different transcriptomes. However, one of the major gene groups commonly regulated by both PR isoforms was the one associated with regulation of cell proliferation. Conclusion: PR plays an inhibitory role in prostate stromal cell proliferation. PMID:23666965

Quality of life analyses from the randomized, open-label, phase III PointBreak study of pemetrexed-carboplatin-bevacizumab followed by maintenance pemetrexed-bevacizumab versus paclitaxel-carboplatin-bevacizumab followed by maintenance bevacizumab in patients with stage IIIB or IV nonsquamous non-small-cell lung cancer.

PubMed

Spigel, David R; Patel, Jyoti D; Reynolds, Craig H; Garon, Edward B; Hermann, Robert C; Govindan, Ramaswamy; Olsen, Mark R; Winfree, Katherine B; Chen, Jian; Liu, Jingyi; Guba, Susan C; Socinski, Mark A; Bonomi, Philip

2015-02-01

Treatment impact on quality of life (QoL) informs treatment management decisions in advanced nonsquamous non-small-cell lung cancer (NS NSCLC). QoL outcomes from the phase III PointBreak trial are reported. Chemonaive patients (n = 939) with stage IIIB/IV nonsquamous non-small-cell lung cancer and Eastern Cooperative Oncology Group performance status 0 to 1 were randomized (1:1) to pemetrexed-carboplatin-bevacizumab (pemetrexed arm) or paclitaxel-carboplatin-bevacizumab (paclitaxel arm). Patients without progressive disease received maintenance pemetrexed-bevacizumab (pemetrexed arm) or bevacizumab (paclitaxel arm). QoL was assessed using Functional Assessment of Cancer Therapy (FACT)-General (FACT-G), FACT-Lung (FACT-L), and FACT/Gynecologic Oncology Group-Neurotoxicity (FACT-Ntx) instruments. Subscale scores, total scores, and trial outcome indices were analyzed using linear mixed-effects models. Post hoc analyses examined the association between baseline FACT scores and overall survival (OS). Mean score differences in change from baseline significantly favored the pemetrexed arm for the neurotoxicity subscale score, FACT-Ntx total scores, and FACT-Ntx trial outcome index. They occurred at cycle 2 (p < 0.001) and persisted through induction cycles 2 to 4 and six maintenance cycles. Investigator-assessed, qualitative, drug-related differences in grade 2 (1.6% versus 10.6%) and grade 3 (0.0% versus 4.1%) sensory neuropathy and grade 3/4 fatigue (10.9% versus 5.0%, p = 0.0012) were observed between the pemetrexed and paclitaxel arms. Baseline FACT-G, FACT-L, and FACT-Ntx scores were significant prognostic factors for OS (p < 0.001). Randomized patients reported similar changes in QoL, except for less change from baseline in neurotoxicity on the pemetrexed arm; investigators reported greater neurotoxicity on the paclitaxel arm and greater fatigue on the pemetrexed arm. Higher baseline FACT scores were favorable prognostic factors for OS.

Impedance measurements on a spiral-wound nickel/metal hydride cell cycled in a simulated Leo orbit

NASA Technical Reports Server (NTRS)

Reid, Margaret A.

1993-01-01

A spiral-wound size C cell was cycled at 25 C in a low earth orbit (LEO) regime at 50 percent depth of discharge (DOD) with approximately five percent over-charge. The nominal capacity was 3.5 AH. The cell was cycled for 2000 cycles. Capacity checks and impedance measurements over the complete range of state of charge were made upon receipt and after 500, 1000, and 2000 cycles. The capacity of the cell was essentially unchanged until after the impedance measurements at 2000 cycles. Only small changes in the impedance parameters were observed, but there was somewhat more scatter in the data after 2000 cycles. When the cell was returned to LEO cycling after 2000 cycles, only 38 percent of the capacity could be obtained. It is believed that the cell failed because of an equipment failure at the end of the final impedance measurements which allowed an over-discharge.

Expressed in high metastatic cells (Ehm2) is a positive regulator of keratinocyte adhesion and motility: The implication for wound healing.

PubMed

Bosanquet, David C; Ye, Lin; Harding, Keith G; Jiang, Wen G

2013-08-01

Multiple factors have been shown to delay dermal wound healing. These resultant wounds pose a significant problem in terms of morbidity and healthcare spend. Recently, an increasing volume of research has focused on the molecular perturbations underlying non-healing wounds. This study investigates the effect of a novel cancer promoter, Ehm2, in wound healing. Ehm2 belongs to the FERM family of proteins, known to be involved in membrane-cytoskeletal interactions, and has been shown to promote cancer metastasis in melanoma, prostate cancer and breast cancer. Ehm2 mRNA levels were analysed using qRT-PCR, standardised to GAPDH, from either acute or chronic wounds, and normal skin. IHC analysis was also undertaken from wound edge biopsies. An anti-Ehm2 transgene was created and transfected into the HaCaT cell line. The effect of Ehm2 knockdown on migration, adhesion, growth, cell cycle progression and apoptosis was analysed using standard laboratory methods. Western Blot analysis was used to investigate potential downstream protein interactions. Ehm2 is expressed nearly three times higher in acute wound tissues, compared to chronic wound tissues. Increased Ehm2 expression is found in wounds undergoing healing, especially at the leading wound edge. In vitro, Ehm2 knockdown reduces cellular adhesion, migration and motility, without affecting growth, cell cycle and apoptosis. Finally, Ehm2 knockdown results in reduced NWasp protein expression. These results suggest Ehm2 may be an important player in the wound healing process, and show that Ehm2 knockdown downregulates the expression of NWasp, through which it may have its effect on cellular migration. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

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

Ding, Li; College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158; Huang, Yong

2014-03-07

Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressedmore » cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.« less

S.A.M., the Italian Martian Simulation Chamber

NASA Astrophysics Data System (ADS)

Galletta, G.; Ferri, F.; Fanti, G.; D'Alessandro, M.; Bertoloni, G.; Pavarin, D.; Bettanini, C.; Cozza, P.; Pretto, P.; Bianchini, G.; Debei, S.

2006-12-01

The Martian Environment Simulator (SAM “Simulatore di Ambiente Marziano”) is a interdisciplinary project of Astrobiology done at University of Padua. The research is aimed to the study of the survival of the microorganisms exposed to the “extreme” planetary environment. The facility has been designed in order to simulate Mars’ environmental conditions in terms of atmospheric pressure, temperature cycles and UV radiation dose. The bacterial cells, contained into dedicated capsules, will be exposed to thermal cycles simulating diurnal and seasonal Martian cycles. The metabolism of the different biological samples will be analysed at different phases of the experiment, to study their survival and eventual activity of protein synthesis (mortality, mutations and capability of DNA reparing). We describe the experimental facility and provide the perspectives of the biological experiments we will perform in order to provide hints on the possibility of life on Mars either autochthonous or imported from Earth.

Hypoxia induces p53 accumulation in the S-phase and accumulation of hypophosphorylated retinoblastoma protein in all cell cycle phases of human melanoma cells.

PubMed Central

Danielsen, T.; Hvidsten, M.; Stokke, T.; Solberg, K.; Rofstad, E. K.

1998-01-01

Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of the cell populations (0.60+/-0.10) showed hypophosphorylated pRb. Thus, pRb is probably not the only mediator of the hypoxia-induced cell cycle block seen in all cells and all cell cycle phases. Moreover, the cell cycle-dependent induction of p53 by hypoxia suggests that the primary function of p53 accumulation during hypoxia is other than to arrest the cells. Images Figure 4 Figure 7 PMID:9862563

Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

PubMed

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.

Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

PubMed Central

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain. PMID:17901127

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.

Docosahexaenoic acid inhibits monocrotaline-induced pulmonary hypertension via attenuating endoplasmic reticulum stress and inflammation.

PubMed

Chen, Rui; Zhong, Wei; Shao, Chen; Liu, Peijing; Wang, Cuiping; Wang, Zhongqun; Jiang, Meiping; Lu, Yi; Yan, Jinchuan

2018-02-01

Endoplasmic reticulum (ER) stress and inflammation contribute to pulmonary hypertension (PH) pathogenesis. Previously, we confirmed that docosahexaenoic acid (DHA) could improve hypoxia-induced PH. However, little is known about the link between DHA and monocrotaline (MCT)-induced PH. Our aims were, therefore, to evaluate the effects and molecular mechanisms of DHA on MCT-induced PH in rats. Rat PH was induced by MCT. Rats were treated with DHA daily in the prevention group (following MCT injection) and the reversal group (after MCT injection for 2 wk) by gavage. After 4 wk, mean pulmonary arterial pressure (mPAP), right ventricular (RV) hypertrophy index, and morphological and immunohistochemical analyses were evaluated. Rat pulmonary artery smooth muscle cells (PASMCs) were used to investigate the effects of DHA on cell proliferation stimulated by platelet-derived growth factor (PDGF)-BB. DHA decreased mPAP and attenuated pulmonary vascular remodeling and RV hypertrophy, which were associated with suppressed ER stress. DHA blocked the mitogenic effect of PDGF-BB on PASMCs and arrested the cell cycle via inhibiting nuclear factor of activated T cells-1 (NFATc1) expression and activation and regulating cell cycle-related proteins. Moreover, DHA ameliorated inflammation in lung and suppressed macrophage and T lymphocyte accumulation in lung and adventitia of resistance pulmonary arteries. These findings suggest that DHA could protect against MCT-induced PH by reducing ER stress, suppressing cell proliferation and inflammation.

Analysis of Gene Expression Changes in PHA-M Stimulated Lymphocytes - Unraveling PHA Activity as Prerequisite for Dicentric Chromosome Analysis.

PubMed

Beinke, C; Port, M; Ullmann, R; Gilbertz, K; Majewski, M; Abend, M

2018-06-01

Dicentric chromosome analysis (DCA) is the gold standard for individual radiation dose assessment. However, DCA is limited by the time-consuming phytohemagglutinin (PHA)-mediated lymphocyte activation. In this study using human peripheral blood lymphocytes, we investigated PHA-associated whole genome gene expression changes to elucidate this process and sought to identify suitable gene targets as a means of meeting our long-term objective of accelerating cell cycle kinetics to reduce DCA culture time. Human peripheral whole blood from three healthy donors was separately cultured in RPMI/FCS/antibiotics with BrdU and PHA-M. Diluted whole blood samples were transferred into PAXgene tubes at 0, 12, 24 and 36 h culture time. RNA was isolated and aliquots were used for whole genome gene expression screening. Microarray results were validated using qRT-PCR and differentially expressed genes [significantly (FDR corrected) twofold different from the 0 h value reference] were analyzed using several bioinformatic tools. The cell cycle positions and DNA-synthetic activities of lymphocytes were determined by analyzing the correlated total DNA content and incorporated BrdU level with flow cytometry after continued BrdU incubation. From 42,545 transcripts of the whole genome microarray 47.6%, on average, appeared expressed. The number of differentially expressed genes increased linearly from 855 to 2,858 and 4,607 at 12, 24 and 36 h after PHA addition, respectively. Approximately 2-3 times more up- than downregulated genes were observed with several hundred genes differentially expressed at each time point. Earliest enrichment was observed for gene sets related to the nucleus (12 h) followed by genes assigned to intracellular structures such as organelles (24 h) and finally genes related to the membrane and the extracellular matrix were enriched (36 h). Early gene expression changes at 12 h, in particular, were associated with protein classes such as chemokines/cytokines (e.g., CXCL1, CXCL2) and chaperones. Genes coding for biological processes involved in cell cycle control (e.g., MYBL2, RBL1, CCNA, CCNE) and DNA replication (e.g., POLA, POLE, MCM) appeared enriched at 24 h and later, but many more biological processes (42 altogether) showed enrichment as well. Flow cytometry data fit together with gene expression and bioinformatic analyses as cell cycle transition into S phase was observed with interindividual differences from 12 h onward, whereas progression into G 2 as well as into the second G 1 occurred from 36 h onward after activation. Gene set enrichment analysis over time identifies, in particular, two molecular categories of PHA-responsive gene targets (cytokine and cell cycle control genes). Based on that analysis target genes for cell cycle acceleration in lymphocytes have been identified ( CDKN1A/B/C, RBL-1/RBL-2, E2F2, Deaf-1), and it remains undetermined whether the time expenditure for DCA can be reduced by influencing gene expression involved in the regulatory circuits controlling PHA-associated cell cycle entry and/or progression at a specific early cell cycle phase.

KOH concentration effect on cycle life of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Lim, Hong S.; Verzwyvelt, S. A.

1987-01-01

A cycle life test of Ni/H2 cells containing electrolytes of various KOH concentrations and a sintered type nickel electrode was carried out at 23 C using a 45 min accelerated low Earth orbit (LEO) cycle regime at 80 percent depth of discharge. One of three cells containing 26 percent KOH has achieved over 28,000 cycles, and the other two 19,000 cycles, without a sign of failure. Two other cells containing 31 percent KOH electrolyte, which is the concentration presently used in aerospace cells, failed after 2,979 and 3,620 cycles. This result indicates that the cycle life of the present type of Ni/H2 cells may be extended by a factor of 5 to 10 simply by lowering the KOH concentration. Long cycle life of a Ni/H2 battery at high depth-of-discharge operation is desired, particularly for an LEO spacecraft application. Typically, battery life of about 30,000 cycles is required for a five year mission in an LEO. Such a cycle life with presently available cells can be assured only at a very low depth-of-discharge operation. Results of testing already show that the cycle life of an Ni/H2 cell is tremendously improved by simply using an electrolyte of low KOH concentration.

The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

PubMed

Mizejewski, G J

2016-09-01

The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles

PubMed Central

Wheeler, Richard John

2015-01-01

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point “snapshot” of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes—cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)—as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. PMID:26543196

Angular-dependent light scattering from cancer cells in different phases of the cell cycle.

PubMed

Lin, Xiaogang; Wan, Nan; Weng, Lingdong; Zhou, Yong

2017-10-10

Cancer cells in different phases of the cell cycle result in significant differences in light scattering properties. In order to harvest cancer cells in particular phases of the cell cycle, we cultured cancer cells through the process of synchronization. Flow cytometric analysis was applied to check the results of cell synchronization and prepare for light scattering measurements. Angular-dependent light scattering measurements of cancer cells arrested in the G1, S, and G2 phases have been performed. Based on integral calculations for scattering intensities from 5° to 10° and from 110° to 150°, conclusions have been reached. Clearly, the sizes of the cancer cells in different phases of the cell cycle dominated the forward scatter. Accompanying the increase of cell size with the progression of the cell cycle, the forward scattering intensity also increased. Meanwhile, the DNA content of cancer cells in every phase of the cell cycle is responsible for light scattering at large scatter angles. The higher the DNA content of cancer cells was, the greater the positive effect on the high-scattering intensity. As expected, understanding the relationships between the light scattering from cancer cells and cell cycles will aid in the development of cancer diagnoses. Also, it may assist in the guidance of antineoplastic drugs clinically.

Stochastic multi-scale models of competition within heterogeneous cellular populations: Simulation methods and mean-field analysis.

PubMed

Cruz, Roberto de la; Guerrero, Pilar; Spill, Fabian; Alarcón, Tomás

2016-10-21

We propose a modelling framework to analyse the stochastic behaviour of heterogeneous, multi-scale cellular populations. We illustrate our methodology with a particular example in which we study a population with an oxygen-regulated proliferation rate. Our formulation is based on an age-dependent stochastic process. Cells within the population are characterised by their age (i.e. time elapsed since they were born). The age-dependent (oxygen-regulated) birth rate is given by a stochastic model of oxygen-dependent cell cycle progression. Once the birth rate is determined, we formulate an age-dependent birth-and-death process, which dictates the time evolution of the cell population. The population is under a feedback loop which controls its steady state size (carrying capacity): cells consume oxygen which in turn fuels cell proliferation. We show that our stochastic model of cell cycle progression allows for heterogeneity within the cell population induced by stochastic effects. Such heterogeneous behaviour is reflected in variations in the proliferation rate. Within this set-up, we have established three main results. First, we have shown that the age to the G1/S transition, which essentially determines the birth rate, exhibits a remarkably simple scaling behaviour. Besides the fact that this simple behaviour emerges from a rather complex model, this allows for a huge simplification of our numerical methodology. A further result is the observation that heterogeneous populations undergo an internal process of quasi-neutral competition. Finally, we investigated the effects of cell-cycle-phase dependent therapies (such as radiation therapy) on heterogeneous populations. In particular, we have studied the case in which the population contains a quiescent sub-population. Our mean-field analysis and numerical simulations confirm that, if the survival fraction of the therapy is too high, rescue of the quiescent population occurs. This gives rise to emergence of resistance to therapy since the rescued population is less sensitive to therapy. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

Modeling Bi-modality Improves Characterization of Cell Cycle on Gene Expression in Single Cells

PubMed Central

Danaher, Patrick; Finak, Greg; Krouse, Michael; Wang, Alice; Webster, Philippa; Beechem, Joseph; Gottardo, Raphael

2014-01-01

Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%–17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome. PMID:25032992

Rapid Ammonia Gas Transport Accounts for Futile Transmembrane Cycling under NH3/NH4+ Toxicity in Plant Roots1[C][W

PubMed Central

Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J.

2013-01-01

Futile transmembrane NH3/NH4+ cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4+ toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4+) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope 13N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4+ ion. Influx of 13NH3/13NH4+, which exceeded 200 µmol g–1 h–1, was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4+), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g–1 h–1). Efflux of 13NH3/13NH4+ responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions. PMID:24134887

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.

[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.

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.

A role for RNA post-transcriptional regulation in satellite cell activation

PubMed Central

2012-01-01

Background Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretm and Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors. Results An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate. Conclusions The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation. PMID:23046558

Localization of FtsZ in Helicobacter pylori and Consequences for Cell Division

PubMed Central

Specht, Mara; Dempwolff, Felix; Schätzle, Sarah; Thomann, Ralf

2013-01-01

Of the various kinds of cell division, the most common mode is binary fission, the division of a cell into two morphologically identical daughter cells. However, in the case of asymmetric cell division, Caulobacter crescentus produces two morphologically and functionally distinct cell types. Here, we have studied cell cycle progression of the human pathogen Helicobacter pylori using a functional green fluorescent protein (GFP) fusion of FtsZ protein and membrane staining. In small cells, representing newly divided cells, FtsZ localizes to a single cell pole. During the cell cycle, spiral intermediates are formed until an FtsZ ring is positioned with very little precision, such that central as well as acentral rings can be observed. Daughter cells showed considerably different sizes, suggesting that H. pylori divides asymmetrically. Fluorescence recovery after photobleaching (FRAP) analyses demonstrate that the H. pylori FtsZ ring is about as dynamic as that of Escherichia coli but that polar assemblies show less turnover. Strikingly, our results demonstrate that H. pylori cell division follows a different route from that in E. coli and Bacillus subtilis. It is also different from that in C. crescentus, where cytokinesis regulation proteins like MipZ play a role. Therefore, this report provides the first cell-biological analysis of FtsZ dynamics in the human pathogen H. pylori and even in epsilonproteobacteria to our knowledge. In addition, analysis of the filament architecture of H. pylori and E. coli FtsZ filaments in the heterologous system of Drosophila melanogaster S2 Schneider cells revealed that both have different filamentation properties in vivo, suggesting a unique intrinsic characteristic of each protein. PMID:23335414

Performance of Li-Ion Cells Under Battery Voltage Charge Control

NASA Technical Reports Server (NTRS)

Rao, Gopalakrishna M.; Vaidyanathan, Hari; Day, John H. (Technical Monitor)

2001-01-01

A study consisting of electrochemical characterization and Low-Earth-Orbit (LEO) cycling of Li-Ion cells from three vendors was initiated in 1999 to determine the cycling performance and to infuse the new technology in the future NASA missions. The 8-cell batteries included in this evaluation are prismatic cells manufactured by Mine Safety Appliances Company (MSA), cylindrical cells manufactured by SAFT and prismatic cells manufactured by Yardney Technical Products, Inc. (YTP). The three batteries were cycle tested in the LEO regime at 40% depth of discharge, and under a charge control technique that consists of battery voltage clamp with a current taper. The initial testing was conducted at 20 C; however, the batteries were cycled also intermittently at low temperatures. YTP 20 Ah cells consisted of mixed-oxide (Co and Ni) positive, graphitic carbon negative, LIPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was 32 V. The low temperature cycling tests started after 4575 cycles at 20 C. The cells were not capable of cycling. at low temperature since the charge acceptance at battery level was poor. There was a cell in the battery that showed too high an end-of-charge (EOC) voltage thereby limiting the ability to charge the rest of the cells in the battery. The battery has completed 6714 cycles. SAFT 12 Ah cells consisted of mixed-oxide (Co and NO positive, graphitic carbon negative, LiPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was for 30.8 V. The low temperature cycling tests started after 4594 cycles at 20 C. A cell that showed low end of discharge (EOD) and EOC voltages and three other cells that showed higher EOC voltages limited the charge acceptance at the selected voltage limit during charge. The cells were capable of cycling at 10 C and 0 C but the charge voltage limit had to be increased to 34.3 V (4.3 V per cell). The low temperature cycling may have induced poor chargeability since the voltage had to be increased to achieve the required charge input. The battery has completed 6226 cycles. MSA 10 Ah cells consisted of Co oxide positive, graphitic carbon negative, LiPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was 30.8 V. The low temperature cycling tests were started after 2182 cycles at 20 C. The cells were capable of cycling at 10 C and 0 C. Like SAFT, the voltage limit on charge had to be increased to 36 V (4.5 V per cell). There was a cell (cell S/N 13) in the battery that showed poor performance features such as low EOD voltage and high EOC voltage. The battery has completed 3441 cycles. A reconditioning procedure that consisted of C15 charge to a taper current of C/100 and C/20 discharge improved the voltage behavior of SAFT and MSA cells with no significant effect on YTP cells. We have demonstrated that the charge operation with VT clamp at battery rather than at cell level is feasible for onboard Li-Ion battery operation.

Inhibitor effects during the cell cycle in Chlamydomonas reinhardtii. Determination of transition points in asynchronous cultures

PubMed Central

1975-01-01

A wide variety of inhibitors (drugs, antibiotics, and antimetabolites) will block cell division within an ongoing cell cycle in autotrophic cultures of Chlamydomonas reinhardtii. To determine when during the cell cycle a given inhibitor is effective in preventing cell division, a technique is described which does not rely on the use of synchronous cultures. The technique permits the measurement of transition points, the cell cycle stage at which the subsequent cell division becomes insensitive to the effects of an inhibitor. A map of transition points in the cell cycle reveals that they are grouped into two broad periods, the second and fourth quarters. In general, inhibitors which block organellar DNA, RNA, and protein synthesis have second-quarter transition points, while those which inhibit nuclear cytoplasmic macromolecular synthesis have fourth-quarter transition points. The specific grouping of these transition points into two periods suggests that the synthesis of organellar components is completed midway through the cell cycle and that the synthesis of nonorganellar components required for cell division is not completed until late in the cell cycle. PMID:1176526

Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

PubMed Central

Zhou, Tong; Chou, Jeff; Mullen, Thomas E.; Elkon, Rani; Zhou, Yingchun; Simpson, Dennis A.; Bushel, Pierre R.; Paules, Richard S.; Lobenhofer, Edward K.; Hurban, Patrick; Kaufmann, William K.

2007-01-01

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. PMID:17404513

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.

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.

Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

PubMed

Wani, Willayat Yousuf; Kandimalla, Ramesh J L; Sharma, Deep Raj; Kaushal, Alka; Ruban, Anand; Sunkaria, Aditya; Vallamkondu, Jayalakshmi; Chiarugi, Alberto; Reddy, P Hemachandra; Gill, Kiran Dip

2017-07-01

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G 0 /G 1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Decoupling of Nuclear Division Cycles and Cell Size during the Coenocytic Growth of the Ichthyosporean Sphaeroforma arctica.

PubMed

Ondracka, Andrej; Dudin, Omaya; Ruiz-Trillo, Iñaki

2018-06-18

Coordination of the cell division cycle with the growth of the cell is critical to achieve cell size homeostasis [1]. Mechanisms coupling the cell division cycle with cell growth have been described across diverse eukaryotic taxa [2-4], but little is known about how these processes are coordinated in organisms that undergo more complex life cycles, such as coenocytic growth. Coenocytes (multinucleate cells formed by sequential nuclear divisions without cytokinesis) are commonly found across the eukaryotic kingdom, including in animal and plant tissues and several lineages of unicellular eukaryotes [5]. Among the organisms that form coenocytes are ichthyosporeans, a lineage of unicellular holozoans that are of significant interest due to their phylogenetic placement as one of the closest relatives of animals [6]. Here, we characterize the coenocytic cell division cycle in the ichthyosporean Sphaeroforma arctica. We observe that, in laboratory conditions, S. arctica cells undergo a uniform and easily synchronizable coenocytic cell cycle, reaching up to 128 nuclei per cell before cellularization and release of daughter cells. Cycles of nuclear division occur synchronously within the coenocyte and in regular time intervals (11-12 hr). We find that the growth of cell volume is dependent on concentration of nutrients in the media; in contrast, the rate of nuclear division cycles is constant over a range of nutrient concentrations. Together, the results suggest that nuclear division cycles in the coenocytic growth of S. arctica are driven by a timer, which ensures periodic and synchronous nuclear cycles independent of the cell size and growth. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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.

Identification of Cell Cycle-regulated Genes in Fission YeastD⃞

PubMed Central

Peng, Xu; Karuturi, R. Krishna Murthy; Miller, Lance D.; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T.; Balasubramanian, Mohan K.; Liu, Jianhua

2005-01-01

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC. PMID:15616197

Loss of Ptpn11 (Shp2) drives satellite cells into quiescence

PubMed Central

Griger, Joscha; Schneider, Robin; Lahmann, Ines; Schöwel, Verena; Keller, Charles; Spuler, Simone; Nazare, Marc; Birchmeier, Carmen

2017-01-01

The equilibrium between proliferation and quiescence of myogenic progenitor and stem cells is tightly regulated to ensure appropriate skeletal muscle growth and repair. The non-receptor tyrosine phosphatase Ptpn11 (Shp2) is an important transducer of growth factor and cytokine signals. Here we combined complex genetic analyses, biochemical studies and pharmacological interference to demonstrate a central role of Ptpn11 in postnatal myogenesis of mice. Loss of Ptpn11 drove muscle stem cells out of the proliferative and into a resting state during muscle growth. This Ptpn11 function was observed in postnatal but not fetal myogenic stem cells. Furthermore, muscle repair was severely perturbed when Ptpn11 was ablated in stem cells due to a deficit in stem cell proliferation and survival. Our data demonstrate a molecular difference in the control of cell cycle withdrawal in fetal and postnatal myogenic stem cells, and assign to Ptpn11 signaling a key function in satellite cell activity. DOI: http://dx.doi.org/10.7554/eLife.21552.001 PMID:28463680

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

PubMed

Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

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.

Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

PubMed Central

hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

2013-01-01

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435

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

Comparative analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in mammals

PubMed Central

Gawriluk, Thomas R.; Simkin, Jennifer; Thompson, Katherine L.; Biswas, Shishir K.; Clare-Salzler, Zak; Kimani, John M.; Kiama, Stephen G.; Smith, Jeramiah J.; Ezenwa, Vanessa O.; Seifert, Ashley W.

2016-01-01

Why mammals have poor regenerative ability has remained a long-standing question in biology. In regenerating vertebrates, injury can induce a process known as epimorphic regeneration to replace damaged structures. Using a 4-mm ear punch assay across multiple mammalian species, here we show that several Acomys spp. (spiny mice) and Oryctolagus cuniculus completely regenerate tissue, whereas other rodents including MRL/MpJ ‘healer' mice heal similar injuries by scarring. We demonstrate ear-hole closure is independent of ear size, and closure rate can be modelled with a cubic function. Cellular and genetic analyses reveal that injury induces blastema formation in Acomys cahirinus. Despite cell cycle re-entry in Mus musculus and A. cahirinus, efficient cell cycle progression and proliferation only occurs in spiny mice. Together, our data unite blastema-mediated regeneration in spiny mice with regeneration in other vertebrates such as salamanders, newts and zebrafish, where all healthy adults regenerate in response to injury. PMID:27109826

Insulin-like growth factor II messenger RNA-binding protein-3 is an independent prognostic factor in uterine leiomyosarcoma.

PubMed

Yasutake, Nobuko; Ohishi, Yoshihiro; Taguchi, Kenichi; Hiraki, Yuka; Oya, Masafumi; Oshiro, Yumi; Mine, Mari; Iwasaki, Takeshi; Yamamoto, Hidetaka; Kohashi, Kenichi; Sonoda, Kenzo; Kato, Kiyoko; Oda, Yoshinao

2018-04-01

The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS). We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study. IMP3 expression in ULMS could be a marker of a poor prognosis. © 2017 John Wiley & Sons Ltd.

Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.

PubMed

Alves, Tiago C; Pongratz, Rebecca L; Zhao, Xiaojian; Yarborough, Orlando; Sereda, Sam; Shirihai, Orian; Cline, Gary W; Mason, Graeme; Kibbey, Richard G

2015-11-03

Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell. Copyright © 2015 Elsevier Inc. All rights reserved.

Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

PubMed

Cooper, Stephen

2017-11-01

Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

PubMed

Koyama, Fernanda C; Chakrabarti, Debopam; Garcia, Célia R S

2009-05-01

The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.

Cell Growth Characteristics, Differentiation Frequency, and Immunophenotype of Adult Ear Mesenchymal Stem Cells

PubMed Central

Staszkiewicz, Jaroslaw; Frazier, Trivia P.; Rowan, Brian G.; Bunnell, Bruce A.; Chiu, Ernest S.; Gimble, Jeffrey M.

2010-01-01

Ear mesenchymal stem cells (EMSCs) represent a readily accessible population of stem-like cells that are adherent, clonogenic, and have the ability to self-renew. Previously, we have demonstrated that they can be induced to differentiate into adipocyte, osteocyte, chondrocyte, and myocyte lineages. The purpose of the current study was to characterize the growth kinetics of the cells and to determine their ability to form colonies of fibroblasts, adipocytes, osteocytes, and chondrocytes. In addition, the immunophenotypes of freshly isolated and culture-expanded cells were evaluated. From 1 g of tissue, we were able to isolate an average of 7.8 × 106 cells exhibiting a cell cycle length of ∼2–3 days. Colony-forming unit (CFU) assays indicated high proliferation potential, and confirmed previously observed multipotentiality of the cells. Fluorescence-activated cell sorting (FACS) showed that EMSCs were negative for hematopoietic markers (CD4, CD45), proving that they did not derive from circulating hematopoietic cells. The FACS analyses also showed high expression of stem cell antigen-1 (Sca-1) with only a minor population of cells expressing CD117, thus identifying Sca-1 as the more robust stem cell biomarker. Additionally, flow cytometry data revealed that the expression patterns of hematopoietic, stromal, and stem cell markers were maintained in the passaged EMSCs, consistent with the persistence of an undifferentiated state. This study indicates that EMSCs provide an alternative model for in vitro analyses of adult mesenchymal stem cells (MSCs). Further studies will be necessary to determine their utility for tissue engineering and regenerative medical applications. PMID:19400629

The Abbreviated Pluripotent Cell Cycle

PubMed Central

Kapinas, Kristina; Grandy, Rodrigo; Ghule, Prachi; Medina, Ricardo; Becker, Klaus; Pardee, Arthur; Zaidi, Sayyed K.; Lian, Jane; Stein, Janet; van Wijnen, Andre; Stein, Gary

2013-01-01

Human embryonic stem cells and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory andstructural. The primary temporal context that the pluripotent self-renewal cell cycle of human embryonic stem cells (hESCs) is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the ESC cell cycle. This supports the requirements of pluripotent cells to self propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated embryonic stem cell cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. PMID:22552993

Proteomic analysis of the bacterial cell cycle

PubMed Central

Grünenfelder, Björn; Rummel, Gabriele; Vohradsky, Jiri; Röder, Daniel; Langen, Hanno; Jenal, Urs

2001-01-01

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. PMID:11287652

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

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

PubMed

Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

2015-05-01

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

PubMed Central

1996-01-01

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point. PMID:8642331

Targeted Approaches to Overcoming Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2011-08-01

NM_001012271 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog AF053305 CDC20 Cell division cycle 20 homolog BG256659 CDC25B Cell division cycle...by benzimidazoles 1 homolog), BIRC5/ Survivin, CDCA8 (cell division cycle-associated protein 8), AURKB (aurora kinase B), CDC25B (cell division cycle

Circadian clock regulation of the cell cycle in the zebrafish intestine.

PubMed

Peyric, Elodie; Moore, Helen A; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally.

Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

PubMed Central

Peyric, Elodie; Moore, Helen A.; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally. PMID:24013905

ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

PubMed

Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

2016-08-02

ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

Identification of herpesvirus proteins that contribute to G1/S arrest.

PubMed

Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

2014-04-01

Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins from three different herpesviruses that contribute to this block. Several of the proteins we identified had previously unknown functions or were structural components of the virion. Subsets of these proteins from Epstein-Barr virus were studied for their effects on the cell cycle regulatory proteins p53 and p21, thereby identifying two proteins that induce p53 and one that induces p21 (BGLF2). We identified interactions of BGLF2 with two human proteins, both of which regulate p21, suggesting that BGLF2 induces p21 by interfering with the functions of these two host proteins. Our study indicates that multiple herpesvirus proteins contribute to the cell proliferation block, including components of the incoming virions.

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

PubMed Central

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K.; Keyomarsi, Khandan

2016-01-01

ABSTRACT Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen. PMID:27049344

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

PubMed

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

2016-06-17

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

High-throughput synchronization of mammalian cell cultures by spiral microfluidics.

PubMed

Lee, Wong Cheng; Bhagat, Ali Asgar S; Lim, Chwee Teck

2014-01-01

The development of mammalian cell cycle synchronization techniques has greatly advanced our understanding of many cellular regulatory events and mechanisms specific to different phases of the cell cycle. In this chapter, we describe a high-throughput microfluidic-based approach for cell cycle synchronization. By exploiting the relationship between cell size and its phase in the cell cycle, large numbers of synchronized cells can be obtained by size fractionation in a spiral microfluidic channel. Protocols for the synchronization of primary cells such as mesenchymal stem cells, and immortal cell lines such as Chinese hamster ovarian cells (CHO-CD36) and HeLa cells are provided as examples.

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis

PubMed Central

Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J. O.; Bakal, Chris

2015-01-01

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. PMID:26333836

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.

PubMed

Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J O; Bakal, Chris

2015-09-01

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. © 2015 The Authors.

The TCA cycle is not required for selection or survival of multidrug-resistant Salmonella

PubMed Central

Ricci, Vito; Loman, Nick; Pallen, Mark; Ivens, Alasdair; Fookes, Maria; Langridge, Gemma C.; Wain, John; Piddock, Laura J. V.

2012-01-01

Objectives The initial aim of this study was to use a systems biology approach to analyse a ciprofloxacin-selected multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium, L664. Methods The whole genome sequence and transcriptome of L664 were analysed. Site-directed mutagenesis to recreate each mutation was carried out, followed by phenotypic characterization and mutation frequency analysis. As a mutation in the TCA cycle was detected we tested the controversial hypothesis regarding the bacterial response to bactericidal antibiotics, put forward by Kohanski et al. (Cell 2007; 130: 797–810 and Mol Cell 2010; 37: 311–20), that exposure of bacteria to agents such as ciprofloxacin produces reactive oxygen species (ROS), which transiently increase the mutation rate giving rise to MDR bacteria. Results L664 contained a mutation in ramR that conferred MDR. A mutation in tctA affected the TCA cycle and conferred the inability to grow on minimal agar. The virulence of L664 was not attenuated. Ciprofloxacin exposure produced ROS in L664 and SL1344 (tctA::aph), but it was reduced and occurred later. There were no significant differences in the rates of killing or mutations per generation to antibiotic resistance between the strains. Conclusions Whilst we confirm production of ROS in response to ciprofloxacin, we have no data to support the hypothesis that this leads to selection of MDR strains. Our results indicate that the mutations in tctA and glgA were random as they did not pre-exist in the parental strain, and that the mutation in tctA did not provide a survival advantage or disadvantage in the presence of antibiotic. PMID:22186876

Phylogenetic and Metagenomic Analyses of Substrate-Dependent Bacterial Temporal Dynamics in Microbial Fuel Cells

PubMed Central

Zhang, Husen; Chen, Xi; Braithwaite, Daniel; He, Zhen

2014-01-01

Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate. PMID:25202990

Phylogenetic and metagenomic analyses of substrate-dependent bacterial temporal dynamics in microbial fuel cells.

PubMed

Zhang, Husen; Chen, Xi; Braithwaite, Daniel; He, Zhen

2014-01-01

Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

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

Redox Changes During the Cell Cycle in the Embryonic Root Meristem of Arabidopsis thaliana.

PubMed

de Simone, Ambra; Hubbard, Rachel; de la Torre, Natanael Viñegra; Velappan, Yazhini; Wilson, Michael; Considine, Michael J; Soppe, Wim J J; Foyer, Christine H

2017-12-20

The aim of this study was to characterize redox changes in the nuclei and cytosol occurring during the mitotic cell cycle in the embryonic roots of germinating Arabidopsis seedlings, and to determine how redox cycling was modified in mutants with a decreased capacity for ascorbate synthesis. Using an in vivo reduction-oxidation (redox) reporter (roGFP2), we show that transient oxidation of the cytosol and the nuclei occurred at G1 in the synchronized dividing cells of the Arabidopsis root apical meristem, with reduction at G2 and mitosis. This redox cycle was absent from low ascorbate mutants in which nuclei were significantly more oxidized than controls. The cell cycle-dependent increase in nuclear size was impaired in the ascorbate-deficient mutants, which had fewer cells per unit area in the root proliferation zone. The transcript profile of the dry seeds and size of the imbibed seeds was strongly influenced by low ascorbate but germination, dormancy release and seed aging characteristics were unaffected. These data demonstrate the presence of a redox cycle within the plant cell cycle and that the redox state of the nuclei is an important factor in cell cycle progression. Controlled oxidation is a key feature of the early stages of the plant cell cycle. However, sustained mild oxidation restricts nuclear functions and impairs progression through the cell cycle leading to fewer cells in the root apical meristem. Antioxid. Redox Signal. 27, 1505-1519.

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.

A genome-wide resource of cell cycle and cell shape genes of fission yeast

PubMed Central

Hayles, Jacqueline; Wood, Valerie; Jeffery, Linda; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Salas-Pino, Silvia; Heichinger, Christian; Nurse, Paul

2013-01-01

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape. PMID:23697806

Systems-level feedback regulation of cell cycle transitions in Ostreococcus tauri.

PubMed

Kapuy, Orsolya; Vinod, P K; Bánhegyi, Gábor; Novák, Béla

2018-05-01

Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic position in the eukaryotic tree of life. In this study, we focus on the regulatory network of its cell division cycle. We propose a mathematical modelling framework to integrate the existing knowledge of cell cycle network of O. tauri. We observe that feedback loop regulation of both G1/S and G2/M transitions in O. tauri is conserved, which can make the transition bistable. This is essential to make the transition irreversible as shown in other eukaryotic organisms. By performing sequence analysis, we also predict the presence of the Greatwall/PP2A pathway in the cell cycle of O. tauri. Since O. tauri cell cycle machinery is conserved, the exploration of the dynamical characteristic of the cell division cycle will help in further understanding the regulation of cell cycle in higher eukaryotes. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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

Serial Charging Test on High Capacity Li-Ion Cells for the Orbiter Advanced Hydraulic Power System

NASA Technical Reports Server (NTRS)

Jeevarajan, Judith A.; Irlbeck, Brad

2006-01-01

Although it looks like module level voltage drives the cutoff for charge, the actual cutoff is due to unbalanced cell voltages that drive the module voltage up. Individual cell voltage drives the cutoff for discharge Low resistance cells are the first to reach the low-voltage cutoff Cell-to-Cell voltage differences are generally small and show similar trends for each cycle Increase for a distinct window during charge and at the end of discharge Increase in max to min cell voltage difference with time/cycles Decrease in max to min cell voltage difference during high current pulses with time/cycles Individual cell voltage trends (with respect to other cells) are very repeatable from cycle to cycle, although voltage slowly degrades with time/cycles (resistance growth) Much more difference observed near end of discharge Little change in order of cell voltage (cell with highest voltage to cell with lowest voltage) Temp sensor on the side of cell (between 2 cells) shows much greater rise during discharge than for single cell tests (18 C vs 5 C) Conclusion: Serial Charging of this string of cells is feasible as it has only a minor impact on useful capacity

Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367

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.

Pharmacodynamic Modeling of Cell Cycle Effects for Gemcitabine and Trabectedin Combinations in Pancreatic Cancer Cells

PubMed Central

Miao, Xin; Koch, Gilbert; Ait-Oudhia, Sihem; Straubinger, Robert M.; Jusko, William J.

2016-01-01

Combinations of gemcitabine and trabectedin exert modest synergistic cytotoxic effects on two pancreatic cancer cell lines. Here, systems pharmacodynamic (PD) models that integrate cellular response data and extend a prototype model framework were developed to characterize dynamic changes in cell cycle phases of cancer cell subpopulations in response to gemcitabine and trabectedin as single agents and in combination. Extensive experimental data were obtained for two pancreatic cancer cell lines (MiaPaCa-2 and BxPC-3), including cell proliferation rates over 0–120 h of drug exposure, and the fraction of cells in different cell cycle phases or apoptosis. Cell cycle analysis demonstrated that gemcitabine induced cell cycle arrest in S phase, and trabectedin induced transient cell cycle arrest in S phase that progressed to G2/M phase. Over time, cells in the control group accumulated in G0/G1 phase. Systems cell cycle models were developed based on observed mechanisms and were used to characterize both cell proliferation and cell numbers in the sub G1, G0/G1, S, and G2/M phases in the control and drug-treated groups. The proposed mathematical models captured well both single and joint effects of gemcitabine and trabectedin. Interaction parameters were applied to quantify unexplainable drug-drug interaction effects on cell cycle arrest in S phase and in inducing apoptosis. The developed models were able to identify and quantify the different underlying interactions between gemcitabine and trabectedin, and captured well our large datasets in the dimensions of time, drug concentrations, and cellular subpopulations. PMID:27895579

Elastic-plastic finite-element analyses of thermally cycled single-edge wedge specimens

NASA Technical Reports Server (NTRS)

Kaufman, A.

1982-01-01

Elastic-plastic stress-strain analyses were performed for single-edge wedge alloys subjected to thermal cycling in fluidized beds. Three cases (NASA TAZ-8A alloy under one cycling condition and 316 stainless steel alloy under two cycling conditions) were analyzed by using the MARC nonlinear, finite-element computer program. Elastic solutions from MARC showed good agreement with previously reported solutions that used the NASTRAN and ISO3DQ computer programs. The NASA TAZ-8A case exhibited no plastic strains, and the elastic and elastic-plastic analyses gave identical results. Elastic-plastic analyses of the 316 stainless steel alloy showed plastic strain reversal with a shift of the mean stresses in the compressive direction. The maximum equivalent total strain ranges for these cases were 13 to 22 percent greater than that calculated from elastic analyses.

Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

PubMed

Krüger, Angela V; Jelier, Rob; Dzyubachyk, Oleh; Zimmerman, Timo; Meijering, Erik; Lehner, Ben

2015-02-15

Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential chromatin regulators during the early development of an animal. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of mTOR's Catalytic Site by PKI-587 Is a Promising Therapeutic Option for Gastroenteropancreatic Neuroendocrine Tumor Disease.

PubMed

Freitag, Helma; Christen, Friederike; Lewens, Florentine; Grass, Irina; Briest, Franziska; Iwaszkiewicz, Sara; Siegmund, Britta; Grabowski, Patricia

2017-01-01

The characteristic clinical heterogeneity and mostly slow-growing behavior of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) cause problems in finding appropriate treatments. Thus, the current therapy options are not satisfactory. PKI-587 is a highly potent, novel dual inhibitor of PI3K and mTORC1/C2. We assessed the effects of PKI-587 in different GEP-NEN tumor models, including the poorly differentiated cell line LCC-18, and compared them with those of the established mTORC1 inhibitor everolimus. We treated BON, QGP-1, KRJ-I, and LCC-18 cell lines with increasing concentrations of the inhibitor PKI-587, and compared the results with those of everolimus and DMSO. We assessed the impact of the treatments on viability (WST-1 assay), on apoptotic processes (caspase 3/7 assay, JC-1), and on cell cycle regulation (flow cytometry). We determined alterations in signaling mediators by phosphor-specific Western blot analysis and conducted multiplexed gene expression analysis (nCounter® technology). In all cell lines, PKI-587 dose-dependently inhibited proliferation, whereas everolimus was less effective. Treatment with PKI-587 led to cell cycle arrest and induction of apoptosis and successfully suppressed activity of the direct mTORC1 target 4E-BP1, a crucial factor for tumor genesis only partially inhibited by everolimus. Gene expression analyses revealed relevant changes of RAS, MAPK, STAT, and PI3K pathway genes after treatment. Treatment-dependent and cell line-characteristic effects on AKT/Rb/E2F signaling regarding cell cycle control and apoptosis are extensively discussed in this paper. PI3K/mTOR dual targeting is a promising new therapeutic approach in neuroendocrine tumor disease that should be evaluated in further clinical trials. © 2016 The Author(s) Published by S. Karger AG, Basel.

Overcoming the response plateau in multiple myeloma: A novel bortezomib-based strategy for secondary induction and high-yield CD34+ stem cell mobilization

PubMed Central

Niesvizky, Ruben; Mark, Tomer M.; Ward, Maureen; Jayabalan, David S.; Pearse, Roger N.; Manco, Megan; Stern, Jessica; Christos, Paul J.; Mathews, Lena; Shore, Tsiporah B.; Zafar, Faiza; Pekle, Karen; Xiang, Zhaoying; Ely, Scott; Skerret, Donna; Chen-Kiang, Selina; Coleman, Morton; Lane, Maureen E.

2014-01-01

Purpose This phase 2 study evaluated bortezomib-based secondary induction and stem cell mobilization in 38 transplant-eligible myeloma patients who had an incomplete and stalled response to, or had relapsed after, previous immunomodulatory drug-based induction. Experimental design Patients received up to six 21-day cycles of bortezomib plus dexamethasone, with added liposomal doxorubicin for patients not achieving partial response or better by cycle 2 or very good partial response or better (≥VGPR) by cycle 4 (DoVeD), followed by bortezomib, high-dose cyclophosphamide, and filgrastim mobilization. Gene expression/signaling pathway analyses were conducted in purified CD34+ cells post-bortezomib-based mobilization and compared against patients who received only filgrastim ± cyclophosphamide. Plasma samples were similarly analyzed for quantification of associated protein markers. Results The response rate to DoVeD relative to the pre-DoVeD baseline was 61%, including 39% ≥VGPR. Deeper responses were achieved in 10 of 27 patients who received bortezomib-based mobilization; post-mobilization response rate was 96%, including 48% ≥VGPR, relative to the pre-DoVeD baseline. Median CD34+ cell yield was 23.2 × 106 cells/kg (median of 1 apheresis session). After a median follow-up of 46.6 months, median progression-free survival was 47.1 months from DoVeD initiation;5-year overall survival rate was 76.4%. Grade ≥3 adverse events included thrombocytopenia (13%), hand-foot syndrome (11%), peripheral neuropathy (8%), and neutropenia (5%). Bortezomib-based mobilization was associated with modulated expression of genes involved in stem cell migration. Conclusion Bortezomib-based secondary induction and mobilization could represent an alternative strategy for elimination of tumor burden in immunomodulatory drug-resistant patients that does not impact stem cell yield. PMID:23357980

Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

PubMed

Young, Kira; Borikar, Sneha; Bell, Rebecca; Kuffler, Lauren; Philip, Vivek; Trowbridge, Jennifer J

2016-10-17

Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging. © 2016 Young et al.

Rapid DNA replication origin licensing protects stem cell pluripotency

PubMed Central

Matson, Jacob Peter; Dumitru, Raluca; Coryell, Philip; Baxley, Ryan M; Chen, Weili; Twaroski, Kirk; Webber, Beau R; Tolar, Jakub; Bielinsky, Anja-Katrin; Purvis, Jeremy E

2017-01-01

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance. PMID:29148972

Architecture and inherent robustness of a bacterial cell-cycle control system.

PubMed

Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

2008-08-12

A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

Microarray Analysis of Differential Gene Expression Profile Between Human Fetal and Adult Heart.

PubMed

Geng, Zhimin; Wang, Jue; Pan, Lulu; Li, Ming; Zhang, Jitai; Cai, Xueli; Chu, Maoping

2017-04-01

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

PubMed Central

Sorokin, Dmitry V; Stixová, Lenka; Sehnalová, Petra; Legartová, Soňa; Suchánková, Jana; Šimara, Pavel; Kozubek, Stanislav; Matula, Pavel; Skalníková, Magdalena; Raška, Ivan; Bártová, Eva

2015-01-01

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase. PMID:26208041

Operando lithium plating quantification and early detection of a commercial LiFePO4 cell cycled under dynamic driving schedule

NASA Astrophysics Data System (ADS)

Anseán, D.; Dubarry, M.; Devie, A.; Liaw, B. Y.; García, V. M.; Viera, J. C.; González, M.

2017-07-01

Lithium plating is considered one of the most detrimental phenomenon in lithium ion batteries (LIBs), as it increases cell degradation and might lead to safety issues. Plating induced LIB failure presents a major concern for emerging applications in transportation and electrical energy storage. Hence, the necessity to operando monitor, detect and analyze lithium plating becomes critical for safe and reliable usage of LIB systems. Here, we report in situ lithium plating analyses for a commercial graphite||LiFePO4 cell cycled under dynamic stress test (DST) driving schedule. We designed a framework based on incremental capacity (IC) analysis and mechanistic model simulations to quantify degradation modes, relate their effects to lithium plating occurrence and assess cell degradation. The results show that lithium plating was induced by large loss of active material on the negative electrode that eventually led the electrode to over-lithiate. Moreover, when lithium plating emerged, we quantified that the loss of lithium inventory pace was increased by a factor of four. This study illustrates the benefits of the proposed framework to improve lithium plating analysis. It also discloses the symptoms of lithium plating formation, which prove valuable for novel, online strategies on early lithium plating detection.

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

PubMed

Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

2008-05-30

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

Single-Cell in Situ RNA Analysis With Switchable Fluorescent Oligonucleotides.

PubMed

Xiao, Lu; Guo, Jia

2018-01-01

Comprehensive RNA analyses in individual cells in their native spatial contexts promise to transform our understanding of normal physiology and disease pathogenesis. Here we report a single-cell in situ RNA analysis approach using switchable fluorescent oligonucleotides (SFO). In this method, transcripts are first hybridized by pre-decoding oligonucleotides. These oligonucleotides subsequently recruit SFO to stain their corresponding RNA targets. After fluorescence imaging, all the SFO in the whole specimen are simultaneously removed by DNA strand displacement reactions. Through continuous cycles of target staining, fluorescence imaging, and SFO removal, a large number of different transcripts can be identified by unique fluorophore sequences and visualized at the optical resolution. To demonstrate the feasibility of this approach, we show that the hybridized SFO can be efficiently stripped by strand displacement reactions within 30 min. We also demonstrate that this SFO removal process maintains the integrity of the RNA targets and the pre-decoding oligonucleotides, and keeps them hybridized. Applying this approach, we show that transcripts can be restained in at least eight hybridization cycles with high analysis accuracy, which theoretically would enable the whole transcriptome to be quantified at the single molecule sensitivity in individual cells. This in situ RNA analysis technology will have wide applications in systems biology, molecular diagnosis, and targeted therapies.

Environmental and economic assessment of a cracked ammonia fuelled alkaline fuel cell for off-grid power applications

NASA Astrophysics Data System (ADS)

Cox, Brian; Treyer, Karin

2015-02-01

Global mobile telecommunication is possible due to millions of Base Transceiver Stations (BTS). Nearly 1 million of these are operating off-grid, typically powered by diesel generators and therefore leading to significant CO2 emissions and other environmental burdens. A novel type of Alkaline Fuel Cell (AFC) powered by cracked ammonia is being developed for replacement of these generators. This study compares the environmental and economic performance of the two systems by means of Life Cycle Assessment (LCA) and Levelised Cost of Electricity (LCOE), respectively. Results show that the production of ammonia dominates the LCA results, and that renewable ammonia production pathways greatly improve environmental performance. Sensitivity analyses reveal that the fuel cell parameters that most affect system cost and environmental burdens are cell power density and lifetime and system efficiency. Recycling of anode catalyst and electrode substrate materials is found to have large impacts on environmental performance, though without large cost incentives. For a set of target parameter values and fossil sourced ammonia, the AFC is calculated to produce electricity with life cycle CO2 eq emissions of 1.08 kg kWh-1, which is 23% lower than a diesel generator with electricity costs that are 14% higher in the same application.

The Myxococcus xanthus Spore Cuticula Protein C Is a Fragment of FibA, an Extracellular Metalloprotease Produced Exclusively in Aggregated Cells

PubMed Central

Lee, Bongsoo; Mann, Petra; Grover, Vidhi; Treuner-Lange, Anke; Kahnt, Jörg; Higgs, Penelope I.

2011-01-01

Myxococcus xanthus is a soil bacterium with a complex life cycle involving distinct cell fates, including production of environmentally resistant spores to withstand periods of nutrient limitation. Spores are surrounded by an apparently self-assembling cuticula containing at least Proteins S and C; the gene encoding Protein C is unknown. During analyses of cell heterogeneity in M. xanthus, we observed that Protein C accumulated exclusively in cells found in aggregates. Using mass spectrometry analysis of Protein C either isolated from spore cuticula or immunoprecipitated from aggregated cells, we demonstrate that Protein C is actually a proteolytic fragment of the previously identified but functionally elusive zinc metalloprotease, FibA. Subpopulation specific FibA accumulation is not due to transcriptional regulation suggesting post-transcriptional regulation mechanisms mediate its heterogeneous accumulation patterns. PMID:22174937

Elevated germline mutation rate in teenage fathers.

PubMed

Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd

2015-03-22

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'.

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.

Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest

PubMed Central

Carén, Helena; Stricker, Stefan H.; Bulstrode, Harry; Gagrica, Sladjana; Johnstone, Ewan; Bartlett, Thomas E.; Feber, Andrew; Wilson, Gareth; Teschendorff, Andrew E.; Bertone, Paul; Beck, Stephan; Pollard, Steven M.

2015-01-01

Summary Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stem cell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stem cells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Here we find only a subset of GSC cultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy for GBM. PMID:26607953

Discovering new mTOR inhibitors for cancer treatment through virtual screening methods and in vitro assays

NASA Astrophysics Data System (ADS)

Wang, Ling; Chen, Lei; Yu, Miao; Xu, Li-Hui; Cheng, Bao; Lin, Yong-Sheng; Gu, Qiong; He, Xian-Hui; Xu, Jun

2016-01-01

Mammalian target of rapamycin (mTOR) is an attractive target for new anticancer drug development. We recently developed in silico models to distinguish mTOR inhibitors and non-inhibitors. In this study, we developed an integrated strategy for identifying new mTOR inhibitors using cascaded in silico screening models. With this strategy, fifteen new mTOR kinase inhibitors including four compounds with IC50 values below 10 μM were discovered. In particular, compound 17 exhibited potent anticancer activities against four tumor cell lines, including MCF-7, HeLa, MGC-803, and C6, with IC50 values of 1.90, 2.74, 3.50 and 11.05 μM. Furthermore, cellular studies and western blot analyses revealed that 17 induces cell death via apoptosis by targeting both mTORC1 and mTORC2 within cells and arrests the cell cycle of HeLa at the G1/G0-phase. Finally, multi-nanosecond explicit solvent simulations and MM/GBSA analyses were carried out to study the inhibitory mechanisms of 13, 17, and 40 for mTOR. The potent compounds presented here are worthy of further investigation.

Suppression of RRM2 inhibits cell proliferation, causes cell cycle arrest and promotes the apoptosis of human neuroblastoma cells and in human neuroblastoma RRM2 is suppressed following chemotherapy.

PubMed

Li, Junfeng; Pang, Jinglin; Liu, Yongdong; Zhang, Jing; Zhang, Chuanguang; Shen, Gang; Song, Lili

2018-07-01

Ribonucleotide reductase regulatory subunit M2 (RRM2) is a rate‑limiting enzyme for DNA synthesis and repair. RRM2 has vital roles in controlling the progression of cancer. In the present study, we investigated the RRM2 level in neuroblastoma tissues, analyzed its relationship with clinicopathological characteristics of neuroblastoma patients, and explored the effect of RRM2 on the biological functions of neuroblastoma cells. RRM2 levels in 67 pairs of neuroblastoma and matched adjacent non‑cancerous tissues were detected by qRT‑PCR, and its association with patient clinicopathological features was assessed. Using RRM2 siRNA, the role of RRM2 in cell viability was detected by CCK‑8 assay, and the effects on cell cycle distribution and cell apoptosis were detected by flow cytometry. Hoechst 33342 staining was also performed. For RRM2 protein detection in cells and tissues, western blot analyses were employed. Our results revealed that RRM2 expression was significant higher in neuroblastoma tissues than that noted in adjacent non‑cancerous tissues at both the mRNA and protein levels. The increased RRM2 level was significantly associated with clinical stage. RRM2 levels were suppressed in stage III and IV tumors in the chemotherapy subgroup, compared with levels noted in tumors in the preoperative non‑chemotherapy subgroup. RRM2 siRNA significantly inhibited cell viability in the SH‑5Y5Y cells, induced cell arrest in the G0/G1 phase, and enhanced cell apoptosis. Taken together, overexpression of RRM2 is associated with the genesis and progression of neuroblastoma, and may be a potential chemotherapeutic target.

HDAC inhibitors, MS-275 and salermide, potentiates the anticancer effect of EF24 in human pancreatic cancer cells.

PubMed

Yar Saglam, Atiye Seda; Yilmaz, Akin; Onen, Hacer Ilke; Alp, Ebru; Kayhan, Handan; Ekmekci, Abdullah

2016-01-01

Histone deacetylases (HDACs) play a major role in the regulation of chromatin structure and gene expression by changing acetylation status of histone and non-histone proteins. MS-275 (entinostat, MS) is a well-known benzamide-based HDACI and Salermide (SAL), a reverse amide compound HDACI, have antiproliferative effects on several human cancer cells. In this study, we aimed to investigate the effects of HDACIs (MS and SAL) alone and/or combined use with EF24 (EF), a novel synthetic curcumin analog, on human pancreatic cancer cell line (BxPC-3). In vitro, BxPC-3 cells were exposed to varying concentrations of MS, SAL with or without EF, and their effects on cell viability, acetylated Histone H3 and H4 levels, cytotoxicity, and cleaved caspase 3 levels, and cell cycle distribution were measured. The viability of BxPC-3 cells decreased significantly after treatment with EF, MS and SAL treatments. MS and SAL treatment increased the acetylation of histone H3 and H4 in a dose dependent manner. MS and SAL alone or combined with EF were increased the number of cells in G1 phase. In addition, treatment with agents significantly decreased the ratio of cell in G2/M phase. There were significant dose-dependent increases at cleaved Caspase 3 levels after MS treatment but not after SAL treatment. Our results showed that HDAC inhibitors (MS and SAL), when combined with EF, may effectively reduce pancreatic cancer cell (BxPC-3) progression and stop the cell cycle at G1 phase. Further molecular analyses are needed to understand the fundamental molecular consequences of HDAC inhibition in pancreas cancer cells.

Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells.

PubMed

Jung, Ha-Na; Zerin, Tamanna; Podder, Biswajit; Song, Ho-Yeon; Kim, Yong-Sik

2014-06-01

In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 μg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 μg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 μg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding antioxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intracellular reactive oxygen species and alteration of gene expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

PubMed Central

Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

2016-01-01

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis. PMID:26493868

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

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.

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis.

PubMed

Hu, Shen; Le, Zhang; Krylov, Sergey; Dovichi, Norman J

2003-07-15

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.

Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

PubMed

Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido

2007-09-15

When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

Antitumoral activity of 1,2-diaminocyclohexane derivatives in breast, colon and skin human cancer cells.

PubMed

Morales, Fátima; Ramírez, Alberto; Morata-Tarifa, Cynthia; Navarro, Saúl A; Marchal, Juan A; Campos, Joaquín M; Conejo-García, Ana

2017-03-01

Cancer is among the leading causes of death worldwide. Medical interest has focused on macrocyclic polyamines because of their properties as antitumor agents. Results/Methodology: We have designed and synthesized a series of 1,2-diaminocyclohexane derivatives with notable in vitro antiproliferative activities against the MCF-7, HCT-116 and A375 cancer cell lines. Cell cycle and apoptosis analyses were also carried out. Our results show that all the compounds are potent cytotoxic agents, especially against the A375 cell line. The selective activity of the macrocyclic derivative against A375, via apoptosis, supposes a great advantage for future therapeutic use. This exemplifies the potential of 1,2-diaminocyclohexane derivatives to qualify as lead structures for future anticancer drug development due to their easy syntheses and noteworthy bioactivity.

Repressive histone methylation regulates cardiac myocyte cell cycle exit.

PubMed

El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

2018-05-22

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.

Cytotoxic constituents from the mangrove endophytic Pestalotiopsis sp. induce G0/G1 cell cycle arrest and apoptosis in human cancer cells.

PubMed

Zhou, Jing; Li, Gang; Deng, Qin; Zheng, Dongyao; Yang, Xiaobo; Xu, Jing

2017-10-31

Chemical examination of Chinese mangrove Rhizophora mucronata endophytic Pestalotiopsis sp., yielded  11 known metabolites with various structure types, including demethylincisterol A 3 (1), dankasterone B (2), (22E, 24R)-ergosta-7,9(11), 22-triene-3β, 5α, 6α-triol (3), ergosta-5,7,22-trien-3-ol (4), 5, 8-epidioxy-5, 8-ergosta-6, 22E-dien-3-ol (5), stigmastan-3-one (6), stigmast-4-en-3-one (7), stigmast-4-en-6 -ol-3-one (8), flufuran (9), (2-cis, 4-trans)-abscisic acid (10), similanpyrone B (11). Their structures were unambiguously elucidated on the basis of extensive NMR spectroscopic and mass spectrometric analyses. Compounds 1, 4, 6-9 showed significant in vitro cytotoxicity against the human cancer cell lines Hela, A549 and HepG, of which compound 1 was the most potential with IC 50 values reaching nM degree ranging from 0.17 to 14.16 nM. Flow cytometric investigation demonstrated that compound 1 mainly inhibited cell cycle at G 0 /G 1 phase in a dose-dependent manner with a significant induction of apoptosis on the three tested cell lines. The involvement of the mitochondria in compound 1 induced apoptosis was investigated using MMP. We suggested that R. mucronata endophytic Pestalotiopsis sp. contained a potential anticancer compound demethylincisterol A 3 .

APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

PubMed

Local, Andrea; Zhang, Hongying; Benbatoul, Khalid D; Folger, Peter; Sheng, Xia; Tsai, Cheng-Yu; Howell, Stephen B; Rice, William G

2018-06-01

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G 0 -G 1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253) 3 ]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253) 3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253) 3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR . ©2018 American Association for Cancer Research.