Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

PubMed Central

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka

2017-01-01

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next. PMID:28906251

Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

PubMed

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka; Stearns, Tim

2017-09-14

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

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.

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

PubMed Central

Irons, R D

1981-01-01

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

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

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.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

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

Rubin, D.B.; Drab, E.A.; Ward, W.F.

1988-11-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and anmore » increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous (3H)thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro.« less

DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

PubMed Central

Yedjou, Clement G.; Tchounwou, Hervey M.; Tchounwou, Paul B.

2015-01-01

In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO3)2] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO3)2 for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO3)2-treated cells, indicative of membrane rupture by Pb(NO3)2 compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO3)2 exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO3)2 exposure caused cell cycle arrest at the G0/G1 checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO3)2 inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G0/G1 checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO3)2 exposure and its associated adverse health effects. PMID:26703663

DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

PubMed

Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

2015-12-22

In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

Chromatin Structure and the Cell Cycle

PubMed Central

Pederson, Thoru

1972-01-01

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

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.

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.

Transcriptome changes and cAMP oscillations in an archaeal cell cycle.

PubMed

Baumann, Anke; Lange, Christian; Soppa, Jörg

2007-06-11

The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.

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

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

PubMed

Ma, Zhaowu; Yu, Guanghui

2010-02-15

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

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

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

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

2013-05-25

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

Nucleosome architecture throughout the cell cycle

PubMed Central

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

2016-01-01

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

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.

Capacity decline of ambient temperature secondary Li-TiS2 cells

NASA Technical Reports Server (NTRS)

Subbarao, S.; Shen, D. H.; Huang, C.-K.; Deligiannis, F.; Halpert, G.; Peled, E.

1990-01-01

The main objective of the study described was to identify the causes responsible for the capacity losses observed during cycling of secondary Li-TiS2 cells. Experimental Li-TiS2 cells were fabricated and tested for their cycle life performance. The open circuit voltage of the cells was monitored during the rest period between the charging and discharging. The polarization at the Li and TiS2 electrodes was also monitored during cycling. Cycled cells were disassembled and the cathodes were analyzed by various analytical techniques. The results of the study indicate that the observed capacity loss is almost entirely due to the increased polarization of the TiS2 electrode with cycling. The electrolyte was found to degrade during cycling and the degradation products were found to deposit at the TiS2 electrode, which probably lead to the higher polarization.

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

NASA Astrophysics Data System (ADS)

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

2014-09-01

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

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.

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

PubMed Central

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

2018-01-01

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

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

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

Nanosecond pulsed electric fields and the cell cycle

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.

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

Do lipids shape the eukaryotic cell cycle?

PubMed

Furse, Samuel; Shearman, Gemma C

2018-01-01

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

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

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

Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey

2007-12-31

Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. Thesemore » cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.« less

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.

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

PubMed

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

2014-09-05

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

Cycling behavior of NCM523/graphite lithium-ion cells in the 3–4.4 V range: Diagnostic studies of full cells and harvested electrodes

DOE PAGES

Gilbert, James A.; Bareño, Javier; Spila, Timothy; ...

2016-09-22

Energy density of full cells containing layered-oxide positive electrodes can be increased by raising the upper cutoff voltage above the current 4.2 V limit. In this article we examine aging behavior of cells, containing LiNi 0.5Co 0.2Mn 0.3O 2 (NCM523)-based positive and graphite-based negative electrodes, which underwent up to ~400 cycles in the 3-4.4 V range. Electrochemistry results from electrodes harvested from the cycled cells were obtained to identify causes of cell performance loss; these results were complemented with data from X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectroscopy (SIMS) measurements. Our experiments indicate that the full cell capacitymore » fade increases linearly with cycle number and results from irreversible lithium loss in the negative electrode solid electrolyte interphase (SEI) layer. The accompanying electrode potential shift reduces utilization of active material in both electrodes and causes the positive electrode to cycle at higher states-of-charge. Here, full cell impedance rise on aging arises primarily at the positive electrode and results mainly from changes at the electrode-electrolyte interface; the small growth in negative electrode impedance reflects changes in the SEI layer. Our results indicate that cell performance loss could be mitigated by modifying the electrode-electrolyte interfaces through use of appropriate electrode coatings and/or electrolyte additives.« less

CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

PubMed Central

Van't Hof, Jack

1965-01-01

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G 1, S, and G 2 periods of interphase.1 The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours. PMID:5857253

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

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

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

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

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

Martinotti, Simona; Ranzato, Elia, E-mail: ranzato@unipmn.it; Parodi, Monica

Malignant mesothelioma (MMe) is a poor-prognosis tumor in need of innovative therapies. In a previous in vivo study, we showed synergistic anti-MMe properties of the ascorbate/epigallocatechin-3-gallate/gemcitabine combination. We have now focused on the mechanism of action, showing the induction of apoptosis and cell cycle arrest through measurements of caspase 3, intracellular Ca{sup 2+}, annexin V, and DNA content. StellArray™ PCR technology and Western immunoblotting revealed DAPK2-dependent apoptosis, upregulation of cell cycle promoters, downregulation of cell cycle checkpoints and repression of NFκB expression. The complex of data indicates that the mixture is synergistic in inducing cell cycle deregulation and non-inflammatory apoptosis,more » suggesting its possible use in MMe treatment. - Highlights: • Ascorbate/epigallocathechin-gallate/gemcitabine has been tested on mesothelioma cells • A synergistic mechanism has been shown for cell cycle arrest and apoptosis • PCR-array analysis has revealed the de-regulation of apoptosis and cell cycle genes • Maximum upregulation has been found for the Death-Associated Protein Kinase-2 gene • Data suggest that the mixture could be used as a clinical treatment.« less

Induction of cell death in renal cell carcinoma with combination of D-fraction and vitamin C.

PubMed

Alexander, Bobby; Fishman, Andrew I; Eshghi, Majid; Choudhury, Muhammad; Konno, Sensuke

2013-09-01

Although several conventional therapeutic options for advanced renal cell carcinoma (RCC) are currently available, the unsatisfactory outcomes demand establishing more effective interventions. D-fraction (PDF), a bioactive proteoglucan of Maitake mushroom, demonstrates anticancer and immunomodulatory activities, which are also shown to be potentiated by vitamin C (VC). We thus hypothesized that a combination of PDF and VC (PDF + VC) could be an alternative approach to more effectively inhibit the growth of RCC. We examined the dose-dependent effects of PDF + VC on RCC cell viability and also performed biochemical assays to explore the growth regulatory mechanism. Human RCC, ACHN cell line, was employed and exposed to varying concentrations of PDF or VC and their combinations. Cell viability at specified times was determined by MTT assay. Lipid peroxidation assay, cell cycle analysis, and Western blot analysis were also performed. PDF or VC alone led to the significant reduction in cell viability at 72 hours with PDF >500 µg/mL and VC ≥300 µM. When various combinations of PDF and VC were tested, the combination of the ineffective concentrations of PDF (300 µg/mL) and VC (200 µM) resulted in ~90% cell death in 24 hours. Lipid peroxidation assay then indicated significantly (~2.5 fold) elevated oxidative stress with this PDF + VC. Cell cycle analysis also indicated a G1 cell cycle arrest following a 6-hour PDF + VC treatment. Western blots further revealed a downregulation of Bcl2, an upregulation of Bax, and proteolytic activation of PARP (poly[ADP-ribose] polymerase) in PDF + VC-treated cells, indicating induction of apoptosis. The present study demonstrates that the combination of PDF and VC can become highly cytotoxic, inducing severe cell death in ACHN cells. This cytotoxic mechanism appears to be primarily attributed to oxidative stress, accompanied by a G1 cell cycle arrest. Such cell death induced by PDF + VC could be more likely linked to apoptosis, as indicated by the modulation of apoptosis regulators (Bcl2, Bax, and PARP). Therefore, as PDF and VC may work synergistically to induce apoptotic cell death, they may have clinical implications in an alternative, improved therapeutic modality for advanced RCC.

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.

Cell-cycle-dependent drug-resistant quiescent cancer cells induce tumor angiogenesis after chemotherapy as visualized by real-time FUCCI imaging.

PubMed

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

2017-03-04

We previously demonstrated that quiescent cancer cells in a tumor are resistant to conventional chemotherapy as visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). We also showed that proliferating cancer cells exist in a tumor only near nascent vessels or on the tumor surface as visualized with FUCCI and green fluorescent protein (GFP)-expressing tumor vessels. In the present study, we show the relationship between cell-cycle phase and chemotherapy-induced tumor angiogenesis using in vivo FUCCI real-time imaging of the cell cycle and nestin-driven GFP to detect nascent blood vessels. We observed that chemotherapy-treated tumors, consisting of mostly of quiescent cancer cells after treatment, had much more and deeper tumor vessels than untreated tumors. These newly-vascularized cancer cells regrew rapidly after chemotherapy. In contrast, formerly quiescent cancer cells decoyed to S/G 2 phase by a telomerase-dependent adenovirus did not induce tumor angiogenesis. The present results further demonstrate the importance of the cancer-cell position in the cell cycle in order that chemotherapy be effective and not have the opposite effect of stimulating tumor angiogenesis and progression.

Cell-cycle-dependent drug-resistant quiescent cancer cells induce tumor angiogenesis after chemotherapy as visualized by real-time FUCCI imaging

PubMed Central

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2017-01-01

ABSTRACT We previously demonstrated that quiescent cancer cells in a tumor are resistant to conventional chemotherapy as visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). We also showed that proliferating cancer cells exist in a tumor only near nascent vessels or on the tumor surface as visualized with FUCCI and green fluorescent protein (GFP)-expressing tumor vessels. In the present study, we show the relationship between cell-cycle phase and chemotherapy-induced tumor angiogenesis using in vivo FUCCI real-time imaging of the cell cycle and nestin-driven GFP to detect nascent blood vessels. We observed that chemotherapy-treated tumors, consisting of mostly of quiescent cancer cells after treatment, had much more and deeper tumor vessels than untreated tumors. These newly-vascularized cancer cells regrew rapidly after chemotherapy. In contrast, formerly quiescent cancer cells decoyed to S/G2 phase by a telomerase-dependent adenovirus did not induce tumor angiogenesis. The present results further demonstrate the importance of the cancer-cell position in the cell cycle in order that chemotherapy be effective and not have the opposite effect of stimulating tumor angiogenesis and progression. PMID:27715464

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.

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.

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

PubMed

Bieler, Jonathan; Cannavo, Rosamaria; Gustafson, Kyle; Gobet, Cedric; Gatfield, David; Naef, Felix

2014-07-15

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Cycling-Induced Changes in the Entropy Profiles of Lithium Cobalt Oxide Electrodes

DOE PAGES

Hudak, N. S.; Davis, L. E.; Nagasubramanian, G.

2014-12-09

Entropy profiles of lithium cobalt oxide (LiCoO2) electrodes were measured at various stages in the cycle life to examine performance degradation and cycling-induced changes, or lack thereof, in thermodynamics. LiCoO 2 electrodes were cycled at C/2 rate in half-cells (vs. lithium anodes) up to 20 cycles or C/5 rate in full cells (vs. MCMB anodes) up to 500 cycles. The electrodes were then subjected to entropy measurements (∂E/∂T, where E is open-circuit potential and T is temperature) in half-cells at regular intervals over the approximate range 0.5 ≤ x ≤ 1 in LixCoO 2. Despite significant losses in capacity uponmore » cycling, neither cycling rate resulted in any change to the overall shape of the entropy profile relative to an uncycled electrode, indicating retention of the basic LiCoO 2 structure, lithium insertion mechanism, and thermodynamics. This confirms that cycling-induced performance degradation in LiCoO 2 electrodes is primarily caused by kinetic barriers that increase with cycling. In the case of electrodes cycled at C/5, there was a subtle, quantitative, and gradual change in the entropy profile in the narrow potential range of the hexagonal-to-monoclinic phase transition. The observed change is indicative of a decrease in the intralayer lithium ordering that occurs at these potentials, and it demonstrates that a cyclinginduced structural disorder accompanies the kinetic degradation mechanisms.« less

Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex

NASA Technical Reports Server (NTRS)

Cai, L.; Hayes, N. L.; Nowakowski, R. S.

1997-01-01

We have analyzed clonal cell proliferation in the ventricular zone (VZ) of the early developing mouse neocortex with a replication-incompetent retrovirus encoding human placental alkaline phosphatase (AP). The retrovirus was injected into the lateral ventricles on embryonic day 11 (E11), i.e., at the onset of neuronogenesis. Three days postinjection, on E14, a total of 259 AP-labeled clones of various sizes were found in 7 fetal brains. There are approximately 7 cell cycles between E11 and E14 (), and there is a 1-2 cell cycle delay between retroviral injection and the production of a retrovirally labeled "founder" cell; thus, we estimate that the "age" of the clones was about 5-6 cell cycles. Almost one-half of the clones (48.3%) identified were pure proliferating clones containing cells only in the VZ. Another 18.5% contained both proliferating and postproliferative cells, and 33.2% contained only postproliferative cells. It was striking that over 90% of the clonally related proliferating cells occurred in clusters of two or more apparently contiguous cells, and about 73% of the proliferating cells occurred in clusters of three or more cells. Regardless of the number of cells in the clone, these clusters were tightly packed and confined to a single level of the VZ. This clustering of proliferating cells indicates that clonally related cells maintain neighbor-neighbor relationships as they undergo interkinetic nuclear migration and progress through several cell cycles, and, as a result, the ventricular zone is a mosaic of small clusters of clonally related and synchronously cycling cells. In addition, cells in the intermediate zone and the cortical plate were also frequently clustered, indicating that they became postproliferative at a similar time and that the output of the VZ is influenced by its mosaic structure.

Structural characterization of the cell division cycle in Strigomonas culicis, an endosymbiont-bearing trypanosomatid.

PubMed

Brum, Felipe Lopes; Catta-Preta, Carolina Moura Costa; de Souza, Wanderley; Schenkman, Sergio; Elias, Maria Carolina; Motta, Maria Cristina Machado

2014-02-01

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis.

PubMed

Xu, Deyang; Huang, Weihua; Li, Yang; Wang, Hua; Huang, Hai; Cui, Xiaofeng

2012-03-01

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

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

PubMed Central

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

2015-01-01

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

The potential role of ribosomal protein S5 on cell cycle arrest and initiation of murine erythroleukemia cell differentiation.

PubMed

Matragkou, Christina N; Papachristou, Eleni T; Tezias, Sotirios S; Tsiftsoglou, Asterios S; Choli-Papadopoulou, Theodora; Vizirianakis, Ioannis S

2008-07-01

Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL-C14 and MEL-C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G(1)/G(0) phase, and (c) modulate the level of cyclin-dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer-treated MEL cells. 2008 Wiley-Liss, Inc.

Tributyltin induces cell cycle arrest at G1 phase in the yeast Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Akiyama, Koichi; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2014-04-01

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

KOH concentration effect on the cycle life of nickel-hydrogen cells. Part 4: Results of failure analyses

NASA Technical Reports Server (NTRS)

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

1989-01-01

KOH concentration effects on cycle life of a Ni/H2 cell have been studied by carrying out a cycle life test of ten Ni/H2 boiler plate cells which contain electrolytes of various KOH concentrations. Failure analyses of these cells were carried out after completion of the life test which accumulated up to 40,000 cycles at an 80 percent depth of discharge over a period of 3.7 years. These failure analyses included studies on changes of electrical characteristics of test cells and component analyses after disassembly of the cell. The component analyses included visual inspections, dimensional changes, capacity measurements of nickel electrodes, scanning electron microscopy, BET surface area measurements, and chemical analyses. Results have indicated that failure mode and change in the nickel electrode varied as the concentration was varied, especially, when the concentration was changed from 31 percent or higher to 26 percent or lower.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

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

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Aging and insulin signaling differentially control normal and tumorous germline stem cells.

PubMed

Kao, Shih-Han; Tseng, Chen-Yuan; Wan, Chih-Ling; Su, Yu-Han; Hsieh, Chang-Che; Pi, Haiwei; Hsu, Hwei-Jan

2015-02-01

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Inhibitor of DNA binding 1 regulates cell cycle progression of endothelial progenitor cells through induction of Wnt2 expression

PubMed Central

Xia, Xi; Yu, Yang; Zhang, Li; Ma, Yang; Wang, Hong

2016-01-01

Endothelial injury is a risk factor for atherosclerosis. Endothelial progenitor cell (EPC) proliferation contributes to vascular injury repair. Overexpression of inhibitor of DNA binding 1 (Id1) significantly promotes EPC proliferation; however, the underlying molecular mechanism remains to be fully elucidated. The present study investigated the role of Id1 in cell cycle regulation of EPCs, which is closely associated with proliferation. Overexpression of Id1 increased the proportion of EPCs in the S/G2M phase and significantly increased cyclin D1 expression levels, while knockdown of Id1 arrested the cell cycle progression of EPCs in the G1 phase and inhibited cyclin D1 expression levels. In addition, it was demonstrated that Id1 upregulated wingless-type mouse mammary tumor virus integration site family member 2 (Wnt2) expression levels and promoted β-catenin accumulation and nuclear translocation. Furthermore, Wnt2 knockdown counteracted the effects of Id1 on cell cycle progression of EPCs. In conclusion, the results of the present study indicate that Id1 promoted Wnt2 expression, which accelerated cell cycle progression from G1 to S phase. This suggests that Id1 may promote cell cycle progression of EPCs, and that Wnt2 may be important in Id1 regulation of the cell cycle of EPCs. PMID:27432753

Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells.

PubMed Central

Ligueros, M.; Jeoung, D.; Tang, B.; Hochhauser, D.; Reidenberg, M. M.; Sonenberg, M.

1997-01-01

The antiproliferative effects of gossypol on human MCF-7 mammary cancer cells and cyclin D1-transfected HT-1060 human fibrosarcoma cells were investigated by cell cycle analysis and effects on the cell cycle regulatory proteins Rb and cyclin D1. Flow cytometry of MCF-7 cells at 24 h indicated that 10 microM gossypol inhibited DNA synthesis by producing a G1/S block. Western blot analysis using anti-human Rb antibodies and anti-human cyclin D1 antibodies in MCF-7 cells and high- and low-expression cyclin D1-transfected fibrosarcoma cells indicated that, after 6 h exposure, gossypol decreased the expression levels of these proteins in a dose-dependent manner. Gossypol also decreased the ratio of phosphorylated to unphosphorylated Rb protein in human mammary cancer and fibrosarcoma cell lines. Gossypol (10 microM) treated also decreased cyclin D1-associated kinase activity on histone H1 used as a substrate in MCF-7 cells. These results suggest that gossypol might suppress growth by modulating the expression of cell cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9218727

Development of ambient temperature secondary lithium cells

NASA Technical Reports Server (NTRS)

Subbarao, S.; Shen, D. H.; Dawson, S.; Deligiannis, F.; Taraszkiewicz, J.; Halpert, G.

1988-01-01

JPL is developing ambient temperature secondary lithium cells for future spacecraft applications. Prior studies on experimental laboratory type Li-TiS2 cells yielded promising results in terms of cycle life and rate capability. To further assess the performance of this cell, 5 Ah engineering model cells were developed. Initially baseline cells were designed and fabricated. Each cell had 15 cathodes and 16 anodes and the ratio of anode to cathode capacity is 6:1. A solution of 1.5 molar LiAsF6 in 2Me-THF was used as the electrolyte. Cells were evaluated for their cycle life at C/1 and C/5 discharge rates and 100 percent depth of discharge. The cells were cycled between voltage limits 1.7 and 2.8 volts. The rate of charge in all cases is C/10. The results obtained indicate that cells can operate at C/10 to C/2 discharge rates and have an initial energy density of 70 Wh/kg. Cells delivered more than 100 cycles at C/2 discharge rate. The details of cell design, the test program, and the results obtained are described.

Development of ambient temperature secondary lithium cells

NASA Technical Reports Server (NTRS)

Subbarao, S.; Shen, D. H.; Dawson, S.; Deligiannis, F.; Taraszkiewicz, J.; Halpert, Gerald

1987-01-01

JPL is developing ambient temperature secondary lithium cells for future spacecraft applications. Prior studies on experimental laboratory type Li-TiS2 cells yielded promising results in terms of cycle life and rate capability. To further assess the performance of this cell, 5 Ah engineering model cells were developed. Initially baseline cells were designed and fabricated. Each cell had 15 cathodes and 16 anodes and the ratio of anode to cathode capacity is 6:1. A solution of 1.5 molar LiAsF6 in 2Me-THF was used as the electrolyte. Cells were evaluated for their cycle life at C/1 and C/5 discharge rates and 100 percent depth of discharge. The cells were cycled between voltage limits 1.7 and 2.8 volts. The rate of charge in all cases is C/10. The results obtained indicate that cells can operate at C/10 to C/2 discharge rates and have an initial energy density of 70 Wh/kg. Cells delivered more than 100 cycles at C/2 discharge rate. The details of cell design, the test program, and the results obtained are described.

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

PubMed Central

Beaumont, Kimberley A.; Anfosso, Andrea; Ahmed, Farzana

2015-01-01

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761

Investigation of welded interconnection of large area wraparound contacted silicon solar cells

NASA Technical Reports Server (NTRS)

Lott, D. R.

1984-01-01

An investigation was conducted to evaluate the welding and temperature cycle testing of large area 5.9 x 5.9 wraparound silicon solar cells utilizing printed circuit substrates with SSC-155 interconnect copper metals and the LMSC Infrared Controlled weld station. An initial group of 5 welded modules containing Phase 2 developmental 5.9 x 5.9 cm cells were subjected to cyclical temperatures of + or 80 C at a rate of 120 cycles per day. Anomalies were noted in the adhesion of the cell contact metallization; therefore, 5 additional modules were fabricated and tested using available Phase I cells with demonstrated contact integrity. Cycling of the later module type through 12,000 cycles indicated the viability of this type of lightweight flexible array concept. This project demonstrated acceptable use of an alternate interconnect copper in combination with large area wraparound cells and emphasized the necessity to implement weld pull as opposed to solder pull procedures at the cell vendors for cells that will be interconnected by welding.

Cancer cells mimic in vivo spatial-temporal cell-cycle phase distribution and chemosensitivity in 3-dimensional Gelfoam® histoculture but not 2-dimensional culture as visualized with real-time FUCCI imaging.

PubMed

Yano, Shuya; Miwa, Shinji; Mii, Sumiyuki; Hiroshima, Yukihiko; Uehara, Fuminaru; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Zhao, Ming; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M

2015-01-01

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We previously reported monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor, intravitally in live mice, using a fluorescence ubiquitination-based cell-cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after cessation of chemotherapy. These results suggested why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. In the present report, we used FUCCI imaging and Gelfoam® collagen-sponge-gel histoculture, to demonstrate in real time, that the cell-cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is highly similar, whereby only the surface cells proliferate and interior cells are quiescent in G0/G1. This is in contrast to 2D culture where most cancer cells cycle. Similarly, the cancer cells responded similarly to toxic chemotherapy in Gelfoam® culture as in vivo, and very differently than cancer cells in 2D culture which were much more chemosensitive. Gelfoam® culture of FUCCI-expressing cancer cells offers the opportunity to image the cell cycle of cancer cells continuously and to screen for novel effective therapies to target quiescent cells, which are the majority in a tumor and which would have a strong probability to be effective in vivo.

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

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

PubMed

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

2016-02-01

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

Thermal and Cycle-Life Behavior of Commercial Li-ion and Li-Polymer Cells

NASA Technical Reports Server (NTRS)

Zimmerman, Albert H.; Quinzio, M. V.

2001-01-01

Accelerated and real-time LEO cycle-life test data will be presented for a range of commercial Li-ion and Li-polymer (gel type) cells indicating the ranges of performance that can be obtained, and the performance screening tests that must be done to assure long life. The data show large performance variability between cells, as well as a highly variable degradation signature during non-cycling periods within the life tests. High-resolution Dynamic Calorimetry data will be presented showing the complex series of reactions occurring within these Li cells as they are cycled. Data will also be presented for cells being tested using an Adaptive Charge Control Algorithm (ACCA) that continuously adapts itself to changes in cell performance, operation, or environment to both find and maintain the optimum recharge over life. The ACCA has been used to prevent all unneeded overcharge for Li cells, NiCd cells and NiH2 cells. While this is important for all these cell types, it is most critical for Li-ion cells, which are not designed with electrochemical tolerance for overcharge.

Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

PubMed

Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

2016-05-01

Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

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

PubMed

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

2004-05-01

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

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

PubMed

Zuma, Aline Araujo; Mendes, Isabela Cecília; Reignault, Lissa Catherine; Elias, Maria Carolina; de Souza, Wanderley; Machado, Carlos Renato; Motta, Maria Cristina M

2014-02-01

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects approximately 8 million people in Latin America. This parasite contains a single nucleus and a kinetoplast, which harbors the mitochondrial DNA (kDNA). DNA topoisomerases act during replication, transcription and repair and modulate DNA topology by reverting supercoiling in the DNA double-strand. In this work, we evaluated the effects promoted by camptothecin, a topoisomerase I inhibitor that promotes protozoan proliferation impairment, cell cycle arrest, ultrastructure alterations and DNA lesions in epimastigotes of T. cruzi. The results showed that inhibition of cell proliferation was reversible only at the lowest drug concentration (1μM) used. The unpacking of nuclear heterochromatin and mitochondrion swelling were the main ultrastructural modifications observed. Inhibition of parasite proliferation also led to cell cycle arrest, which was most likely caused by nuclear DNA lesions. Following camptothecin treatment, some of the cells restored their DNA, whereas others entered early apoptosis but did not progress to late apoptosis, indicating that the protozoa stay alive in a "senescence-like" state. This programmed cell death may be associated with a decrease in mitochondrial membrane potential and an increase in the production of reactive oxygen species. Taken together, these results indicate that the inhibition of T. cruzi proliferation is related to events capable of affecting cell cycle, DNA organization and mitochondrial activity. Copyright © 2014. Published by Elsevier B.V.

A generalized model for multi-marker analysis of cell cycle progression in synchrony experiments.

PubMed

Mayhew, Michael B; Robinson, Joshua W; Jung, Boyoun; Haase, Steven B; Hartemink, Alexander J

2011-07-01

To advance understanding of eukaryotic cell division, it is important to observe the process precisely. To this end, researchers monitor changes in dividing cells as they traverse the cell cycle, with the presence or absence of morphological or genetic markers indicating a cell's position in a particular interval of the cell cycle. A wide variety of marker data is available, including information-rich cellular imaging data. However, few formal statistical methods have been developed to use these valuable data sources in estimating how a population of cells progresses through the cell cycle. Furthermore, existing methods are designed to handle only a single binary marker of cell cycle progression at a time. Consequently, they cannot facilitate comparison of experiments involving different sets of markers. Here, we develop a new sampling model to accommodate an arbitrary number of different binary markers that characterize the progression of a population of dividing cells along a branching process. We engineer a strain of Saccharomyces cerevisiae with fluorescently labeled markers of cell cycle progression, and apply our new model to two image datasets we collected from the strain, as well as an independent dataset of different markers. We use our model to estimate the duration of post-cytokinetic attachment between a S.cerevisiae mother and daughter cell. The Java implementation is fast and extensible, and includes a graphical user interface. Our model provides a powerful and flexible cell cycle analysis tool, suitable to any type or combination of binary markers. The software is available from: http://www.cs.duke.edu/~amink/software/cloccs/. michael.mayhew@duke.edu; amink@cs.duke.edu.

Cell output, cell cycle duration and neuronal specification: a model of integrated mechanisms of the neocortical proliferative process

NASA Technical Reports Server (NTRS)

Caviness, V. S. Jr; Goto, T.; Tarui, T.; Takahashi, T.; Bhide, P. G.; Nowakowski, R. S.

2003-01-01

The neurons of the neocortex are generated over a 6 day neuronogenetic interval that comprises 11 cell cycles. During these 11 cell cycles, the length of cell cycle increases and the proportion of cells that exits (Q) versus re-enters (P) the cell cycle changes systematically. At the same time, the fate of the neurons produced at each of the 11 cell cycles appears to be specified at least in terms of their laminar destination. As a first step towards determining the causal interrelationships of the proliferative process with the process of laminar specification, we present a two-pronged approach. This consists of (i) a mathematical model that integrates the output of the proliferative process with the laminar fate of the output and predicts the effects of induced changes in Q and P during the neuronogenetic interval on the developing and mature cortex and (ii) an experimental system that allows the manipulation of Q and P in vivo. Here we show that the predictions of the model and the results of the experiments agree. The results indicate that events affecting the output of the proliferative population affect both the number of neurons produced and their specification with regard to their laminar fate.

The Molecules of the Cell Membrane.

ERIC Educational Resources Information Center

Bretscher, Mark S.

1985-01-01

Cell membrane molecules form a simple, two-dimensional liquid controlling what enters and leaves the cell. Discusses cell membrane molecular architecture, plasma membranes, epithelial cells, cycles of endocytosis and exocytosis, and other topics. Indicates that some cells internalize, then recycle, membrane area equivalent to their entire surface…

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.

Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8+ T Cell Recognition across All Three Phases of the Lytic Cycle

PubMed Central

Quinn, Laura L.; Zuo, Jianmin; Abbott, Rachel J. M.; Shannon-Lowe, Claire; Tierney, Rosemary J.; Hislop, Andrew D.; Rowe, Martin

2014-01-01

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

Local homogeneity of cell cycle length in developing mouse cortex

NASA Technical Reports Server (NTRS)

Cai, L.; Hayes, N. L.; Nowakowski, R. S.

1997-01-01

We have measured the amount of variation in the length of the cell cycle for cells in the pseudostratified ventricular epithelium (PVE) of the developing cortex of mice on embryonic day 14. Our measurements were made in three cortical regions (i.e., the neocortex, archicortex, and periarchicortex) using three different methods: the cumulative labeling method (CLM), the percent labeled mitoses (PLM) method, and a comparison of the time needed for the PLM to ascend from 0 to 100% with the time needed for the PLM to descend from 100 to 0%. These 3 different techniques provide different perspectives on the cytokinetic parameters. Theoretically, CLM gives an estimate for a maximum value of the total length of the cell cycle (TC), whereas PLM gives an estimate of a minimum value of TC. The difference between these two estimates indicates that the range for TC is +/-1% of the mean TC for periarchicortex, +/-7% for neocortex, and +/-8% for archicortex. This was confirmed by a lengthening of the PLM descent time in comparison with its ascent time. The sharpness of the transitions and the flatness of the plateau of the PLM curves indicate that 99% of the proliferating cells are within this narrow estimated range for TC; hence, only approximately 1% deviate outside of a relatively restricted range from the average TC of the population. In the context of the possible existence within the cortical PVE of two populations with markedly dissimilar cell cycle kinetics from the mean, one such population must comprise approximately 99% of the total population, and the other, if it exists, is only approximately 1% of the total. This seems to be true for all three cortical regions. The narrow range of TC indicates a homogeneity in the cell cycle length for proliferating cells in three different cortical regions, despite the fact that progenitor cells of different lineages may be present. It further predicts the existence of almost synchronous interkinetic nuclear movements of the proliferating cells in the ventricular zone during early development of the cerebral cortex.

Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

PubMed

Kaldis, Philipp; Aleem, Eiman

2005-11-01

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

Multiparameter Cell Cycle Analysis.

PubMed

Jacobberger, James W; Sramkoski, R Michael; Stefan, Tammy; Woost, Philip G

2018-01-01

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

Alternative therapeutic approach to renal-cell carcinoma: induction of apoptosis with combination of vitamin K3 and D-fraction.

PubMed

Degen, Michael; Alexander, Bobby; Choudhury, Muhammad; Eshghi, Majid; Konno, Sensuke

2013-12-01

Because of a dismal prognosis for advanced renal-cell carcinoma (RCC), an alternative therapeutic approach, using vitamin K3 (VK3) and D-fraction (DF) was investigated. VK3 is a synthetic VK derivative and DF is a bioactive mushroom extract, and they have been shown to have antitumor activity. We examined if the combination of VK3 and DF would exhibit the improved anticancer effect on RCC in vitro. Human RCC, ACHN cell line, were treated with varying concentrations of VK3, DF, or a combination of the two. Cell viability was assessed at 72 hours by MTT assay. To explore the possible anticancer mechanism, studies on cell cycle, chromatin modifications, and apoptosis were conducted. VK3 alone led to a ~20% reduction in cell viability at 4 μM, while DF alone induced a 20% to 45% viability reduction at ≥ 500 μg/mL. A combination of VK3 (4 μM) and DF (300 μg/mL) led to a drastic >90% viability reduction, however. Cell cycle analysis indicated that VK3/DF treatment induced a G1 cell cycle arrest, accompanied by the up-regulation of p21(WAF1) and p27(Kip1). Histone deacetylase (HDAC) was also significantly (~60%) inactivated, indicating chromatin modifications. In addition, Western blot analysis revealed that the up-regulation of Bax and activation of poly-(ADP-ribose)-polymerase (PARP) were seen in VK3/DF-treated cells, indicating induction of apoptosis. The combination of VK3 and DF can lead to a profound reduction in ACHN cell viability, through a p21(WAF1)-mediated G1 cell cycle arrest, and ultimately induces apoptosis. Therefore, the combination of VK3/DF may have clinical implications as an alternative, improved therapeutic modality for advanced RCC.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy.

PubMed

Baithalu, Rubina Kumari; Singh, S K; Gupta, Chhavi; Raja, Anuj K; Saxena, Abhishake; Kumar, Yogendra; Singh, R; Agarwal, S K

2013-08-01

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3β-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3β-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23μm) and LLC (range 25-55μm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3β-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

A novel electrolyte salt additive for lithium-ion batteries with voltages greater than 4.7 V

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

Li, Yunchao; Wan, Shun; Veith, Gabriel M.

2016-11-07

Here, lithium bis(2-methyl-2-fluoromalonato)borate (LiBMFMB), as an additive in conventional electrolyte for LiNi 0.5Mn 1.5O 4, exhibits improved coulombic efficiencies and cycling stability. Cyclic voltammograms indicate the cells with additive form good SEIs during the first cycle whereas no additive cell needs more cycles to form a functional SEI. XPS reveals LiBMFMB could reduce the decomposition of LiPF 6 salt and solvents, resulting in thinner SEI.

Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

PubMed Central

Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

2016-01-01

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle. PMID:27180572

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation.

PubMed

Liu, Tai-Hang; Wu, Yun-Fei; Dong, Xiao-Long; Pan, Cai-Xia; Du, Guo-Yu; Yang, Ji-Gui; Wang, Wei; Bao, Xi-Yan; Chen, Peng; Pan, Min-Hui; Lu, Cheng

2017-05-03

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.

Lithium/disulfide battery R and D

NASA Astrophysics Data System (ADS)

Kaun, T. D.; Deluca, W.; Lee, J.; Redey, L.; Nelson, P. A.

The focus of molten-salt cell R and D in the past year at Argonne National Laboratory has been on developing an understanding of the excellent performance and stability of a lithium/disulfide cell using LiCl-LiBr-KBr electrolyte. For further improvement, we have initiated development of a rod-electrode cell design and design of cells which can tolerate overdischarge and overcharge abuse. Earlier Li/FeS2 cells offered performance quite below expectations and had high capacity decline rates: 0.10 to 0.25 percent per cycle. Approaches for reducing the capacity decline rates of the earlier cells also reduced cell performance. However, our improved Li/FeS2 cell tests indicate good prospects for attaining cell development goals of specific energy of 200 Wh/kg at a 4-h discharge rate, a specific power of 200 W/kg at 80 percent depth of discharge, and a cycle life of 1000 cycles.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

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

"Fuel Gage" for Electric Vehicles

NASA Technical Reports Server (NTRS)

Rowlette, J. J.

1984-01-01

Gas-emmission and time-integrated-current measurements indicate battery charge state. Tests indicate possibility of monitoring state of charge of lead/acid batteries at any stage in charging cycle by measuring charging current and either gas evolution or electrode potential. Data then processed by microcomputer. Uses include cell voltage, cell pressure, cell temperature and rate of gas recombination on catalyst.

Solar power satellite system definition study. Volume 4: Silicon solar cell annealing test, phase 1

NASA Technical Reports Server (NTRS)

Walker, F.

1979-01-01

Laser annealing tests were conducted on ten 50 micron cells. Two were control cells that were not irradiated. These showed no loss in output due to exposure to the laser. Two cells were broken in handling. Six cells were successfully tested. All cells tested without breakage showed some recovery. One cell was subjected to two cycles and showed recovery on both cycles. Cells that were moderately degraded appeared to recover more completely than those more severly degraded. Exposure times ranged from two to ten seconds at 500 degrees centigrade. There was some indication that longer exposure was beneficial.

Sodium-sulfur technology evaluation at Argonne National Laboratory

NASA Astrophysics Data System (ADS)

Mulcahey, T. P.; Tummillo, A. F.; Hogrefe, R. L.; Christianson, C. C.; Biwer, R.; Webster, C. E.; Lee, J.; Miller, J. F.; Marr, J. J.; Smaga, J. A.

The Analysis and Diagnostics Laboratory (ADL) at Argonne National Laboratory has completed evaluation of the Ford Aerospace and Communication Corp. (FACC) technology in the form of four load-levelling (LL) cells, five electric vehicle (EV) cells, and a sub-battery of 89 series connected EV cells. The ADL also has initiated evaluation of the Chloride Silent Power Limited (CSPL) sodium-sulfur (PB) battery technology in the form of 8 individual cells. The evaluation of the FACC-LL cells consisted of an abbreviated performance characterization followed by life-cycle tests on two individual cells and life-cycle tests only on the two other individual cells. The evaluation indicated that the technology was improving, but long-term (life) reliability was not yet adequate for utility applications. The cells exhibited individual cycle lives ranging from 659 to over 1366 cycles, which is equivalent to 2 1/2 to 5 1/2 years in utility use. It was also found that full-cell capacity could only be maintained by applying a special charge regime, regularly or periodically, that consisted of a constant-current followed by a constant-voltage.

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells.

PubMed

Jeng, J H; Wang, Y J; Chang, W H; Wu, H L; Li, C H; Uang, B J; Kang, J J; Lee, J J; Hahn, L J; Lin, B R; Chang, M C

2004-01-01

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (delta Psim) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 microM) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05-0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.

Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cells.

PubMed

Alja, Štraser; Filipič, Metka; Novak, Matjaž; Žegura, Bojana

2013-08-21

The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1-0.5 µg/mL, 24-96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle

PubMed Central

Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A.; Moore, Christina A.; Vella, Stephen A.; Hortua Triana, Miryam A.; Liu, Jing; Garcia, Celia R. S.; Pace, Douglas A.; Moreno, Silvia N. J.

2015-01-01

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca2+ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca2+ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca2+ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca2+ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca2+ influx. This is the first study showing, in real time, Ca2+ signals preceding egress and their direct link with motility, an essential virulence trait. PMID:26374900

The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

PubMed Central

Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

2013-01-01

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

Thermal Characterization Study of Lithium-Ion Cells

NASA Technical Reports Server (NTRS)

Britton, Doris L.; Miller, Thomas B.; Bennett, William R.

2007-01-01

The primary challenge in designing a full scale lithium-ion (Li-ion) battery system is safety under both normal operating as well as abusive conditions. The normal conditions involve expected charge/discharge cycles and it is known that heat evolves in batteries during those cycles. This is a major concern in the design for high power applications and careful thermal management is necessary to alleviate this concern. An emerging thermal measurement technology, such as the electrochemical calorimetric of batteries, will aid in the development of advanced, safe battery system. To support this technology, several "commercial-off-the-shelf" (COTS) Li-ion cells with different chemistries and designs are being evaluated for different cycling regimes at a given operating temperature. The Accelerated Rate Calorimeter (ARC)-Arbin cycler setup is used to measure the temperature, voltage, and current of the cells at different charge/discharge rates. Initial results demonstrated good cell cyclability. During the cycle testing, the cell exhibited an endothermic cooling in the initial part of the charge cycle. The discharge portion of the cycle is exothermic during the entire discharge period. The presence of an endothermic reaction indicates a significant entropy effect during the beginning of charge cycle. Further studies will be performed to understand the thermal characteristics of the Li-ion cells at the different operating conditions. The effects on the thermal response on cell aging and states-of-charge will also be identified.

DEVELOPMENTAL NEUROTOXICITY OF ORGANOPHOSPHATES TARGETS CELL CYCLE AND APOPTOSIS, REVEALED BY TRANSCRIPTIONAL PROFILES IN VIVO AND IN VITRO

PubMed Central

Slotkin, Theodore A.; Seidler, Frederic J.

2012-01-01

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1–4, in doses straddling the threshold for barely-detectable cholinesterase, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20–25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60–70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme. PMID:22222554

Developmental neurotoxicity of organophosphates targets cell cycle and apoptosis, revealed by transcriptional profiles in vivo and in vitro.

PubMed

Slotkin, Theodore A; Seidler, Frederic J

2012-03-01

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1-4, in doses straddling the threshold for barely-detectable cholinesterase inhibition, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20-25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60-70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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

1990-12-15

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

SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression.

PubMed

Gonyo, P; Bergom, C; Brandt, A C; Tsaih, S-W; Sun, Y; Bigley, T M; Lorimer, E L; Terhune, S S; Rui, H; Flister, M J; Long, R M; Williams, C L

2017-12-14

The chaperone protein and guanine nucleotide exchange factor SmgGDS (RAP1GDS1) is a key promoter of cancer cell proliferation and tumorigenesis. SmgGDS undergoes nucleocytoplasmic shuttling, suggesting that it has both cytoplasmic and nuclear functions that promote cancer. Previous studies indicate that SmgGDS binds cytoplasmic small GTPases and promotes their trafficking to the plasma membrane. In contrast, little is known about the functions of SmgGDS in the nucleus, or how these nuclear functions might benefit cancer cells. Here we show unique nuclear localization and regulation of gene transcription pathways by SmgGDS. Strikingly, SmgGDS depletion significantly reduces expression of over 600 gene products that are targets of the DREAM complex, which is a transcription factor complex that regulates expression of proteins controlling the cell cycle. The cell cycle regulators E2F1, MYC, MYBL2 (B-Myb) and FOXM1 are among the DREAM targets that are diminished by SmgGDS depletion. E2F1 is well known to promote G1 cell cycle progression, and the loss of E2F1 in SmgGDS-depleted cells provides an explanation for previous reports that SmgGDS depletion characteristically causes a G1 cell cycle arrest. We show that SmgGDS localizes in nucleoli, and that RNAi-mediated depletion of SmgGDS in cancer cells disrupts nucleolar morphology, signifying nucleolar stress. We show that nucleolar SmgGDS interacts with the RNA polymerase I transcription factor upstream binding factor (UBF). The RNAi-mediated depletion of UBF diminishes nucleolar localization of SmgGDS and promotes proteasome-mediated degradation of SmgGDS, indicating that nucleolar sequestration of SmgGDS by UBF stabilizes SmgGDS protein. The ability of SmgGDS to interact with UBF and localize in the nucleolus is diminished by expressing DiRas1 or DiRas2, which are small GTPases that bind SmgGDS and act as tumor suppressors. Taken together, our results support a novel nuclear role for SmgGDS in protecting malignant cells from nucleolar stress, thus promoting cell cycle progression and tumorigenesis.

Live-cell imaging visualizes frequent mitotic skipping during senescence-like growth arrest in mammary carcinoma cells exposed to ionizing radiation.

PubMed

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-06-01

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Dynamics of cell proliferation in the adult dentate gyrus of two inbred strains of mice

NASA Technical Reports Server (NTRS)

Hayes, N. L.; Nowakowski, R. S.

2002-01-01

The output potential of proliferating populations in either the developing or the adult nervous system is critically dependent on the length of the cell cycle (T(c)) and the size of the proliferating population. We developed a new approach for analyzing the cell cycle, the 'Saturate and Survive Method' (SSM), that also reveals the dynamic behaviors in the proliferative population and estimates of the size of the proliferating population. We used this method to analyze the proliferating population of the adult dentate gyrus in 60 day old mice of two inbred strains, C57BL/6J and BALB/cByJ. The results show that the number of cells labeled by exposure to BUdR changes dramatically with time as a function of the number of proliferating cells in the population, the length of the S-phase, cell division, the length of the cell cycle, dilution of the S-phase label, and cell death. The major difference between C57BL/6J and BALB/cByJ mice is the size of the proliferating population, which differs by a factor of two; the lengths of the cell cycle and the S-phase and the probability that a newly produced cell will die within the first 10 days do not differ in these two strains. This indicates that genetic regulation of the size of the proliferating population is independent of the genetic regulation of cell death among those newly produced cells. The dynamic changes in the number of labeled cells as revealed by the SSM protocol also indicate that neither single nor repeated daily injections of BUdR accurately measure 'proliferation.'.

Temporal remodeling of the cell cycle accompanies differentiation in the Drosophila germline.

PubMed

Hinnant, Taylor D; Alvarez, Arturo A; Ables, Elizabeth T

2017-09-01

Development of multicellular organisms relies upon the coordinated regulation of cellular differentiation and proliferation. Growing evidence suggests that some molecular regulatory pathways associated with the cell cycle machinery also dictate cell fate; however, it remains largely unclear how the cell cycle is remodeled in concert with cell differentiation. During Drosophila oogenesis, mature oocytes are created through a series of precisely controlled division and differentiation steps, originating from a single tissue-specific stem cell. Further, germline stem cells (GSCs) and their differentiating progeny remain in a predominantly linear arrangement as oogenesis proceeds. The ability to visualize the stepwise events of differentiation within the context of a single tissue make the Drosophila ovary an exceptional model for study of cell cycle remodeling. To describe how the cell cycle is remodeled in germ cells as they differentiate in situ, we used the Drosophila Fluorescence Ubiquitin-based Cell Cycle Indicator (Fly-FUCCI) system, in which degradable versions of GFP::E2f1 and RFP::CycB fluorescently label cells in each phase of the cell cycle. We found that the lengths of the G1, S, and G2 phases of the cell cycle change dramatically over the course of differentiation, and identified the 4/8-cell cyst as a key developmental transition state in which cells prepare for specialized cell cycles. Our data suggest that the transcriptional activator E2f1, which controls the transition from G1 to S phase, is a key regulator of mitotic divisions in the early germline. Our data support the model that E2f1 is necessary for proper GSC proliferation, self-renewal, and daughter cell development. In contrast, while E2f1 degradation by the Cullin 4 (Cul4)-containing ubiquitin E3 ligase (CRL4) is essential for developmental transitions in the early germline, our data do not support a role for E2f1 degradation as a mechanism to limit GSC proliferation or self-renewal. Taken together, these findings provide further insight into the regulation of cell proliferation and the acquisition of differentiated cell fate, with broad implications across developing tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae.

PubMed

Plachta, Michal; Halas, Agnieszka; McIntyre, Justyna; Sledziewska-Gojska, Ewa

2015-05-01

Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80 ± 14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell cycle constraints on capsulation and bacteriophage susceptibility.

PubMed

Ardissone, Silvia; Fumeaux, Coralie; Bergé, Matthieu; Beaussart, Audrey; Théraulaz, Laurence; Radhakrishnan, Sunish Kumar; Dufrêne, Yves F; Viollier, Patrick H

2014-11-25

Despite the crucial role of bacterial capsules in pathogenesis, it is still unknown if systemic cues such as the cell cycle can control capsule biogenesis. In this study, we show that the capsule of the synchronizable model bacterium Caulobacter crescentus is cell cycle regulated and we unearth a bacterial transglutaminase homolog, HvyA, as restriction factor that prevents capsulation in G1-phase cells. This capsule protects cells from infection by a generalized transducing Caulobacter phage (φCr30), and the loss of HvyA confers insensitivity towards φCr30. Control of capsulation during the cell cycle could serve as a simple means to prevent steric hindrance of flagellar motility or to ensure that phage-mediated genetic exchange happens before the onset of DNA replication. Moreover, the multi-layered regulatory circuitry directing HvyA expression to G1-phase is conserved during evolution, and HvyA orthologues from related Sinorhizobia can prevent capsulation in Caulobacter, indicating that alpha-proteobacteria have retained HvyA activity.

Identification of miR-133b and RB1CC1 as independent predictors for biochemical recurrence and potential therapeutic targets for prostate cancer.

PubMed

Li, Xia; Wan, Xuechao; Chen, Hongbing; Yang, Shu; Liu, Yiyang; Mo, Wenjuan; Meng, Delong; Du, Wenting; Huang, Yan; Wu, Hai; Wang, Jingqiang; Li, Tao; Li, Yao

2014-05-01

We aimed to investigate the contribution of microRNA-133b (miR-133b) in prostate cancer cell proliferation, cell cycle, and apoptosis. We also examined expression of miR-133b in prostate cancer tissues, and evaluated the prognostic significance of miR-133b, as well as its target gene RB1CC1 in patients with prostate cancer after radical prostatectomy. miR-133b mimics (miR-133bm) and anti-miR-133b were transfected into LNCaP and PC-3 cells. CCK-8 was used to look at cell proliferation, flow cytometric analysis was carried out to study cell cycle, and apoptosis was determined by caspase-3 activity. miR-133b expression was assessed by real-time reverse transcription PCR and in situ hybridization in prostatic cell lines and 178 prostate tissue samples, respectively. The protein level of RB1CC1 was examined by Western blot and immunohistochemistry in prostatic cell lines and prostate tissue samples, respectively. Overexpression of miR-133b in LNCaP cells boosted cell proliferation and cell-cycle progression, but inhibited apoptosis; in contrast, miR-133bm promoted cell apoptosis, but suppressed cell proliferation and cell-cycle progression in PC-3 cells. In LNCaP cells, silencing of RB1CC1, a target of miR-133b, inhibited cell apoptosis, and promoted cell-cycle progression. Moreover, miR-133b expression was significantly inversely correlated with RB1CC1 expression in prostate cancer tissues. Multivariate Cox analysis indicated that miR-133b and RB1CC1 might be two independent prognostic factors of biochemical recurrence. miR-133b might enhance tumor-promoting properties in less aggressive LNCaP cells, whereas this miR may act as a tumor suppressor in more aggressive PC-3 cells. miR-133b and RB1CC1 were independent prognostic indicators for prostate cancer. ©2014 AACR.

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress

PubMed Central

Laggner, Maria; Pollreisz, Andreas; Schmidinger, Gerald; Schmidt-Erfurth, Ursula; Chen, Ying-Ting

2017-01-01

Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC’s stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H2DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation. PMID:28700649

A generalized model for multi-marker analysis of cell cycle progression in synchrony experiments

PubMed Central

Mayhew, Michael B.; Robinson, Joshua W.; Jung, Boyoun; Haase, Steven B.; Hartemink, Alexander J.

2011-01-01

Motivation: To advance understanding of eukaryotic cell division, it is important to observe the process precisely. To this end, researchers monitor changes in dividing cells as they traverse the cell cycle, with the presence or absence of morphological or genetic markers indicating a cell's position in a particular interval of the cell cycle. A wide variety of marker data is available, including information-rich cellular imaging data. However, few formal statistical methods have been developed to use these valuable data sources in estimating how a population of cells progresses through the cell cycle. Furthermore, existing methods are designed to handle only a single binary marker of cell cycle progression at a time. Consequently, they cannot facilitate comparison of experiments involving different sets of markers. Results: Here, we develop a new sampling model to accommodate an arbitrary number of different binary markers that characterize the progression of a population of dividing cells along a branching process. We engineer a strain of Saccharomyces cerevisiae with fluorescently labeled markers of cell cycle progression, and apply our new model to two image datasets we collected from the strain, as well as an independent dataset of different markers. We use our model to estimate the duration of post-cytokinetic attachment between a S.cerevisiae mother and daughter cell. The Java implementation is fast and extensible, and includes a graphical user interface. Our model provides a powerful and flexible cell cycle analysis tool, suitable to any type or combination of binary markers. Availability: The software is available from: http://www.cs.duke.edu/~amink/software/cloccs/. Contact: michael.mayhew@duke.edu; amink@cs.duke.edu PMID:21685084

The cyclin-dependent kinase inhibitor p57Kip2 regulates cell cycle exit, differentiation, and migration of embryonic cerebral cortical precursors.

PubMed

Tury, Anna; Mairet-Coello, Georges; DiCicco-Bloom, Emanuel

2011-08-01

Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57(Kip2) and p27(Kip1), control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57(Kip2) remain poorly defined. Using in vivo and culture approaches, we show p57(Kip2) overexpression at E14.5-15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57(Kip2)-deficient precursors. Studies at later ages indicate p57(Kip2) overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57(Kip2) overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57(Kip2) was twice as effective as p27(Kip1) in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57(Kip2) alone inhibits in vivo migration. Furthermore, p57(Kip2) effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27(Kip1) studies report cell cycle-independent functions. These observations suggest p57(Kip2) coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27(Kip1).

The influence of day/night cycles on biomass yield and composition of Neochloris oleoabundans.

PubMed

de Winter, Lenneke; Cabanelas, Iago Teles Dominguez; Martens, Dirk E; Wijffels, René H; Barbosa, Maria J

2017-01-01

Day/night cycles regulate the circadian clock of organisms to program daily activities. Many species of microalgae have a synchronized cell division when grown under a day/night cycle, and synchronization might influence biomass yield and composition. Therefore, the aim of this study was to study the influence of day/night cycle on biomass yield and composition of the green microalgae Neochloris oleoabundans . Hence, we compared continuous turbidostat cultures grown under continuous light with cultures grown under simulated day/night cycles. Under day/night cycles, cultures were synchronized as cell division was scheduled in the night, whereas under continuous light cell division occurred randomly synchronized cultures were able to use the light 10-15% more efficiently than non-synchronized cultures. Our results indicate that the efficiency of light use varies over the cell cycle and that synchronized cell division provides a fitness benefit to microalgae. Biomass composition under day/night cycles was similar to continuous light, with the exception of starch content. The starch content was higher in cultures under continuous light, most likely because the cells never had to respire starch to cover for maintenance during dark periods. Day/night cycles were provided in a 'block' (continuous light intensity during the light period) and in a 'sine' (using a sine function to simulate light intensities from sunrise to sunset). There were no differences in biomass yield or composition between these two ways of providing light (in a 'block' or in a 'sine'). The biomass yield and composition of N. oleoabundans were influenced by day/night cycles. These results are important to better understand the relations between research done under continuous light conditions and with day/night cycle conditions. Our findings also imply that more research should be done under day/night cycles.

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

PubMed

Zakian, V A; Brewer, B J; Fangman, W L

1979-08-01

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.

Radiation Dose-effects on Cell Cycle, Apoptosis, and Marker Expression of Ataxia Telangiectasia-Heterozygous Human Breast Epithelial Cells

NASA Technical Reports Server (NTRS)

Cruz, A.; Bors, K.; Jansen, H.; Richmond, R.

2003-01-01

Ataxia-telangiectasia (A-T) is a radiation-sensitive genetic condition. AT-heterozygous human mammary epithelial cells (HMEC) were irradiated using a Cs137 source in order to compare cell cycle, apoptosis, and marker expression responses across 3 radiation doses. No differences in cell cycle and apoptosis were found with any of the radiation doses used (30, 60, and 90 rads) compared with the unirradiated control (0 rad). At the same doses, however, differences were found in marker expression, such as keratin 18 (kl8), keratin 14 (k14), insulin-like growth factor I receptor (IGF-IR), and connexin 43 (cx43). This may indicate that radiation sensitivity in the heterozygous state may be initiated through signal transduction responses.

The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells.

PubMed

Berrak, Özge; Akkoç, Yunus; Arısan, Elif Damla; Çoker-Gürkan, Ajda; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin

2016-02-01

Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver

PubMed Central

Sui, Zhenhua; Nowak, Roberta B.; Bacconi, Andrea; Kim, Nancy E.; Liu, Hui; Li, Jie; Wickrema, Amittha; An, Xiu-li

2014-01-01

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3−/− erythroblasts, and cell-cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3−/− embryos. Notably, enucleating Tmod3−/− erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3−/− late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3−/− fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation. PMID:24159174

Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response.

PubMed

Cazzalini, Ornella; Scovassi, A Ivana; Savio, Monica; Stivala, Lucia A; Prosperi, Ennio

2010-01-01

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response. 2010 Elsevier B.V. All rights reserved.

Protein PSMD8 may mediate microgravity-induced cell cycle arrest

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Xu, Dan; Wu, Di; Chen, Xiaoning

Microgravity environment of space can induce a serial of changes in cells, such as morphology alterations, cytoskeleton disorder and cell cycle disturbance. Our previous study of simulated-microgravity on zebrafish (Danio rerio) embryos demonstrated 26s proteasome non-ATPase regulatory subunit 8 (PSMD8) might be a microgravity sensitive gene. However, functional study on PSMD8 is very limited and it has not been cloned in zebrafish till now. In this study, we tried to clone PSMD8 gene in zebrafish, quantify its protein expression level in zebrafish embryos after simulated microgravity and identify its possible function in cell cycle regulation. A rotary cell culture system (RCCS) designed by national aeronautics and apace administration (NASA) of America was used to simulate microgravity. The full-length of psmd8 gene in zebrafish was cloned. Preliminary analysis on its sequence and phylogenetic tree construction were carried out subsequently. Quantitative analysis by western blot showed that PSMD8 protein expression levels were significantly increased 1.18 and 1.22 times after 24-48hpf and 24-72hpf simulated microgravity, respectively. Moreover, a significant delay on zebrafish embryo development was found in simulated-microgravity exposed group. Inhibition of PSMD8 protein in zebrafish embryonic cell lines ZF4 could block cell cycle in G1 phase, which indicated that PSMD8 may play a role in cell cycle regulation. Interestingly, simulated-microgravity could also block ZF4 cell in G1 phase. Whether it is PSMD8 mediated cell cycle regulation result in the zebrafish embryo development delay after simulated microgravity exposure still needs further study. Key Words: PSMD8; Simulated-microgravity; Cell cycle; ZF4 cell line

Ultrastructural and histochemical study on the interrenal cells of the male stickleback (Gasterosteus aculeatus, Teleostea), in relation to the reproductive annual cycle

PubMed Central

CIVININI, ANNALENA; PADULA, DANIELA; GALLO, VALENTINA P.

2001-01-01

The steroidogenic interrenal cells in the adrenal homologue of the male stickleback (Gasterosteus aculeatus) were studied in relation to the reproductive cycle by means of histological and ultrastructural observations, and using histochemical methods for the localisation of 3β-hydroxysteroid dehydrogenase (3βHSD) and 17β-hydroxysteroid dehydrogenase (17βHSD). To determine the various stages of the reproductive cycle, the testes were also examined by histological and histochemical methods (3βHSD). The results indicate that in this teleost the interrenal cells can undergo a cycle in which phases characterised by different cytological aspects are observed. During this cycle there is a renewal of organelles, in particular mitochondria and SER. Periodic degenerative processes are also found. Organelle cytology showed that the cell cycle has at least 3 different aspects during the year. An analogy with some cytological aspects of the adrenal zonation in mammals is possible. It is postulated that the interrenal gland activity could substitute or supplement androgen production by the testes. PMID:11554507

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

Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp; Yamauchi, Motohiro; Oka, Yasuyoshi

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% andmore » 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.« less

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.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

PubMed Central

Zhang, Lixia; Kendrick, Christina; Jülich, Dörthe; Holley, Scott A.

2010-01-01

Summary Cell division, differentiation and morphogenesis are coordinated during embryonic development and frequently in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and utilize the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is substantially driven by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1−/−. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for normal segmental arrangement of epithelial borders and internal mesenchymal cells. PMID:18480162

(PECASE 08) - ION-Conducting Network Membranes Using Tapered Block Copolymers

DTIC Science & Technology

2015-07-08

iron phosphate ( LiFePO4 ) as an active material for the cathode. The composite cathode was prepared by mixing P(S-EO) with carbon black and LiFePO4 ...salt- doping ratio of [EO]:[Li] = 12:1. Example cycle-life data for the Li/P(S-EO)/ LiFePO4 cell is shown in Figure 1. The specific discharge...rates, indicating good cycling stability. This investigation currently is in progress. 1 Figure 1: Cycle-life data for the Li/P(S-EO)/ LiFePO4 cell

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

PubMed

Boso, Guney; Somia, Nikunj V

2015-01-01

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

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

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

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

2006-02-03

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

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

PubMed

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

2006-02-03

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

c-Myc plays a key role in TADs-induced apoptosis and cell cycle arrest in human hepatocellular carcinoma cells.

PubMed

Zhang, Dongdong; Qi, Junpeng; Liu, Rui; Dai, Bingling; Ma, Weina; Zhan, Yingzhuan; Zhang, Yanmin

2015-01-01

Cancer cell growth is complicated progression which is regulated and controlled by multiple factors including cell cycle, migration and apoptosis. In present study, we report that TADs, a novel derivative of taspine, has an essential role in resisting hepatocellular carcinoma growth (including arrest cell cycle) and migration, and inducing cell apoptosis. Our findings demonstrated that the TADs showed good inhibition on the hepatoma cell growth and migration, and good action on apoptosis induction. Using genome-wide microarray analysis, we found the down-regulated growth and apoptosis factors, and selected down-regulated genes were confirmed by Western blot. Knockdown of a checkpoint c-Myc by siRNA significantly attenuated tumor inhibition and apoptosis effects of TADs. Moreover, our results indicated TADs could simultaneously increase cyclin D1 protein levels and decrease amount of cyclin E, cyclin B1 and cdc2 of the cycle proteins, and also TADs reduced Bcl-2 expression, and upregulated Bad, Bak and Bax activities. In conclusion, these results illustrated that TADs is a key factor in growth and apoptosis signaling inhibitor, has potential in cancer therapy.

Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

PubMed

Zan, Xinyi; Cui, Fengjie; Li, Yunhong; Yang, Yan; Wu, Di; Sun, Wenjing; Ping, Lifeng

2015-05-01

HEG-5 is a novel polysaccharide-protein purified from the fermented mycelia of Hericium erinaceus CZ-2. The present study aims to investigate the effects of HEG-5 on proliferation, cell cycle and apoptosis of human gastric cancer cells SGC-7901. Here, we first uncover that HEG-5 significantly inhibited the proliferation and colony formation of SGC-7901 cells by promoting apoptosis and cell cycle arrest at S phase. RT-PCR and Western blot analysis suggested that HEG-5 could decrease the expressions of Bcl2, PI3K and AKT1, while increase the expressions of Caspase-8, Caspase-3, p53, CDK4, Bax and Bad. These findings indicated that the Caspase-8/-3-dependent, p53-dependent mitochondrial-mediated and PI3k/Akt signaling pathways involved in the molecular events of HEG-5 induced apoptosis and cell cycle arrest. Thus, our study provides in vitro evidence that HEG-5 may be taken as a potential candidate for treating gastric cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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

Suzuki, Kanayo; Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp; Tanaka, Satoshi

2014-01-03

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

Effect of storage and LEO cycling on manufacturing technology IPV nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.

1987-01-01

Yardney Manufacturing Technology (MANTECH) 50 A-hr space weight individual pressure vessel nickel-hydrogen cells were evaluated. This consisted of investigating: the effect of storage and charge/discharge cycling on cell performance. For the storage test the cells were precharged with hydrogen, by the manufacturer, to a pressure of 14.5 psia. After undergoing activation and acceptance tests, the cells were discharged at C/10 rate (5A) to 0.1 V or less. The terminals were then shorted. The cells were shipped to NASA Lewis Research Center where they were stored at room temperature in the shorted condition for 1 year. After storage, the acceptance tests were repeated at NASA Lewis. A comparison of test results indicate no significant degradation in electrical performance due to 1 year storage. For the cycle life test the regime was a 90 minute low earth orbit at deep depths of discharge (80 and 60 percent). At the 80 percent DOD the three cells failed on the average at cycle 741. Failure for this test was defined to occur when the cell voltage degraded to 1 V prior to completion of the 35 min discharge. The DOD was reduced to 60 percent. The cycle life test was continued.

Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells

PubMed Central

Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D.; Yu, Jun; Zhu, Lihua Julie

2017-01-01

ABSTRACT The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells. PMID:28630280

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.

Life cycle test results of a bipolar nickel hydrogen battery

NASA Technical Reports Server (NTRS)

Cataldo, R. L.

1985-01-01

A history is given of low Earth orbit (LEO) laboratory test data on a 6.5 ampere-hour bipolar nickel hydrogen battery designed and built at the NASA Lewis Research Center. The bipolar concept is a means of achieving the goal of producing an acceptable battery, of higher energy density, able to withstand the demands of low-Earth-orbit regimes. Over 4100 LEO cycles were established on a ten cell battery. It seems that any perturbation on normal cycling effects the cells performance. Explanations and theories of the battery's behavior are varied and widespread among those closely associated with it. Deep discharging does provide a reconditioning effect and further experimentation is planned in this area. The battery watt-hour efficiency is about 75 percent and the time averaged, discharge voltage is about 1.26 volts for all cells at both the C/4 and LEO rate. Since a significant portion of the electrode capacity has degraded, the LEO cycle discharges are approaching depths of 90 to 100 percent of the high rate capacity. Therefore, the low end-of-discharge voltages occur precipitously after the knee of the discharge curve and is more an indication of electrode capacity and is a lesser indicator of overall cell performance.

shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

PubMed

Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

2015-03-01

EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

PubMed

Kuriyama, Shigeki; Hitomi, Misuzu; Yoshiji, Hitoshi; Nonomura, Takako; Tsujimoto, Tatsuhiro; Mitoro, Akira; Akahane, Takami; Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Masaki, Tsutomu; Uchida, Naohito

2005-08-01

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein-47 and collagen type I in rat hepatic stellate cells in vitro.

PubMed

Xiang, Xian-Hong; Jiang, Tian-Peng; Zhang, Shuai; Song, Jie; Li, Xing; Yang, Jian-Yong; Zhou, Shi

2015-07-01

Pirfenidone (esbiret) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose-dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)-47 and collagen type I in a cultured rat hepatic stellate cell line (HSC-T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit-8 assay and the cell cycle was measured using flow cytometry. HSP-47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose-dependent inhibition of proliferation in HSC-T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC-T6 cells exhibited dose-dependent decreases in HSP-47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC-T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP-47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.

Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

PubMed

Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

2015-01-01

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

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

PubMed

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

2015-09-08

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

The long non-coding RNA GAS5 differentially regulates cell cycle arrest and apoptosis through activation of BRCA1 and p53 in human neuroblastoma

PubMed Central

Mazar, Joseph; Rosado, Amy; Shelley, John; Marchica, John; Westmoreland, Tamarah J

2017-01-01

The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death. PMID:28035057

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

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses

PubMed Central

2014-01-01

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels. PMID:24479647

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

PubMed Central

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

2010-01-01

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

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

PubMed

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

2007-01-01

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

Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells.

PubMed

Morley, Karen L; Ferguson, Peter J; Koropatnick, James

2007-06-18

Tangeretin and nobiletin are citrus flavonoids that are among the most effective at inhibiting cancer cell growth in vitro and in vivo. The antiproliferative activity of tangeretin and nobiletin was investigated in human breast cancer cell lines MDA-MB-435 and MCF-7 and human colon cancer line HT-29. Both flavonoids inhibited proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at G1 in all three cell lines. At concentrations that resulted in significant inhibition of proliferation and cell cycle arrest, neither flavonoid induced apoptosis or cell death in any of the tumor cell lines. To test the ability of arrested cells to recover, cells that were incubated with tangeretin and nobiletin for 4 days were then cultured in flavonoid-free medium for an additional 4 days. Cells resumed proliferation similar to untreated control within a day of flavonoid removal. Cell cycle distribution was similar to that of control within 4 days of flavonoid removal. These data indicate that, in these cell lines at concentrations that inhibit proliferation up to 80% over 4 days, tangeretin and nobiletin are cytostatic and significantly suppress proliferation by cell cycle arrest without apoptosis. Such an agent could be expected to spare normal tissues from toxic side effects. Thus, tangeretin and nobiletin could be effective cytostatic anticancer agents. Inhibition of proliferation of human cancers without inducing cell death may be advantageous in treating tumors as it would restrict proliferation in a manner less likely to induce cytotoxicity and death in normal, non-tumor tissues.

Cell cycle constraints on capsulation and bacteriophage susceptibility

PubMed Central

Ardissone, Silvia; Fumeaux, Coralie; Bergé, Matthieu; Beaussart, Audrey; Théraulaz, Laurence; Radhakrishnan, Sunish Kumar; Dufrêne, Yves F; Viollier, Patrick H

2014-01-01

Despite the crucial role of bacterial capsules in pathogenesis, it is still unknown if systemic cues such as the cell cycle can control capsule biogenesis. In this study, we show that the capsule of the synchronizable model bacterium Caulobacter crescentus is cell cycle regulated and we unearth a bacterial transglutaminase homolog, HvyA, as restriction factor that prevents capsulation in G1-phase cells. This capsule protects cells from infection by a generalized transducing Caulobacter phage (φCr30), and the loss of HvyA confers insensitivity towards φCr30. Control of capsulation during the cell cycle could serve as a simple means to prevent steric hindrance of flagellar motility or to ensure that phage-mediated genetic exchange happens before the onset of DNA replication. Moreover, the multi-layered regulatory circuitry directing HvyA expression to G1-phase is conserved during evolution, and HvyA orthologues from related Sinorhizobia can prevent capsulation in Caulobacter, indicating that alpha-proteobacteria have retained HvyA activity. DOI: http://dx.doi.org/10.7554/eLife.03587.001 PMID:25421297

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells.

PubMed

Chen, C; Yang, R L

2013-08-01

MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

Entrainment of the Mammalian Cell Cycle by the Circadian Clock: Modeling Two Coupled Cellular Rhythms

PubMed Central

Gérard, Claude; Goldbeter, Albert

2012-01-01

The cell division cycle and the circadian clock represent two major cellular rhythms. These two periodic processes are coupled in multiple ways, given that several molecular components of the cell cycle network are controlled in a circadian manner. For example, in the network of cyclin-dependent kinases (Cdks) that governs progression along the successive phases of the cell cycle, the synthesis of the kinase Wee1, which inhibits the G2/M transition, is enhanced by the complex CLOCK-BMAL1 that plays a central role in the circadian clock network. Another component of the latter network, REV-ERBα, inhibits the synthesis of the Cdk inhibitor p21. Moreover, the synthesis of the oncogene c-Myc, which promotes G1 cyclin synthesis, is repressed by CLOCK-BMAL1. Using detailed computational models for the two networks we investigate the conditions in which the mammalian cell cycle can be entrained by the circadian clock. We show that the cell cycle can be brought to oscillate at a period of 24 h or 48 h when its autonomous period prior to coupling is in an appropriate range. The model indicates that the combination of multiple modes of coupling does not necessarily facilitate entrainment of the cell cycle by the circadian clock. Entrainment can also occur as a result of circadian variations in the level of a growth factor controlling entry into G1. Outside the range of entrainment, the coupling to the circadian clock may lead to disconnected oscillations in the cell cycle and the circadian system, or to complex oscillatory dynamics of the cell cycle in the form of endoreplication, complex periodic oscillations or chaos. The model predicts that the transition from entrainment to 24 h or 48 h might occur when the strength of coupling to the circadian clock or the level of growth factor decrease below critical values. PMID:22693436

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

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

Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi

2014-01-17

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

Antitumorigenic effect of atmospheric-pressure dielectric barrier discharge on human colorectal cancer cells via regulation of Sp1 transcription factor

NASA Astrophysics Data System (ADS)

Han, Duksun; Cho, Jin Hyoung; Lee, Ra Ham; Bang, Woong; Park, Kyungho; Kim, Minseok S.; Shim, Jung-Hyun; Chae, Jung-Il; Moon, Se Youn

2017-02-01

Human colorectal cancer cell lines (HT29 and HCT116) were exposed to dielectric barrier discharge (DBD) plasma at atmospheric pressure to investigate the anticancer capacity of the plasma. The dose- and time-dependent effects of DBDP on cell viability, regulation of transcription factor Sp1, cell-cycle analysis, and colony formation were investigated by means of MTS assay, DAPI staining, propidium iodide staining, annexin V-FITC staining, Western blot analysis, RT-PCR analysis, fluorescence microscopy, and anchorage-independent cell transformation assay. By increasing the duration of plasma dose times, significant reductions in the levels of both Sp1 protein and Sp1 mRNA were observed in both cell lines. Also, expression of negative regulators related to the cell cycle (such as p53, p21, and p27) was increased and of the positive regulator cyclin D1 was decreased, indicating that the plasma treatment led to apoptosis and cell-cycle arrest. In addition, the sizes and quantities of colony formation were significantly suppressed even though two cancer promoters, such as TPA and epidermal growth factor, accompanied the plasma treatment. Thus, plasma treatment inhibited cell viability and colony formation by suppressing Sp1, which induced apoptosis and cell-cycle arrest in these two human colorectal cancer cell lines.

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

PubMed Central

Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C.

2015-01-01

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. PMID:25564650

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos.

PubMed

Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C

2015-02-01

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. © 2015. Published by The Company of Biologists Ltd.

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

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

Ji, Shuai

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

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

PubMed Central

Kasai, Hirotake

2007-01-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-02-04

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

The Hog1 MAP Kinase Promotes the Recovery from Cell Cycle Arrest Induced by Hydrogen Peroxide in Candida albicans

PubMed Central

Correia, Inês; Alonso-Monge, Rebeca; Pla, Jesús

2017-01-01

Eukaryotic cell cycle progression in response to environmental conditions is controlled via specific checkpoints. Signal transduction pathways mediated by MAPKs play a crucial role in sensing stress. For example, the canonical MAPKs Mkc1 (of the cell wall integrity pathway), and Hog1 (of the HOG pathway), are activated upon oxidative stress. In this work, we have analyzed the effect of oxidative stress induced by hydrogen peroxide on cell cycle progression in Candida albicans. Hydrogen peroxide was shown to induce a transient arrest at the G1 phase of the cell cycle. Specifically, a G1 arrest was observed, although phosphorylation of Mkc1 and Hog1 MAPKs can take place at all stages of the cell cycle. Interestingly, hog1 (but not mkc1) mutants required a longer time compared to wild type cells to resume growth after hydrogen peroxide challenge. Using GFP-labeled cells and mixed cultures of wild type and hog1 cells we were able to show that hog1 mutants progress faster through the cell cycle under standard growth conditions in the absence of stress (YPD at 37°C). Consequently, hog1 mutants exhibited a smaller cell size. The altered cell cycle progression correlates with altered expression of the G1 cyclins Cln3 and Pcl2 in hog1 cells compared to the wild type strain. In addition, Hgc1 (a hypha-specific G1 cyclin) as well as Cln3 displayed a different kinetics of expression in the presence of hydrogen peroxide in hog1 mutants. Collectively, these results indicate that Hog1 regulates the expression of G1 cyclins not only in response to oxidative stress, but also under standard growth conditions. Hydrogen peroxide treated cells did not show fluctuations in the mRNA levels for SOL1, which are observed in untreated cells during cell cycle progression. In addition, treatment with hydrogen peroxide prevented degradation of Sol1, an effect which was enhanced in hog1 mutants. Therefore, in C. albicans, the MAPK Hog1 mediates cell cycle progression in response to oxidative stress, and further participates in the cell size checkpoint during vegetative growth. PMID:28111572

High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development

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

Wu, Yanqing; Zhang, Zhenghong; Liao, Xinghui

The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovariesmore » in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27{sup Kip1} and p21{sup Cip1}, were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure. - Highlights: • HFD induced-obesity leads to abnormal ovarian morphology. • HFD induced-obesity triggers excessive apoptosis in the ovary. • HFD induced-obesity up-regulates cell cycle inhibitors p21{sup Cip1} and p27{sup Kip1} in the ovary. • HFD induced-obesity causes cell cycle arrest in the ovary.« less

Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis

PubMed Central

Horlock-Roberts, Kathleen; Reaume, Chase; Dayer, Guillem; Ouellet, Christine; Cook, Nicholas

2017-01-01

ABSTRACT Giardia intestinalis is a protozoan parasite that causes giardiasis, a form of severe and infectious diarrhea. Despite the importance of the cell cycle in the control of proliferation and differentiation during a giardia infection, it has been difficult to study this process due to the absence of a synchronization procedure that would not induce cellular damage resulting in artifacts. We utilized counterflow centrifugal elutriation (CCE), a size-based separation technique, to successfully obtain fractions of giardia cultures enriched in G1, S, and G2. Unlike drug-induced synchronization of giardia cultures, CCE did not induce double-stranded DNA damage or endoreplication. We observed increases in the appearance and size of the median body in the cells from elutriation fractions corresponding to the progression of the cell cycle from early G1 to late G2. Consequently, CCE could be used to examine the dynamics of the median body and other structures and organelles in the giardia cell cycle. For the cell cycle gene expression studies, the actin-related gene was identified by the program geNorm as the most suitable normalizer for reverse transcription-quantitative PCR (RT-qPCR) analysis of the CCE samples. Ten of 11 suspected cell cycle-regulated genes in the CCE fractions have expression profiles in giardia that resemble those of higher eukaryotes. However, the RNA levels of these genes during the cell cycle differ less than 4-fold to 5-fold, which might indicate that large changes in gene expression are not required by giardia to regulate the cell cycle. IMPORTANCE Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission. PMID:28959734

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

PubMed

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

2014-01-01

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

Effect of positive pulse charge waveforms on cycle life of nickel-zinc cells

NASA Technical Reports Server (NTRS)

Smithrick, J. J.

1980-01-01

Five amp-hour nickel-zinc cells were life cycled to evaluate four different charge methods. Three of the four waveforms investigated were 120 Hz full wave rectified sinusoidal (FWRS), 120 Hz silicon controlled rectified (SCR), and 1 kHz square wave (SW). The fourth, a constant current method, was used as a baseline of comparison. Three sealed Ni-Zn cells connected in series were cycled. Each series string was charged at an average c/20 rate, and discharged at a c/2.5 rate to a 75% rated depth. Results indicate that the relatively inexpensive 120 Hz FWRS charger appears feasible for charging 5 amp-hour nickel-zinc cells with no significant loss in average cycle life when compared to constant current charging. The 1-kHz SW charger could also be used with no significant loss in average cycle life, and suggests the possibility of utilizing the existing electric vehicle chopper controller circuitry for an on-board charger. There was an apparent difference using the 120 Hz SCR charger compared to the others, however, this difference could be due to an inadvertent severe overcharge, which occurred prior to cell failure. The remaining two positive pulse charging waveforms, FWRS and 1 kHz, did not improve the cycle life of 5 amp-hour nickel-zinc cells over that of constant current charging.

The Molecular Basis of Communication within the Cell.

ERIC Educational Resources Information Center

Berridge, Michael J.

1985-01-01

Only a few substances serve as signals within cells; this indicates that internal signal pathways are remarkably universal. The variety of physiological and biochemical processes regulated by known messengers is discussed along with chemical structures, pathways, inositol-lipid cycles, and cell growth regulation. (DH)

Electrochemical properties of lithium iron phosphate cathode material using polymer electrolyte

NASA Astrophysics Data System (ADS)

Kim, Jae-Kwang; Choi, Jae-Won; Cheruvally, Gouri; Shin, Yong-Jo; Ahn, Jou-Hyeon; Cho, Kwon-Koo; Ahn, Hyo-Jun; Kim, Ki-Won

2007-12-01

Carbon-coated lithium iron phosphate (LiFePO4/C) cathode material was synthesized by mechano-chemical activation method. The performance of LiFePO4/C in lithium battery was tested with an electrospun polymer-based electrolyte. Liquid electrolyte of 1M lithium hexafluorophosphate (LiPF6) in ethylene carbonate/dimethyl carbonate (EC/DMC) (1 : 1vol) was incorporated in electrospun poly(vinylidene fluoride-co-hexafluoropropylene) (P(VdF-HFP)) microfibrous membrane to prepare the polymer electrolyte (PE). The cell based on Li|PE|Li FePO4/C exhibited an initial discharge capacity of 142 mAh g-1 at 0.1 C-rate at room temperature. Good cycling performance even under the high current density of 2 C could be obtained. Impedance spectroscopy was applied to investigate the material behavior during 0.1 C-rate charge-discharge cycling. When the fresh cell and the cell after different cycles were compared, impedance resistance was found to decrease with cycling. Impedance study indicated good cycle life for the cell when tested at room temperature.

Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

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

Patel, Shreya, E-mail: Shreya.patel214@gmail.com; Peretz, Jackye, E-mail: Jackye.peretz@gmail.com; Pan, Yuan-Xiang, E-mail: yxpan@illinois.edu

Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 micemore » were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes. • Genistein exposure alters Esr1 and Esr2 expression.« less

Cell cycle arrest and biochemical changes accompanying cell death in harmful dinoflagellates following exposure to bacterial algicide IRI-160AA

NASA Astrophysics Data System (ADS)

Pokrzywinski, Kaytee L.; Tilney, Charles L.; Warner, Mark E.; Coyne, Kathryn J.

2017-03-01

Bacteria may play a role in regulating harmful algal blooms, but little is known about the biochemical and physiological changes associated with cell death induced by algicidal bacteria. Previous work characterized an algicidal exudate (IRI-160AA) produced by Shewanella sp. IRI-160 that is effective against dinoflagellates, while having little to no effect on other phytoplankton species in laboratory culture experiments. The objective of this study was to evaluate biochemical changes associated with cell death and impacts on the cell cycle in three dinoflagellate species (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum) after exposure to IRI-160AA. In this study, IRI-160AA induced cell cycle arrest in all dinoflagellates examined. Several indicators for programmed cell death (PCD) that are often observed in phytoplankton in response to a variety of stressors were also evaluated. Cell death was accompanied by significant increases in DNA degradation, intra- and extracellular ROS concentrations and DEVDase (caspase-3 like) protease activity, which have been associated with PCD in other phytoplankton species. Overall, results of this investigation provide strong evidence that treatment with the bacterial algicide, IRI-160AA results in cell cycle arrest and induces biochemical changes consistent with stress-related cell death responses observed in other phytoplankton.

Cell cycle arrest and biochemical changes accompanying cell death in harmful dinoflagellates following exposure to bacterial algicide IRI-160AA

PubMed Central

Pokrzywinski, Kaytee L.; Tilney, Charles L.; Warner, Mark E.; Coyne, Kathryn J.

2017-01-01

Bacteria may play a role in regulating harmful algal blooms, but little is known about the biochemical and physiological changes associated with cell death induced by algicidal bacteria. Previous work characterized an algicidal exudate (IRI-160AA) produced by Shewanella sp. IRI-160 that is effective against dinoflagellates, while having little to no effect on other phytoplankton species in laboratory culture experiments. The objective of this study was to evaluate biochemical changes associated with cell death and impacts on the cell cycle in three dinoflagellate species (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum) after exposure to IRI-160AA. In this study, IRI-160AA induced cell cycle arrest in all dinoflagellates examined. Several indicators for programmed cell death (PCD) that are often observed in phytoplankton in response to a variety of stressors were also evaluated. Cell death was accompanied by significant increases in DNA degradation, intra- and extracellular ROS concentrations and DEVDase (caspase-3 like) protease activity, which have been associated with PCD in other phytoplankton species. Overall, results of this investigation provide strong evidence that treatment with the bacterial algicide, IRI-160AA results in cell cycle arrest and induces biochemical changes consistent with stress-related cell death responses observed in other phytoplankton. PMID:28332589

Somatostatin Receptor-1 Induces Cell Cycle Arrest and Inhibits Tumor Growth in Pancreatic Cancer

PubMed Central

Li, Min; Wang, Xiaochi; Li, Wei; Li, Fei; Yang, Hui; Wang, Hao; Brunicardi, F. Charles; Chen, Changyi; Yao, Qizhi; Fisher, William E.

2010-01-01

Functional somatostatin receptors (SSTRs) are lost in human pancreatic cancer. Transfection of SSTR-1 inhibited pancreatic cancer cell proliferation in vitro. We hypothesize that stable transfection of SSTR-1 may inhibit pancreatic cancer growth in vivo possibly through cell cycle arrest. In this study, we examined the expression of SSTR-1 mRNA in human pancreatic cancer tissue specimens, and investigated the effect of SSTR-1 overexpression on cell proliferation, cell cycle, and tumor growth in in a subcutaneous nude mouse model. We found that SSTR-1 mRNA was downregulated in the majority of pancreatic cancer tissue specimens. Transfection of SSTR-1 caused cell cycle arrest at the G0/G1 growth phase, with a corresponding decline of cells in the S (mitotic) phase. The overexpression of SSTR-1 significantly inhibited subcutaneous tumor size by 71% and 43% (n=5, p<0.05, t-test), and inhibited tumor weight by 69% and 47%, (n=5, p<0.05, t-test), in Panc-SSTR-1 and MIA-SSTR-1 groups, respectively, indicating the potent inhibitory effect of SSTR-1 on pancreatic cancer growth. Our data demonstrate that overexpression of SSTR-1 significantly inhibits pancreatic cancer growth possibly through cell cycle arrest. This study suggests that gene therapy with SSTR-1 may be a potential adjuvant treatment for pancreatic cancer. PMID:18823376

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

PubMed

Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

1998-10-01

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells.

PubMed

Li, Xiaotong; Xie, Hongqing; Chen, Yajie; Lang, Mingzi; Chen, Yuyin; Shi, Liangen

2018-03-28

Silkworm pupae ( Bombyx mori ) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.

Ultraviolet irradiation at elevated temperatures and thermal cycling in vacuum of FEP-A covered silicon solar cells

NASA Technical Reports Server (NTRS)

Broder, J. D.; Marsik, S. J.

1978-01-01

Experiments were designed and performed on silicon solar cells covered with heat-bonded FEP-A in an effort to explain the rapid degeneration of open-circuit voltage and maximum power observered on cells of this type included in an experiment on the ATS-6 spacecraft. Solar cells were exposed to ultraviolet light in vacuum at temperatures ranging from 30 to 105 C. The samples were then subjected to thermal cycling from 130 to -130 C. Inspection following irradiation indicated that all the covers remained physically intact. However, during the temperature cycling heat-bonded covers showed cracking. The test showed that heat-bonded FEP-A covers embrittle during UV exposure and the embrittlement is dependent upon sample temperature during irradiation. The results of the experiment suggest a probable mechanism for the degradation of the FEP-A cells on ATS-6.

Search, capture and signal: games microtubules and centrosomes play.

PubMed

Schuyler, S C; Pellman, D

2001-01-01

Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.

Characterization of physiochemical properties of polymeric and electrochemical materials for aerospace flight

NASA Technical Reports Server (NTRS)

Rock, M.; Kunigahalli, V.; Khan, S.; Mcnair, A.

1984-01-01

Nickel-cadmium rechargeable batteries are a vital and reliable energy storage source for aerospace applications. As the demand for longer life and more reliable space batteries increases, the understanding and solving of cell aging factors and mechanisms become essential. Over the years, many cell designs and manufacturing process changes have been developed and implemented. Cells fabricated with various design features were life cycled in a simulated low-Earth orbit regime. Following the test program, a comprehensive electrochemical analysis of cell components was undertaken to study cell degradation mechanisms. Discharge voltage degradation or voltage plateau has been observed during orbit cycling, but, its cause and explanation have been the subject of much discussion. A Hg/HgO reference electrode was used to monitor the reference versus each electrode potential during the discharge of a cycled cell. The results indicate that the negative electrode was responsible for the voltage plateau. Cell analysis revealed large crystals of cadmium hydroxide on the surface of the negative electrode and throughout the separator.

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

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

Cunha, Elizabeth S.; Kawahara, Rebeca; Kadowaki, Marina K.

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cellmore » cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.« less

c-Myc plays a key role in TADs-induced apoptosis and cell cycle arrest in human hepatocellular carcinoma cells

PubMed Central

Zhang, Dongdong; Qi, Junpeng; Liu, Rui; Dai, Bingling; Ma, Weina; Zhan, Yingzhuan; Zhang, Yanmin

2015-01-01

Cancer cell growth is complicated progression which is regulated and controlled by multiple factors including cell cycle, migration and apoptosis. In present study, we report that TADs, a novel derivative of taspine, has an essential role in resisting hepatocellular carcinoma growth (including arrest cell cycle) and migration, and inducing cell apoptosis. Our findings demonstrated that the TADs showed good inhibition on the hepatoma cell growth and migration, and good action on apoptosis induction. Using genome-wide microarray analysis, we found the down-regulated growth and apoptosis factors, and selected down-regulated genes were confirmed by Western blot. Knockdown of a checkpoint c-Myc by siRNA significantly attenuated tumor inhibition and apoptosis effects of TADs. Moreover, our results indicated TADs could simultaneously increase cyclin D1 protein levels and decrease amount of cyclin E, cyclin B1 and cdc2 of the cycle proteins, and also TADs reduced Bcl-2 expression, and upregulated Bad, Bak and Bax activities. In conclusion, these results illustrated that TADs is a key factor in growth and apoptosis signaling inhibitor, has potential in cancer therapy. PMID:26045987

Antitumor Effects of Flavopiridol on Human Uterine Leiomyoma In Vitro and in a Xenograft Model

PubMed Central

Lee, Hyun-Gyo; Baek, Jong-Woo; Shin, So-Jin; Kwon, Sang-Hoon; Cha, Soon-Do; Park, Won-Jin; Chung, Rosa; Choi, Eun-Som; Lee, Gun-Ho

2014-01-01

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21cip/wafl and p27kip1 in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma. PMID:24572052

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

PubMed

Ferro, Anabela; Mestre, Tânia; Carneiro, Patrícia; Sahumbaiev, Ivan; Seruca, Raquel; Sanches, João M

2017-05-01

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

xCT expression reduces the early cell cycle requirement for calcium signaling

PubMed Central

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

2009-01-01

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

In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells.

PubMed

Cunneen, Thomas S; Conway, R Max; Madigan, Michele C

2009-04-01

To investigate the effects of mitomycin C and the histone deacetylase inhibitors sodium butyrate and trichostatin on the viability and growth of conjunctival melanoma cell lines and Tenon capsule fibroblasts. Cells were treated with a range of concentrations of sodium butyrate, trichostatin, and mitomycin C. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assays were performed 48 hours after treatment. Treated cells were stained with acridine orange/ethidium bromide to assess for cell death. Cell-cycle changes in histone deacetylase inhibitor-treated melanoma cells were quantified using flow cytometry. All agents induced dose-dependent cell death in the melanoma cell lines; however, sodium butyrate and trichostatin were relatively nontoxic to Tenon capsule fibroblasts. Acridine orange/ethidium bromide staining indicated that sodium butyrate and trichostatin induced apoptotic cell death. At low doses, sodium butyrate and trichostatin induced a G1 cell-cycle block in the melanoma cells. Sodium butyrate and trichostatin induced cell death in melanoma cells, comparable with mitomycin C, with minimal effect on Tenon capsule fibroblasts. In addition, they induced a G1 cell-cycle block. These findings support the need for further investigation into the in vivo efficacy of these agents.

Physical and chemical analysis of a Ni/H2 cell

NASA Technical Reports Server (NTRS)

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

1991-01-01

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

Green synthesis of platinum nanoparticles that induce cell death and G2/M-phase cell cycle arrest in human cervical cancer cells.

PubMed

Alshatwi, Ali A; Athinarayanan, Jegan; Vaiyapuri Subbarayan, Periasamy

2015-01-01

Platinum-based chemotherapeutic drugs, including cisplatin, carboplatin, and oxaliplatin, have been used to manage cancer in spite of dose-dependent side effects, including nephrotoxicity, neurotoxicity and ototoxicity. These disadvantages have prompted the development of new strategies for cancer therapy that utilize functionalized nanoparticles as nanomedicines. In the present investigation, we have synthesized platinum nanoparticles using tea polyphenol (TPP) as both a reducing and surface modifying agent. The crystalline nature and morphology of the prepared TPP-functionalized platinum nanoparticles (TPP@Pt) were analyzed using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The XRD results revealed that the TPP@Pt had a crystalline nature with a face-centered cubic structure. TEM imaging suggested that the TTP@Pt are flower shaped with a well-dispersed 30-60 nm-sized TPP@Pt formation. Cervical cancer cells (SiHa) were then treated with different concentrations of TPP@Pt. The effects of TPP@Pt on cell viability, nuclear morphology and cell cycle distribution were investigated. A cell viability assay revealed that the proliferation of SiHa cells was inhibited by TPP@Pt. Propidium iodide nuclear staining indicated that TPP@Pt induced nuclear fragmentation and chromatin condensation. Treatment with TPP@Pt significantly increased the percentage of cells in the G2/M phase, which indicates induced cell cycle arrest in the G2/M phase and an increased number of cells in the subG0 cell death phase. These findings highlight a potential use of TPP@Pt in cervical cancer treatment.

Repetitive Supra-Physiological Shear Stress Impairs Red Blood Cell Deformability and Induces Hemolysis.

PubMed

Horobin, Jarod T; Sabapathy, Surendran; Simmonds, Michael J

2017-11-01

The supra-physiological shear stress that blood is exposed to while traversing mechanical circulatory assist devices affects the physical properties of red blood cells (RBCs), impairs RBC deformability, and may induce hemolysis. Previous studies exploring RBC damage following exposure to supra-physiological shear stress have employed durations exceeding clinical instrumentation, thus we explored changes in RBC deformability following exposure to shear stress below the reported "hemolytic threshold" using shear exposure durations per minute (i.e., duty-cycles) reflective of that employed by circulatory assist devices. Blood collected from 20 male donors, aged 18-38 years, was suspended in a viscous medium and exposed to an intermittent shear stress protocol of 1 s at 100 Pa, every 60 s for 60 duty-cycles. During the remaining 59 s/min, the cells were left at stasis until the subsequent duty-cycle commenced. At discrete time points (15/30/45/60 duty-cycles), an ektacytometer measured RBC deformability immediately after shear exposure at 100 Pa. Plasma-free hemoglobin, a measurement of hemolysis, was quantified via spectrophotometry. Supra-physiological shear stress impaired RBC properties, as indicated by: (1) decreased maximal elongation of RBCs at infinite shear stress following 15 duty-cycles (P <0.05); (2) increased real-time RBC deformability during application of the supra-physiological shear stress protocol (100 Pa) following exposure to 1 duty-cycle (F (1.891, 32.15) = 12.21, P = 0.0001); and (3) increased plasma-free hemoglobin following 60 duty-cycles (P < 0.01). The present study indicates that exposure of RBCs to short-term, repeated supra-physiological shear stress, impairs RBC deformability, with the extent of impairment exacerbated with each duty-cycle, and ultimately precipitates hemolysis. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Inhibition effects of scorpion venom extracts (Buthus matensii Karsch) on the growth of human breast cancer MCF-7 cells.

PubMed

Li, Weiling; Li, Ye; Zhao, Yuwan; Yuan, Jieli; Mao, Weifeng

2014-01-01

To observe the inhibition effects of the Buthus matensii Karsch (BmK) scorpion venom extracts on the growth of human breast cancer MCF-7 cells, and to explore its mechanisms. Two common tumor cells (SMMC7721, MCF-7) were examined for the one which wasmore sensitivity to scorpion venom by MTT method. Cell cycle was determined by flow cytometry. Immunocytochemistry was applied to detect apoptosis-related protein Caspase-3 and Bcl-2 levels, while the expression of cell cycle-related protein Cyclin D1 was shown by Western blotting. Our data indicated that MCF-7 was the more sensitive cell line to scorpion venom. The extracts of scorpion venom could inhibit the growth and proliferation of MCF-7 cells. Furthermore, the extract of scorpion venom induced apoptosis through Caspase-3 up-regulation while Bcl-2 down-regulation in MCF-7 cells. In addition, the extracts of scorpion venom blocked the cells from G0/G1 phase to S phase and decreased cell cycle-related protein Cyclin D1 level after drug intervention compared with the negative control group. These results showed that the BmK scorpion venom extracts could inhibit the growth of MCF-7 cells by inducing apoptosis and blocking cell cycle in G0/G1 phase. The BmK scorpion venom extracts will be very valuable for the treatment of breast cancer.

Modelling the balance between quiescence and cell death in normal and tumour cell populations.

PubMed

Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

2006-08-01

When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

NASA Astrophysics Data System (ADS)

Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

2010-02-01

Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

A high-resolution transcriptome map of cell cycle reveals novel connections between periodic genes and cancer

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Gomez, Nicholas; Jha, Deepak Kumar; Davis, Ian; Wang, Zefeng

2016-01-01

Progression through the cell cycle is largely dependent on waves of periodic gene expression, and the regulatory networks for these transcriptome dynamics have emerged as critical points of vulnerability in various aspects of tumor biology. Through RNA-sequencing of human cells during two continuous cell cycles (>2.3 billion paired reads), we identified over 1 000 mRNAs, non-coding RNAs and pseudogenes with periodic expression. Periodic transcripts are enriched in functions related to DNA metabolism, mitosis, and DNA damage response, indicating these genes likely represent putative cell cycle regulators. Using our set of periodic genes, we developed a new approach termed “mitotic trait” that can classify primary tumors and normal tissues by their transcriptome similarity to different cell cycle stages. By analyzing >4 000 tumor samples in The Cancer Genome Atlas (TCGA) and other expression data sets, we found that mitotic trait significantly correlates with genetic alterations, tumor subtype and, notably, patient survival. We further defined a core set of 67 genes with robust periodic expression in multiple cell types. Proteins encoded by these genes function as major hubs of protein-protein interaction and are mostly required for cell cycle progression. The core genes also have unique chromatin features including increased levels of CTCF/RAD21 binding and H3K36me3. Loss of these features in uterine and kidney cancers is associated with altered expression of the core 67 genes. Our study suggests new chromatin-associated mechanisms for periodic gene regulation and offers a predictor of cancer patient outcomes. PMID:27364684

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

PubMed

Chen, Wei-Qiang; Xu, Bin; Mao, Jian-Wen; Wei, Feng-Xiang; Li, Ming; Liu, Tao; Jin, Xiao-Bao; Zhang, Li-Rong

2014-01-01

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

N-acetylcysteine normalizes the urea cycle and DNA repair in cells from patients with Batten disease.

PubMed

Kim, June-Bum; Lim, Nary; Kim, Sung-Jo; Heo, Tae-Hwe

2012-12-01

Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Copyright © 2012 John Wiley & Sons, Ltd.

The transcriptome of corona radiata cells from individual MІІ oocytes that after ICSI developed to embryos selected for transfer: PCOS women compared to healthy women.

PubMed

Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David; Nguyen, Kho do; Belling, Kirstine; Høst, Thomas; Mikkelsen, Anne Lis

2014-11-29

Corona radiata cells (CRCs) refer to the fraction of cumulus cells just adjacent to the oocyte. The CRCs are closely connected to the oocyte throughout maturation and their gene expression profiles might reflect oocyte quality. Polycystic ovary syndrome (PCOS) is a common cause of infertility. It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls. All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays. The transcriptomes of CRCs showed no individual genes with significant differential expression between PCOS and controls, but gene set enrichment analysis identified several cell cycle- and DNA replication pathways overexpressed in PCOS CRCs (FDR < 0.05). Five of the genes contributing to the up-regulated cell cycle pathways in the PCOS CRCs were selected for qRT-PCR validation in ten PCOS and ten control CRC samples. qRT-PCR confirmed significant up-regulation in PCOS CRCs of cell cycle progression genes HIST1H4C (FC = 2.7), UBE2C (FC = 2.6) and cell cycle related transcription factor E2F4 (FC = 2.5). The overexpression of cell cycle-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future studies are needed to clarify whether the up-regulated cell cycle pathways in PCOS CRCs have any clinical implications.

Human Cpr (Cell Cycle Progression Restoration) Genes Impart a Far(-) Phenotype on Yeast Cells

PubMed Central

Edwards, M. C.; Liegeois, N.; Horecka, J.; DePinho, R. A.; Sprague-Jr., G. F.; Tyers, M.; Elledge, S. J.

1997-01-01

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G(1) arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G(1) arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles. PMID:9383053

Effects of altered gravity on the cell cycle, actin cytoskeleton and proteome in Physarum polycephalum

NASA Astrophysics Data System (ADS)

He, Jie; Zhang, Xiaoxian; Gao, Yong; Li, Shuijie; Sun, Yeqing

Some researchers suggest that the changes of cell cycle under the effect of microgravity may be associated with many serious adverse physiological changes. In the search for underlying mechanisms and possible new countermeasures, we used the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony to study the effects of altered gravity on the cell cycle, actin cytoskeleton and proteome. In parallel, the cell cycle was analyzed in Physarum incubated (1) in altered gravity for 20 h, (2) in altered gravity for 40 h, (3) in altered gravity for 80 h, and (4) in ground controls. The cell cycle, the actin cytoskeleton, and proteome in the altered gravity and ground controls were examined. The results indicated that the duration of the G2 phase was lengthened 20 min in high aspect ratio vessel (HARV) for 20 h, and prolonged 2 h in altered gravity either for 40 h or for 80 h, whereas the duration of other phases in the cell cycle was unchanged with respect to the control. The microfilaments in G2 phase had a reduced number of fibers and a unique abnormal morphology in altered gravity for 40 h, whereas the microfilaments in other phases of cell cycle were unchanged when compared to controls. Employing classical two-dimensional electrophoresis (2-DE), we examined the effect of the altered gravity on P. polycephalum proteins. The increase in the duration of G2 phase in altered gravity for 40 h was accompanied by changes in the 2-DE protein profiles, over controls. Out of a total of 200 protein spots investigated in G2 phase, which were reproducible in repeated experiments, 72 protein spots were visually identified as specially expressed, and 11 proteins were up-regulated by 2-fold and 28 proteins were down-regulated by 2-fold over controls. Out of a total of three low-expressed proteins in G2 phase in altered gravity for 40 h, two proteins were unknown proteins, and one protein was spherulin 3b by MALDI-TOF mass spectrometry (MS). Our results suggest that a low level of spherulin 3b in G2 phase, which may lead to a reduction of Poly(b-L-malate) (PMLA), may contribute to the lengthened duration of G2 phase in altered gravity for 40 h. Present results indicate that altered gravity results in the prolongation of G2 phase with significantly altered actin cytoskeleton and proteome in P. polycephalum.

A superoxide anion generator, pyrogallol, inhibits the growth of HeLa cells via cell cycle arrest and apoptosis.

PubMed

Kim, Sang Wook; Han, Yong Whan; Lee, Soo Teik; Jeong, Hey Jin; Kim, Seong Hun; Kim, In Hee; Lee, Seung Ok; Kim, Dae Ghon; Kim, Suhn Hee; Kim, Sung Zoo; Park, Woo Hyun

2008-02-01

We investigated the in vitro effects of pyrogallol on cell growth, cell cycle regulation, and apoptosis in HeLa cells. Pyrogallol inhibited the growth of HeLa cells with an IC(50) of approximately 45 microM. Pyrogallol induced arrest during all phases of the cell cycle and also very efficiently resulted in apoptosis in HeLa cells, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation, and PARP cleavage. Pan-caspase inhibitor (Z-VAD) could rescue some HeLa cells from pyrogallol-induced cell death, while caspase-8 and -9 inhibitors unexpectedly enhanced the apoptosis. When we examined the changes of the ROS, H(2)O(2) or O(2)(*-) in pyrogallol-treated cells, H(2)O(2) was slightly increased and O(2)(*-) significantly was increased. In addition, we detected a decreased GSH content in pyrogallol-treated cells. Only pan-caspase inhibitor showing recovery of GSH depletion and reduced intracellular O(2)(*-) level decreased PI staining in pyrogallol-treated HeLa cells, which indicates dead cells. In summary, we have demonstrated that pyrogallol as a generator of ROS, especially O(2) (*-), potently inhibited the growth of HeLa cells through arrests during all phases of the cell cycle and apoptosis. (c) 2007 Wiley-Liss, Inc.

A temperature-sensitive dcw1 mutant of Saccharomyces cerevisiae is cell cycle arrested with small buds which have aberrant cell walls.

PubMed

Kitagaki, Hiroshi; Ito, Kiyoshi; Shimoi, Hitoshi

2004-10-01

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Deltadcw1 Deltadfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Deltadfg5). When DC61 cells were incubated at 37 degrees C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37 degrees C. At 37 degrees C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37 degrees C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37 degrees C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Deltadcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

Enhanced Longevity by Ibuprofen, Conserved in Multiple Species, Occurs in Yeast through Inhibition of Tryptophan Import

PubMed Central

He, Chong; Tsuchiyama, Scott K.; Nguyen, Quynh T.; Plyusnina, Ekaterina N.; Terrill, Samuel R.; Sahibzada, Sarah; Patel, Bhumil; Faulkner, Alena R.; Shaposhnikov, Mikhail V.; Tian, Ruilin; Tsuchiya, Mitsuhiro; Kaeberlein, Matt; Moskalev, Alexey A.; Kennedy, Brian K.; Polymenis, Michael

2014-01-01

The common non-steroidal anti-inflammatory drug ibuprofen has been associated with a reduced risk of some age-related pathologies. However, a general pro-longevity role for ibuprofen and its mechanistic basis remains unclear. Here we show that ibuprofen increased the lifespan of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, indicative of conserved eukaryotic longevity effects. Studies in yeast indicate that ibuprofen destabilizes the Tat2p permease and inhibits tryptophan uptake. Loss of Tat2p increased replicative lifespan (RLS), but ibuprofen did not increase RLS when Tat2p was stabilized or in an already long-lived strain background impaired for aromatic amino acid uptake. Concomitant with lifespan extension, ibuprofen moderately reduced cell size at birth, leading to a delay in the G1 phase of the cell cycle. Similar changes in cell cycle progression were evident in a large dataset of replicatively long-lived yeast deletion strains. These results point to fundamental cell cycle signatures linked with longevity, implicate aromatic amino acid import in aging and identify a largely safe drug that extends lifespan across different kingdoms of life. PMID:25521617

A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

PubMed

Weisskopf, M; Schaffner, W; Jundt, G; Sulser, T; Wyler, S; Tullberg-Reinert, H

2005-10-01

Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel

PubMed Central

Pietrofesa, Ralph A.; Velalopoulou, Anastasia; Lehman, Stacey L.; Arguiri, Evguenia; Solomides, Pantelis; Koch, Cameron J.; Mishra, Om P.; Koumenis, Constantinos; Goodwin, Thomas J.; Christofidou-Solomidou, Melpo

2016-01-01

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest. PMID:27322243

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel.

PubMed

Pietrofesa, Ralph A; Velalopoulou, Anastasia; Lehman, Stacey L; Arguiri, Evguenia; Solomides, Pantelis; Koch, Cameron J; Mishra, Om P; Koumenis, Constantinos; Goodwin, Thomas J; Christofidou-Solomidou, Melpo

2016-06-16

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O₂ for 8 h only (O₂), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O₂ + IR) followed by 16 h of normoxia (ambient air containing 21% O₂ and 5% CO₂) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O₂ + IR exacerbated cell death and DNA damage compared to individual exposures O₂ or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.

X ray sensitivity of diploid skin fibroblasts from patients with Fanconi's anemia

NASA Technical Reports Server (NTRS)

Kale, Ranjini

1989-01-01

Experiments were performed on Fanconi's anemia and normal human fibroblast cell lines growing in culture in an attempt to correlate cell cycle kinetics with genomic damage and determine their bearing on the mechanism of chromosome aberration induction. FA fibroblasts showed a significantly increased susceptibility to chromosomal breakage by x rays in the G2 phase of the cell cycle. No such response was observed in fibroblasts irradiated in the G0 phase. The observed increases in achromatic lesions and in chromatid deletions in FA cells as compared with normal cells appear to indicate that FA cells are deficient in strand break repair and also possibly in base damage excision repair. Experiments are now in progress to further elucidate the mechanisms involved.

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

PubMed Central

Elliott, David M.; Nagarkatti, Mitzi

2016-01-01

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

Aglycone Ebselen and β-d-Xyloside Primed Glycosaminoglycans Co-contribute to Ebselen β-d-Xyloside-Induced Cytotoxicity.

PubMed

Tang, Yang; Zhang, Siqi; Chang, Yajing; Fan, Dacheng; Agostini, Ariane De; Zhang, Lijuan; Jiang, Tao

2018-04-12

Most β-d-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to d-xylose, d-glucose, d-galactose, and d-lactose (8A-D), only Ebselen β-d-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-d-xyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B-D; 3. Both 8A and phenyl-β-d-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans.

Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

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

Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblasticmore » cells.« less

Effects of ginsenosides Rg1 and Rb1 of Panax ginseng on mitosis in root tip cells of Allium cepa.

PubMed

Ng, W Y; Chao, C Y

1981-01-01

The effects of ginsenosides Rg1 and Rb1 of Panax ginseng on mitosis in the onion root tip cells as well as on the rate of DNA synthesis in onion seedlings were studied. Results obtained from the concentration and time course study in bulb and seeding root tip cells indicate that Rg1 promotes and Rb1 inhibits mitosis, both being dose-dependent. The promoting effect of Rg1 on the rate of DNA synthesis was observed at the peak hour which occurs at the same time as that of the control. Rb1 was found to shift the peak hour of DNA synthesis to a later period of the experiment. These results are in agreement with the results obtained from the study of the cell cycle by pulse labeling and autoradiography, which show that Rg1 shortens the mitotic cell cycle and S period while Rb1 lengthens them. They in turn increase and decrease the mitotic indices respectively.

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

PubMed Central

Chen, C.; Yang, R.L.

2013-01-01

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer. PMID:23903687

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells.

PubMed

Qiu, Yueqin; Ma, Xianyong; Yang, Xuefen; Wang, Li; Jiang, Zongyong

2017-04-01

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.

Comparing aging of graphite/LiFePO4 cells at 22 °C and 55 °C - Electrochemical and photoelectron spectroscopy studies

NASA Astrophysics Data System (ADS)

Hellqvist Kjell, Maria; Malmgren, Sara; Ciosek, Katarzyna; Behm, Mårten; Edström, Kristina; Lindbergh, Göran

2013-12-01

Accelerated aging at elevated temperature is commonly used to test lithium-ion battery lifetime, but the effect of an elevated temperature is still not well understood. If aging at elevated temperature would only be faster, but in all other respects equivalent to aging at ambient temperature, cells aged to end-of-life (EOL) at different temperatures would be very similar. The present study compares graphite/LiFePO4-based cells either cycle- or calendar-aged to EOL at 22 °C and 55 °C. Cells cycled at the two temperatures show differences in electrochemical impedance spectra as well as in X-ray photoelectron spectroscopy (XPS) spectra. These results show that lithium-ion cell aging is a complex set of processes. At elevated temperature, the aging is accelerated in process-specific ways. Furthermore, the XPS results of cycle-aged samples indicate increased deposition of oxygenated LiPF6 decomposition products in both the negative and positive electrode/electrolyte interfaces. The decomposition seems more pronounced at elevated temperature, and largely accelerated by cycling, which could contribute to the observed cell impedance increase.

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

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

PubMed

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

2016-06-28

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

Cell Cycle Inhibition To Treat Sleeping Sickness.

PubMed

Epting, Conrad L; Emmer, Brian T; Du, Nga Y; Taylor, Joann M; Makanji, Ming Y; Olson, Cheryl L; Engman, David M

2017-09-19

African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens. IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals. Copyright © 2017 Epting et al.

Multiprobe Study of the Solid Electrolyte Interphase on Silicon-Based Electrodes in Full-Cell Configuration

PubMed Central

Moreau, P.; De Vito, E.; Quazuguel, L.; Boniface, M.; Bordes, A.; Rudisch, C.; Bayle-Guillemaud, P.; Guyomard, D.

2016-01-01

The failure mechanism of silicon-based electrodes has been studied only in a half-cell configuration so far. Here, a combination of 7Li, 19F MAS NMR, XPS, TOF-SIMS, and STEM-EELS, provides an in-depth characterization of the solid electrolyte interphase (SEI) formation on the surface of silicon and its evolution upon aging and cycling with LiNi1/3Mn1/3Co1/3O2 as the positive electrode in a full Li-ion cell configuration. This multiprobe approach indicates that the electrolyte degradation process observed in the case of full Li-ion cells exhibits many similarities to what has been observed in the case of half-cells in previous works, in particular during the early stages of the cycling. Like in the case of Si/Li half-cells, the development of the inorganic part of the SEI mostly occurs during the early stage of cycling while an incessant degradation of the organic solvents of the electrolyte occurs upon cycling. However, for extended cycling, all the lithium available for cycling is consumed because of parasitic reactions and is either trapped in an intermediate part of the SEI or in the electrolyte. This nevertheless does not prevent the further degradation of the organic electrolyte solvents, leading to the formation of lithium-free organic degradation products at the extreme surface of the SEI. At this point, without any available lithium left, the cell cannot function properly anymore. Cycled positive and negative electrodes do not show any sign of particles disconnection or clogging of their porosity by electrolyte degradation products and can still function in half-cell configuration. The failure mechanism for full Li-ion cells appears then very different from that known for half-cells and is clearly due to a lack of cyclable lithium because of parasitic reactions occurring before the accumulation of electrolyte degradation products clogs the porosity of the composite electrode or disconnects the active material particles. PMID:27212791

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

PubMed Central

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

2015-01-01

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

cdc-25.2, a Caenorhabditis elegans ortholog of cdc25, is required for male tail morphogenesis.

PubMed

Oh, Sangmi; Yoon, Sunghee; Youn, Esther; Kawasaki, Ichiro; Shim, Yhong-Hee

2017-01-22

Cell division cycle 25 (Cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression by activating cyclin-dependent kinases (Cdks) which are inactivated by Wee1/Myt1 kinases. It was previously reported that cdc-25.2 promotes oocyte maturation and intestinal cell divisions in Caenorhabditis elegans hermaphrodites. Here, we report a novel function of cdc-25.2 in male tail development which was significantly deformed by cdc-25.2 RNAi depletion and in cdc-25.2 mutant males. The deformation was also observed after RNAi depletion of other cell cycle regulators, cdk-1, cyb-3, cyd-1, and cyl-1. Furthermore, wee-1.3 counteracted cdc-25.2 in male tail development as observed in oocyte maturation and intestine development. The number of cells in ray precursor cell lineages was significantly reduced in cdc-25.2 depleted males. These results indicate that CDC-25.2 is essential for cell divisions in ray precursor cell lineages for proper male tail development. Copyright © 2016 Elsevier Inc. All rights reserved.

Relocation of mitochondria to the prospective dorsal marginal zone during Xenopus embryogenesis

NASA Technical Reports Server (NTRS)

Yost, H. J.; Phillips, C. R.; Boore, J. L.; Bertman, J.; Whalon, B.; Danilchik, M. V.

1995-01-01

Dorsal-ventral axis formation in Xenopus laevis begins with a cytoplasmic rotation during the first cell cycle and culminates in a series of cell interactions and movements during gastrulation and neurulation that lead to the formation of dorsal-anterior structures. Evidence reported here indicates that mitochondria are differentially redistributed along the prospective dorsal-ventral axis as a consequence of the cortical-cytoplasmic rotation during the first cell cycle. This finding reinvigorates a possibility that has been considered for many years: asymmetries in cytoplasmic components and metabolic activities contribute to the development of morphological asymmetries.

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

PubMed

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

2017-04-01

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

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems.

A dual role of p21 in stem cell aging.

PubMed

Ju, Zhenyu; Choudhury, Aaheli Roy; Rudolph, K Lenhard

2007-04-01

A decline in adult stem cell function occurs during aging, likely contributing to the decline in organ homeostasis and regeneration with age. An emerging field in aging research is to analyze molecular pathways limiting adult stem cell function in response to macromolecular damage accumulation during aging. Current data suggest that the p21 cell cycle inhibitor has a dual role in stem cell aging: On one hand, p21 protects adult stem cells from acute genotoxic stress by preventing inappropriate cycling of acutely damaged stem cells. On the other hand, p21 activation impairs stem cell function and survival of aging telomere dysfunctional mice indicating that p21 checkpoint function is disadvantageous in the context of chronic and persistent damage, which accumulates during aging. This article focuses on these dual roles of p21 in aging stem cells.

Overexpression of the growth arrest-specific homeobox gene Gax inhibits proliferation, migration, cell cycle progression, and apoptosis in serum-induced vascular smooth muscle cells.

PubMed

Zheng, H; Xue, S; Hu, Z L; Shan, J G; Yang, W G

2014-03-24

The Gax gene has been implicated in a variety of cell-developmental and biological processes, and aberrant Gax expression is linked to many diseases. In this study, to provide important insights for Gax-based gene therapy in vein graft restenosis and its anti-restenotic mechanism, we used rabbit vascular smooth muscle cells (VSMCs) to investigate the effects of Gax overexpression on proliferation, migration, cell cycle, and apoptosis in a serum-stimulated culture. Rabbit VSMC lines that stably overexpressed Gax were established by transfection with recombinant adenoviral vector Ad5-Gax. The effect of Gax overexpression on in vitro serum-induced VSMCs proliferation, migration, cell cycle, and apoptosis was assessed by MTT, wound healing, and flow cytometry assays, respectively. To investigate the effect of Gax overexpression on PCNA and MMP-2 in serum-induced VSMCs, immunocytochemistry, RT-PCR, and gelatin zymography were performed. The results clearly showed that Gax overexpression decreases PCNA expression in serum-induced VSMCs. Gax overexpression also significantly inhibited cell proliferation by blocking entry into the S-phase of the cell cycle, promoted cell apoptosis, and reduced cell migration activity by downregulating MMP-2 release and activity. These findings indicate that Gax would be an optimal target gene for gene therapy to treat vein graft restenosis.

The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells.

PubMed

Wu, Xin-jiang; Stahl, Thorsten; Hu, Ying; Kassie, Fekadu; Mersch-Sundermann, Volker

2006-03-01

Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.

Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb

PubMed Central

Mi, Da; Carr, Catherine B.; Georgala, Petrina A.; Huang, Yu-Ting; Manuel, Martine N.; Jeanes, Emily; Niisato, Emi; Sansom, Stephen N.; Livesey, Frederick J.; Theil, Thomas; Hasenpusch-Theil, Kerstin; Simpson, T. Ian; Mason, John O.; Price, David J.

2013-01-01

Summary The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct. PMID:23622063

Distinct chronology of neuronal cell cycle re-entry and tau pathology in the 3xTg-AD mouse model and Alzheimer's disease patients.

PubMed

Hradek, Alex C; Lee, Hyun-Pil; Siedlak, Sandra L; Torres, Sandy L; Jung, Wooyoung; Han, Ashley H; Lee, Hyoung-gon

2015-01-01

Cell cycle re-entry in Alzheimer's disease (AD) has emerged as an important pathological mechanism in the progression of the disease. This appearance of cell cycle related proteins has been linked to tau pathology in AD, but the causal and temporal relationship between the two is not completely clear. In this study, we found that hyperphosphorylated retinoblastoma protein (ppRb), a key regulator for G1/S transition, is correlated with a late marker for hyperphosphorylation of tau but not with other early markers for tau alteration in the 3xTg-AD mouse model. However, in AD brains, ppRb can colocalize with both early and later markers for tau alterations, and can often be found singly in many degenerating neurons, indicating the distinct development of pathology between the 3xTg-AD mouse model and human AD patients. The conclusions of this study are two-fold. First, our findings clearly demonstrate the pathological link between the aberrant cell cycle re-entry and tau pathology. Second, the chronological pattern of cell cycle re-entry with tau pathology in the 3xTg-AD mouse is different compared to AD patients suggesting the distinct pathogenic mechanism between the animal AD model and human AD patients.

Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

PubMed

Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

2015-05-12

This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

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

PubMed Central

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

2007-01-01

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

IR spectroscopic characteristics of cell cycle and cell death probed by synchrotron radiation based Fourier transform IR spectromicroscopy

NASA Technical Reports Server (NTRS)

Holman, H. Y.; Martin, M. C.; Blakely, E. A.; Bjornstad, K.; McKinney, W. R.

2000-01-01

Synchrotron radiation based Fourier transform IR (SR-FTIR) spectromicroscopy allows the study of individual living cells with a high signal to noise ratio. Here we report the use of the SR-FTIR technique to investigate changes in IR spectral features from individual human lung fibroblast (IMR-90) cells in vitro at different points in their cell cycle. Clear changes are observed in the spectral regions corresponding to proteins, DNA, and RNA as a cell changes from the G(1)-phase to the S-phase and finally into mitosis. These spectral changes include markers for the changing secondary structure of proteins in the cell, as well as variations in DNA/RNA content and packing as the cell cycle progresses. We also observe spectral features that indicate that occasional cells are undergoing various steps in the process of cell death. The dying or dead cell has a shift in the protein amide I and II bands corresponding to changing protein morphologies, and a significant increase in the intensity of an ester carbonyl C===O peak at 1743 cm(-1) is observed. Copyright John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 57: 329-335, 2000.

Overexpression of high molecular weight FGF-2 forms inhibits glioma growth by acting on cell-cycle progression and protein translation

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

Lemiere, Sylvie; University Bordeaux1, Talence, F-33405; Azar, Rania

2008-12-10

In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at themore » G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation.« less

Long noncoding RNA SNHG7 accelerates prostate cancer proliferation and cycle progression through cyclin D1 by sponging miR-503.

PubMed

Qi, Honggang; Wen, Bifeng; Wu, Qihang; Cheng, Wei; Lou, Jiangyong; Wei, Junjun; Huang, Jianjun; Yao, Xuping; Weng, Guobin

2018-06-01

Increasing evidence has indicated the important roles of long non-coding RNAs (lncRNAs) in tumorigenesis and cellular progression, including prostate cancer. In this study, we aim to investigate the expression level of SNHG7 and its biological functions on prostate cancer cells. Results indicated that SNHG7 expression was significantly up-regulated in prostate cancer tissue and cell lines. Besides, the overexpression of SNHG7 was closely correlated with the poor prognosis. In vitro and in vivo, experiments demonstrated that SNHG7 knockdown markedly inhibited prostate cancer proliferation and cycle-related protein (CDK4, CDK6, Cyclin D1), induced cell cycle arrest at G0/G1 phase and suppressed tumor growth. Moreover, miR-503 was predicted by bioinformatics tools and validated using luciferase reporter assay to both directly inhibited SNHG7 and Cyclin D1 expression by targeting their RNA 3'-UTR. In conclusion, results present that SNHG7 regulates the cycle progression and acts as an oncogenic gene in the prostate cancer tumorigenesis via miR-503/Cyclin D1 pathway, revealing the vital role of lncRNA/miRNA/mRNA axis in prostate cancer carcinogenesis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

PubMed

Nagle, Amrita A; Gan, Fei-Fei; Jones, Gavin; So, Choon-Leng; Wells, Geoffrey; Chew, Eng-Hui

2012-01-01

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2)/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2) phase. G(2) arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2) to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2) phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.

Antitumoral effect of vanadium compounds in malignant melanoma cell lines.

PubMed

Rozzo, Carla; Sanna, Daniele; Garribba, Eugenio; Serra, Maria; Cantara, Alessio; Palmieri, Giuseppe; Pisano, Marina

2017-09-01

In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [V IV O(dhp) 2 ] where dhp - is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [V IV O(mpp) 2 ] where mpp - is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [V IV O(ppp) 2 ] where ppp - is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC 50 ranges from 2.4μM up to 14μM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC 50 of 4.7 and 2.6μM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of HRAS oncogene on cell cycle progression in a cervical cancer-derived cell line.

PubMed

Córdova-Alarcón, Emilio; Centeno, Federico; Reyes-Esparza, Jorge; García-Carrancá, Alejandro; Garrido, Efraín

2005-01-01

Human papillomavirus (HPV) infection is the most prevalent factor in anogenital cancers. However, epidemiological surveys and molecular data indicate that viral presence is not enough to induce cervical cancer, suggesting that cellular factors could play a key role. One of the most important genes involved in cancer development is the RAS oncogene, and activating mutations in this gene have been associated with HPV infection and cervical neoplasia. Thus, we determined the effect of HRAS oncogene expression on cell proliferation in a cell line immortalized by E6 and E7 oncogenes. HPV positive human cervical carcinoma-derived cell lines (HeLa), previously transfected with the HRAS oncogene or the empty vector, were used. We first determined the proliferation rate and cell cycle profile of these cells by using flow cytometry and BrdU incorporation assays. In order to determine the signaling pathway regulated by HRAS and implicated in the alteration of proliferation of these cells, we used specific chemical inhibitors to inactivate the Raf and PI3K pathways. We observed that HeLa cells stably transfected with oncogenic HRAS progressed faster than control cells on the cell cycle by reducing their G1 phase. Additionally, HRAS overexpression accelerated the G1/S transition. Specific chemical inhibitors for PI3K and MEK activities indicated that both PI3K/AKT and RAF/MEK/ERK pathways are involved in the HRAS oncogene-induced reduction of the G1 phase. Our results suggest that the HRAS oncogene could play an important role in the development of cervical cancer, in addition to the presence of HPV, by reducing the G1 phase and accelerating the G1/S transition of infected cells.

Proliferation of murine c-kit(pos) cardiac stem cells stimulated with IGF-1 is associated with Akt-1 mediated phosphorylation and nuclear export of FoxO3a and its effect on downstream cell cycle regulators.

PubMed

Johnson, Ann Mary; Kartha, C C

2014-04-01

Insulin-like growth factor-1 (IGF-1) is known to promote proliferation in many cell types including c-kit(pos) cardiac stem cells (CSCs). Downstream signaling pathways of IGF-1 induced CSC proliferation have not been investigated. An important downstream target of IGF-1/Akt-1 signaling is FoxO3a, a key negative regulator of cell-cycle progression. We studied the effect of IGF-1 on proliferation of c-kit(pos) murine CSCs and found that IGF-1-mediated cell proliferation is associated with FoxO3a phosphorylation and inactivation of its transcriptional activity. PI3 inhibitors LY294002 and Wortmannin abolished the effect of IGF-1 on FoxO3a phosphorylation indicating that FoxO3a phosphorylation is mediated by PI3/Akt-1 pathway. In cells with FoxO3a translocation to the cytoplasm, there is decreased expression of cell-cycle inhibitors such as p27(kip1) and p57(kip2) and increased expression of CyclinD1. Our study provides evidence that IGF-1 induced CSC proliferation could be the result of FoxO3a inactivation and its downstream effect on cell-cycle regulators.

Intact Arabidopsis RPB1 functions in stem cell niches maintenance and cell cycling control.

PubMed

Zhang, Qian-Qian; Li, Ying; Fu, Zhao-Ying; Liu, Xun-Biao; Yuan, Kai; Fang, Ying; Liu, Yan; Li, Gang; Zhang, Xian-Sheng; Chong, Kang; Ge, Lei

2018-05-12

Plant meristem activity depends on accurate execution of transcriptional networks required for establishing optimum functioning of stem cell niches. An Arabidopsis mutant card1-1 (constitutive auxin response with DR5:GFP) that encodes a truncated RPB1 (RNA Polymerase II's largest subunit) with shortened C-terminal domain (CTD) was identified. Phosphorylation of the CTD repeats of RPB1 is coupled to transcription in eukaryotes. Here we uncover that the truncated CTD of RPB1 disturbed cell cycling and enlarged the size of shoot and root meristem. The defects in patterning of root stem cell niche in card1-1 indicates that intact CTD of RPB1 is necessary for fine-tuning the specific expression of genes responsible for cell-fate determination. The gene-edited plants with different CTD length of RPB1, created by CRISPR-CAS9 technology, confirmed that both the full length and the DK-rich tail of RPB1's CTD play roles in the accurate transcription of CYCB1;1 encoding a cell-cycle marker protein in root meristem and hence participate in maintaining root meristem size. Our experiment proves that the intact RPB1 CTD is necessary for stem cell niche maintenance, which is mediated by transcriptional regulation of cell cycling genes. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML

PubMed Central

Bolli, Niccolò; Rhodes, Jennifer; Abdel-Wahab, Omar I.; Levine, Ross; Hedvat, Cyrus V.; Stone, Richard; Khanna-Gupta, Arati; Sun, Hong; Kanki, John P.; Gazda, Hanna T.; Beggs, Alan H.; Cotter, Finbarr E.

2011-01-01

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18hi1727/+ embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies. PMID:21653321

Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21WAF1/CIP1 expression.

PubMed

Tseng, Tsui-Hwa; Chien, Ming-Hsien; Lin, Wea-Lung; Wen, Yu-Ching; Chow, Jyh-Ming; Chen, Chi-Kuan; Kuo, Tsang-Chih; Lee, Wei-Jiunn

2017-02-01

Apigenin (4',5,7-trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA-MB-231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin-dependent kinase-1 (CDK1), which control the G2-to-M phase transition in the cell cycle. In addition, apigenin upregulated p21 WAF1/CIP1 and increased the interaction of p21 WAF1/CIP1 with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21 WAF1/CIP1 promoter region, resulting in the increase of p21 WAF1/CIP1 transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin-treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21 WAF1/CIP1 and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 434-444, 2017. © 2016 Wiley Periodicals, Inc.

Proteomic analysis of blastema formation in regenerating axolotl limbs

PubMed Central

2009-01-01

Background Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs. Results We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. Conclusion Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures. PMID:19948009

A Novel, Non-Apoptotic Role for Scythe/BAT3: A Functional Switch between the Pro- and Anti-Proliferative Roles of p21 during the Cell Cycle

PubMed Central

Yong, Sheila T.; Wang, Xiao-Fan

2012-01-01

Background Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. Methods/Principal Findings Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. Conclusion: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle. PMID:22761665

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

PubMed

Lange, S; Viergutz, T; Simkó, M

2004-10-01

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

Extremely low-frequency electromagnetic fields cause DNA strand breaks in normal cells

PubMed Central

2014-01-01

Background Extremely low frequency electromagnetic fields aren’t considered as a real carcinogenic agent despite the fact that some studies have showed impairment of the DNA integrity in different cells lines. The aim of this study was evaluation of the late effects of a 100 Hz and 5.6 mT electromagnetic field, applied continuously or discontinuously, on the DNA integrity of Vero cells assessed by alkaline Comet assay and by cell cycle analysis. Normal Vero cells were exposed to extremely low frequency electromagnetic fields (100 Hz, 5.6 mT) for 45 minutes. The Comet assay and cell cycle analysis were performed 48 hours after the treatment. Results Exposed samples presented an increase of the number of cells with high damaged DNA as compared with non-exposed cells. Quantitative evaluation of the comet assay showed a significantly (<0.001) increase of the tail lengths, of the quantity of DNA in tail and of Olive tail moments, respectively. Cell cycle analysis showed an increase of the frequency of the cells in S phase, proving the occurrence of single strand breaks. The most probable mechanism of induction of the registered effects is the production of different types of reactive oxygen species. Conclusions The analysis of the registered comet indices and of cell cycle showed that extremely low frequency electromagnetic field of 100 Hz and 5.6 mT had a genotoxic impact on Vero cells. PMID:24401758

Phloretin induces cell cycle arrest and apoptosis of human glioblastoma cells through the generation of reactive oxygen species.

PubMed

Liu, Yuanyuan; Fan, Chenghe; Pu, Lv; Wei, Cui; Jin, Haiqiang; Teng, Yuming; Zhao, Mingming; Yu, Albert Cheung Hoi; Jiang, Feng; Shu, Junlong; Li, Fan; Peng, Qing; Kong, Jian; Pan, Bing; Zheng, Lemin; Huang, Yining

2016-06-01

Phloretin, a flavonoid present in various plants, has been reported to exert anticarcinogenic effects. However, the mechanism of its chemo-preventive effect on human glioblastoma cells is not fully understood. This study aimed to investigate the molecular mechanism of phloretin and its associated chemo-preventive effect in human glioblastoma cells. The results indicate that phloretin inhibited cell proliferation by inducing cell cycle arrest at the G0-G1 phase and induced apoptosis of human glioblastoma cells. Phloretin-induced cell cycle arrest was associated with increased expression of p27 and decreased expression of cdk2, cdk4, cdk6, cyclinD and cyclinE. Moreover, the PI3K/AKT/mTOR signaling cascades were suppressed by phloretin in a dose-dependent manner. In addition, phloretin triggered the mitochondrial apoptosis pathway and generated reactive oxygen species (ROS). This was accompanied by the up-regulation of Bax, Bak and c-PARP and the down-regulation of Bcl-2. The antioxidant agents N-acetyl-L-cysteine and glutathione weakened the effect of phloretin on glioblastoma cells. In conclusion, these results demonstrate that phloretin exerts potent chemo-preventive activity in human glioblastoma cells through the generation of ROS.

Antiproliferative action of Xylopia aethiopica fruit extract on human cervical cancer cells.

PubMed

Adaramoye, Oluwatosin A; Sarkar, Jayanta; Singh, Neetu; Meena, Sanjeev; Changkija, Bendangla; Yadav, Prem P; Kanojiya, Sanjeev; Sinha, Sudhir

2011-10-01

The anticancer potential of Xylopia aethiopica fruit extract (XAFE), and the mechanism of cell death it elicits, was investigated in various cell lines. Treatment with XAFE led to a dose-dependent growth inhibition in most cell lines, with selective cytotoxicity towards cancer cells and particularly the human cervical cancer cell line C-33A. In this study, apoptosis was confirmed by nuclear fragmentation and sub-G(0)/G(1) phase accumulation. The cell cycle was arrested at the G(2)/M phase with a decreased G(0)/G(1) population. A semi-quantitative gene expression study revealed dose-dependent up-regulation of p53 and p21 genes, and an increase in the Bax/Bcl-2 ratio. These results indicate that XAFE could be a potential therapeutic agent against cancer since it inhibits cell proliferation, and induces apoptosis and cell cycle arrest in C-33A cells. Copyright © 2011 John Wiley & Sons, Ltd.

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

PubMed Central

2012-01-01

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

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

PubMed

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

2012-07-12

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

Modeling cell-cycle synchronization during embryogenesis in Xenopus laevis

NASA Astrophysics Data System (ADS)

McIsaac, R. Scott; Huang, K. C.; Sengupta, Anirvan; Wingreen, Ned

2010-03-01

A widely conserved aspect of embryogenesis is the ability to synchronize nuclear divisions post-fertilization. How is synchronization achieved? Given a typical protein diffusion constant of 10 μm^2sec, and an embryo length of 1mm, it would take diffusion many hours to propagate a signal across the embryo. Therefore, synchrony cannot be attained by diffusion alone. We hypothesize that known autocatalytic reactions of cell-cycle components make the embryo an ``active medium'' in which waves propagate much faster than diffusion, enforcing synchrony. We report on robust spatial synchronization of components of the core cell cycle circuit based on a mathematical model previously determined by in vitro experiments. In vivo, synchronized divisions are preceded by a rapid calcium wave that sweeps across the embryo. Experimental evidence supports the hypothesis that increases in transient calcium levels lead to derepression of a negative feedback loop, allowing cell divisions to start. Preliminary results indicate a novel relationship between the speed of the initial calcium wave and the ability to achieve synchronous cell divisions.

A novel flavonoid isolated from Sophora flavescens exhibited anti-angiogenesis activity, decreased VEGF expression and caused G0/G1 cell cycle arrest in vitro.

PubMed

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

2013-05-01

Kushen, the dried root of Sophora flavescens Ait, is a traditional Chinese herbal medicine. Kushen alkaloids have been developed in China as anticancer drugs, and more potent antitumor activities have been identified in kushen flavonoids than in kushen alkaloids. In this study, the anti-angiogenic properties of (2S)-7,2',4'-triihydroxy-5-methoxy-8-dimethylallyl flavanone (Compound 1, a novel flavonoid isolated from Kushen), were examined using the human umbilical vein endothelial cell line (ECV304) in vitro. The results indicated that compound 1 shows anti-angiogenesis activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. Further studies indicated that compound 1 blocks cell cycles in the G0/G1 phase without inducing apoptosis, and down regulates vascular endothelial growth factor (VEGF) expression. The free radical scavenging activity of compound 1 was found through 2',7'-dichlorofluorescin diacetate (DCFH-DA) incubation assay in cells. The anti-angiogenic properties of compound 1 and its antiproliferative effect on endothelial cells without causing apoptosis make it a good candidate for development as a agent against development of tumors.

The MEK1/2 Inhibitor AZD6244 Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

PubMed

Song, Hao; Zhang, Jinna; Ning, Liang; Zhang, Honglai; Chen, Dong; Jiao, Xuelong; Zhang, Kejun

2018-05-08

BACKGROUND [i]BRAF[/i]V600E mutation occurs in approximately 45% of papillary thyroid cancer (PTC) cases, and 25% of anaplastic thyroid cancer (ATC) cases. Vemurafenib/PLX4032, a selective BRAF inhibitor, suppresses extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) signaling and shows beneficial effects in patients with metastatic melanoma harboring the [i]BRAFV600E[/i] mutation. However, the response to vemurafenib is limited in BRAF-mutant thyroid cancer. The present study evaluated the effect of vemurafenib in combination with the selective MEK1/2 inhibitor AZD6244 on cell survival and explored the mechanism underlying the combined effect of vemurafenib and AZD6244 on thyroid cancer cells harboring BRAFV600E. MATERIAL AND METHODS Thyroid cancer 8505C and BCPAP cells harboring the [i]BRAFV600E[/i] mutation were exposed to vemurafenib (0.01, 0.1, and 1 µM) and AZD6244 (0.01, 0.1, and 1 µM) alone or in the indicated combinations for the indicated times. Cell viability was detected by the MTT assay. Cell cycle distribution and induction of apoptosis were detected by flow cytometry. The expression of cyclin D1, P27, (P)-ERK1/2 was evaluated by Western blotting. The effect of vemurafenib or AZD6244 or their combination on the growth of 8505C cells was examined in orthotopic xenograft mouse models [i]in vivo[/i]. RESULTS Vemurafenib alone did not increase cell apoptosis, whereas it decreased cell viability by promoting cell cycle arrest in BCPAP and 8505C cells. AZD6244 alone increased cell apoptosis by inducing cell cycle arrest in BCPAP and 8505C cells. Combination treatment with AZD6244 and vemurafenib significantly decreased cell viability and increased apoptosis in both BCPAP and 8505C cells compared with the effects of each drug alone. AZD6244 alone abolished phospho-ERK1/2 (pERK1/2) expression at 48 h, whereas vemurafenib alone downregulated pERK1/2 at 4-6 h, with rapid recovery of expression, reaching the highest level at 24-48 h. Combined treatment for 48 h completely inhibited pERK1/2 expression. Combination treatment with vemurafenib and AZD6244 inhibited cell growth and induced apoptosis by causing cell-cycle arrest, with the corresponding changes in the expression of the cell cycle regulators p27Kip1 and cyclin D1. Co-administration of vemurafenib and AZD6244 [i]in vivo[/i] had a significant synergistic antitumor effect in a nude mouse model. CONCLUSIONS Vemurafenib activated pERK1/2 and induced vemurafenib resistance in thyroid cancer cells. Combination treatment with vemurafenib and AZD6244 inhibited ERK signaling and caused cell cycle arrest, resulting in cell growth inhibition. Combination treatment in patients with thyroid cancer harboring the [i]BRAFV600E[/i] mutation may overcome vemurafenib resistance and enhance the therapeutic effect.

Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea

PubMed Central

Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-kun; Sheng, Jun; Shi, Wei

2011-01-01

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest. PMID:21673927

Changes of constituents and activity to apoptosis and cell cycle during fermentation of tea.

PubMed

Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-Kun; Sheng, Jun; Shi, Wei

2011-01-01

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest.

Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection

PubMed Central

Bantele, Susanne CS; Ferreira, Pedro; Gritenaite, Dalia; Boos, Dominik; Pfander, Boris

2017-01-01

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here, we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1, respectively. In yeast, this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin, and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice. DOI: http://dx.doi.org/10.7554/eLife.21687.001 PMID:28063255

Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control

PubMed Central

Chávez, Santiago; Eastman, Guillermo; Smircich, Pablo; Becco, Lorena Lourdes; Oliveira-Rizzo, Carolina; Fort, Rafael; Potenza, Mariana; Garat, Beatriz; Sotelo-Silveira, José Roberto

2017-01-01

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control. PMID:29182646

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.

PubMed

Kocsisova, Zuzana; Kornfeld, Kerry; Schedl, Tim

2018-05-30

The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.

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.

Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

PubMed

Adan, Aysun; Baran, Yusuf

2016-05-01

Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

Glycyrrhetinic acid induces G1-phase cell cycle arrest in human non-small cell lung cancer cells through endoplasmic reticulum stress pathway

PubMed Central

ZHU, JIE; CHEN, MEIJUAN; CHEN, NING; MA, AIZHEN; ZHU, CHUNYAN; ZHAO, RUOLIN; JIANG, MIAO; ZHOU, JING; YE, LIHONG; FU, HAIAN; ZHANG, XU

2015-01-01

Glycyrrhetinic acid (GA) is a natural compound extracted from liquorice, which is often used in traditional Chinese medicine. The purpose of the present study was to investigate the antitumor effect of GA in human non-small cell lung cancer (NSCLC), and its underlying mechanisms in vitro. We have shown that GA suppressed the proliferation of A549 and NCI-H460 cells. Flow cytometric analysis showed that GA arrested cell cycle in G0/G1 phase without inducing apoptosis. Western blot analysis indicated that GA mediated G1-phase cell cycle arrest by upregulation of cyclin-dependent kinase inhibitors (CKIs) (p18, p16, p27 and p21) and inhibition of cyclins (cyclin-D1, -D3 and -E) and cyclin-dependent kinases (CDKs) (CDK4, 6 and 2). GA also maintained pRb phosphorylation status, and inhibited E2F transcription factor 1 (E2F-1) in both cell lines. GA upregulated the unfolded proteins, Bip, PERK and ERP72. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggered the unfolded protein response (UPR), which could be the mechanism by which GA inhibited cell proliferation in NSCLC cells. GA then coordinated the induction of ER chaperones, which decreased protein synthesis and induced cell cycle arrest in the G1 phase. This study provides experimental evidence to support the development of GA as a chemotherapeutic agent for NSCLC. PMID:25573651

Localization of estrogen receptor α and progesterone receptor B in bovine cervix and vagina during the follicular and luteal phases of the sexual cycle.

PubMed

Sağsöz, H; Akbalik, M E; Saruhan, B G; Ketani, M A

2011-08-01

The localization and distribution of estrogen receptors (ERα) and progesterone receptors (PR-B) in the cervix and vagina of sexually mature bovines during the follicular and luteal phases of the sexual cycle were studied using immunohistochemistry. The estrous cycle stage of 23 Holstein bovines was assessed by gross and histological appearance of ovaries and blood steroid hormone values. Tissue samples from cervix and vagina were fixed in 10% formaldehyde for routine histological processing. Nuclear staining for ERα and PR-B was observed in the epithelial cells of the surface epithelium, stromal cells and smooth muscle cells. Generally, in the cervix, ERα immunoreactivity was more intense in the epithelial and smooth muscle cells during the follicular phase and in the epithelial cells during the luteal phase (p < 0.05). PR-B immunoreactivity was more intense in the epithelial and smooth muscle cells than in the superficial and deep stromal cells during the follicular and luteal phases (p < 0.05). In the vagina, ERα and PR-B immunoreactivities were more intense in the epithelial cells than in the connective tissue cells and smooth muscle cells during the follicular and luteal phases (p < 0.05). These results indicated that the frequency and intensity of ERα and PR-B immunoreactivity in the cervix and vagina of bovines varied according to the cervical and vaginal cell types and the phases of the sexual cycle.

The Tlx gene regulates the timing of neurogenesis in the cortex.

PubMed

Roy, Kristine; Kuznicki, Kathleen; Wu, Qiang; Sun, Zhuoxin; Bock, Dagmar; Schutz, Gunther; Vranich, Nancy; Monaghan, A Paula

2004-09-22

The tailless (tlx) gene is a forebrain-restricted transcription factor. Tlx mutant animals exhibit a reduction in the size of the cerebral hemispheres and associated structures (Monaghan et al., 1997). Superficial cortical layers are specifically reduced, whereas deep layers are relatively unaltered (Land and Monaghan, 2003). To determine whether the adult laminar phenotype has a developmental etiology and whether it is associated with a change in proliferation/differentiation decisions, we examined the cell cycle and neurogenesis in the embryonic cortex. We found that there is a temporal and regional requirement for the Tlx protein in progenitor cells (PCs). Neurons prematurely differentiate at all rostrocaudal levels up to mid-neurogenesis in mutant animals. Heterozygote animals have an intermediate phenotype indicating there is a threshold requirement for Tlx in early cortical neurogenesis. Our studies indicate that PCs in the ventricular zone are sensitive to loss of Tlx in caudal regions only; however, PCs in the subventricular zone are altered at all rostrocaudal levels in tlx-deficient animals. Furthermore, we found that the cell cycle is shorter from embryonic day 9.5 in tlx-/- embryos. At mid-neurogenesis, the PC population becomes depleted, and late PCs have a longer cell cycle in tlx-deficient animals. Consequently, later generated structures, such as upper cortical layers, the dentate gyrus, and the olfactory bulbs, are severely reduced. These studies indicate that tlx is an essential intrinsic regulator in the decision to proliferate or differentiate in the developing forebrain.

The Tlx Gene Regulates the Timing of Neurogenesis in the Cortex

PubMed Central

Roy, Kristine; Kuznicki, Kathleen; Wu, Qiang; Sun, Zhuoxin; Bock, Dagmar; Schutz, Gunther; Vranich, Nancy; Monaghan, A. Paula

2009-01-01

The tailless (tlx) gene is a forebrain-restricted transcription factor. Tlx mutant animals exhibit a reduction in the size of the cerebral hemispheres and associated structures (Monaghan et al., 1997). Superficial cortical layers are specifically reduced, whereas deep layers are relatively unaltered (Land and Monaghan, 2003). To determine whether the adult laminar phenotype has a developmental etiology and whether it is associated with a change in proliferation/differentiation decisions, we examined the cell cycle and neurogenesis in the embryonic cortex. We found that there is a temporal and regional requirement for the Tlx protein in progenitor cells (PCs). Neurons prematurely differentiate at all rostrocaudal levels up to mid-neurogenesis in mutant animals. Heterozygote animals have an intermediate phenotype indicating there is a threshold requirement for Tlx in early cortical neurogenesis. Our studies indicate that PCs in the ventricular zone are sensitive to loss of Tlx in caudal regions only; however, PCs in the subventricular zone are altered at all rostrocaudal levels in tlx-deficient animals. Furthermore, we found that the cell cycle is shorter from embryonic day 9.5 in tlx−/− embryos. At mid-neurogenesis, the PC population becomes depleted, and late PCs have a longer cell cycle in tlx-deficient animals. Consequently, later generated structures, such as upper cortical layers, the dentate gyrus, and the olfactory bulbs, are severely reduced. These studies indicate that tlx is an essential intrinsic regulator in the decision to proliferate or differentiate in the developing forebrain. PMID:15385616

Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia.

PubMed

Ishikawa, Hiroaki; Kubo, Akiharu; Tsukita, Shoichiro; Tsukita, Sachiko

2005-05-01

Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2(-/-) cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2(-/-) cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2(-/-) cells, however, the formation of primary cilia is completely suppressed, although approximately 25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2(-/-) F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions.

IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells.

PubMed

Dardalhon, V; Jaleco, S; Kinet, S; Herpers, B; Steinberg, M; Ferrand, C; Froger, D; Leveau, C; Tiberghien, P; Charneau, P; Noraz, N; Taylor, N

2001-07-31

Differences in the immunological reactivity of umbilical cord (UC) and adult peripheral blood (APB) T cells are poorly understood. Here, we show that IL-7, a cytokine involved in lymphoid homeostasis, has distinct regulatory effects on APB and UC lymphocytes. Neither naive nor memory APB CD4(+) cells proliferated in response to IL-7, whereas naive UC CD4(+) lymphocytes underwent multiple divisions. Nevertheless, both naive and memory IL-7-treated APB T cells progressed into the G(1b) phase of the cell cycle, albeit at higher levels in the latter subset. The IL-7-treated memory CD4(+) lymphocyte population was significantly more susceptible to infection with an HIV-1-derived vector than dividing CD4(+) UC lymphocytes. However, activation through the T cell receptor rendered UC lymphocytes fully susceptible to HIV-1-based vector infection. These data unveil differences between UC and APB CD4(+) T cells with regard to IL-7-mediated cell cycle progression and HIV-1-based vector infectivity. This evidence indicates that IL-7 differentially regulates lymphoid homeostasis in adults and neonates.

IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells

PubMed Central

Dardalhon, Valérie; Jaleco, Sara; Kinet, Sandrina; Herpers, Bjorn; Steinberg, Marcos; Ferrand, Christophe; Froger, Delphine; Leveau, Christelle; Tiberghien, Pierre; Charneau, Pierre; Noraz, Nelly; Taylor, Naomi

2001-01-01

Differences in the immunological reactivity of umbilical cord (UC) and adult peripheral blood (APB) T cells are poorly understood. Here, we show that IL-7, a cytokine involved in lymphoid homeostasis, has distinct regulatory effects on APB and UC lymphocytes. Neither naive nor memory APB CD4+ cells proliferated in response to IL-7, whereas naive UC CD4+ lymphocytes underwent multiple divisions. Nevertheless, both naive and memory IL-7-treated APB T cells progressed into the G1b phase of the cell cycle, albeit at higher levels in the latter subset. The IL-7-treated memory CD4+ lymphocyte population was significantly more susceptible to infection with an HIV-1-derived vector than dividing CD4+ UC lymphocytes. However, activation through the T cell receptor rendered UC lymphocytes fully susceptible to HIV-1-based vector infection. These data unveil differences between UC and APB CD4+ T cells with regard to IL-7-mediated cell cycle progression and HIV-1-based vector infectivity. This evidence indicates that IL-7 differentially regulates lymphoid homeostasis in adults and neonates. PMID:11470908

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

PubMed Central

Laos, Maarja; Anttonen, Tommi; Kirjavainen, Anna; Hällström, Taija af; Laiho, Marikki; Pirvola, Ulla

2014-01-01

Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with the in vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs. PMID:25063730

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

PubMed

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

2002-03-15

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

Design and operation of interconnectors for solid oxide fuel cell stacks

NASA Astrophysics Data System (ADS)

Winkler, W.; Koeppen, J.

Highly efficient combined cycles with solid oxide fuel cell (SOFC) need an integrated heat exchanger in the stack to reach efficiencies of about 80%. The stack costs must be lower than 1000 DM/kW. A newly developed welded metallic (Haynes HA 230) interconnector with a free stretching planar SOFC and an integrated heat exchanger was tested in thermal cycling operation. The design allowed a cycling of the SOFC without mechanical damage of the electrolyte in several tests. However, more tests and a further design optimization will be necessary. These results could indicate that commercial high-temperature alloys can be used as interconnector material in order to fullfil the cost requirements.

The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. III. Kinetics of cell proliferation in the external granular layer.

PubMed

Lauder, J M

1977-04-22

The effects of early hypo- and hyperthyroidism on the rates of cell acquisition and proliferation have been studied in the external granular layer (EGL) of the developing rat cerebellar cortex at 10 days of age using quantitative autoradiographic methods. Both altered thyroid states reduce the rate of cell acquisition in the EGL, but appear to do so for different reasons. Hyperthyroidism shortens the average length of the cell cycle by decreasing the duration of the pre-DNA synthetic phase (G1), indicating that excess thyroxine may exert a direct effect on the EGL. This action involves the early onset of neuronal differentiation (cessation of proliferation)46 which presumably leads to the observed decrease in the rate of cell acquisition (increased doubling time). Such differentiating cells do not, however, leave the proliferative zone or the EGL prematurely, resulting in a reduced labeling index, mitotic index, and growth fraction as non-dividing cells dilute the proliferating cell population. Hypothyroidism, on the other hand, leads to no significant change in the length of the cell cycle or in the mitotic index, but causes a decreased labeling index and growth fraction, as well as a reduced rate of cell acquisition (increased doubling time). No significant change in the amount of cell death in the EGL could be found to explain this apparent discrepancy between the rate of cell proliferation (cell cycle length) and cell acqusiition. The answer to this puzzle appears to lie in the mitotic index, which is not affected to the same extent as the labeling index, although it is also slightly reduced. If cells were to remain longer in mitosis, this could result in a decreased labeling index and growth fraction but nearly normal mitotic index and cell cycle length (as measured using the % labeled mitoses method), since those cells dropping out of the cycling population would be counted as mitoses...

The Immunomodulatory Capacity of an Epstein-Barr Virus Abortive Lytic Cycle: Potential Contribution to Viral Tumorigenesis

PubMed Central

2018-01-01

Epstein-Barr virus (EBV) is characterized by a bipartite life cycle in which latent and lytic stages are alternated. Latency is compatible with long-lasting persistency within the infected host, while lytic expression, preferentially found in oropharyngeal epithelial tissue, is thought to favor host-to-host viral dissemination. The clinical importance of EBV relates to its association with cancer, which we think is mainly a consequence of the latency/persistency mechanisms. However, studies in murine models of tumorigenesis/lymphomagenesis indicate that the lytic cycle also contributes to cancer formation. Indeed, EBV lytic expression is often observed in established cell lines and tumor biopsies. Within the lytic cycle EBV expresses a handful of immunomodulatory (BCRF1, BARF1, BNLF2A, BGLF5 & BILF1) and anti-apoptotic (BHRF1 & BALF1) proteins. In this review, we discuss the evidence supporting an abortive lytic cycle in which these lytic genes are expressed, and how the immunomodulatory mechanisms of EBV and related herpesviruses Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV) result in paracrine signals that feed tumor cells. An abortive lytic cycle would reconcile the need of lytic expression for viral tumorigenesis without relaying in a complete cycle that would induce cell lysis to release the newly formed infective viral particles. PMID:29601503

Human immunodeficiency virus type 1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25.

PubMed

Huard, Sylvain; Elder, Robert T; Liang, Dong; Li, Ge; Zhao, Richard Y

2008-03-01

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.

Recombinant Escherichia coli Trx-JZTX-III represses the proliferation of mouse hepatocellular carcinoma cells through induction of cell cycle arrest.

PubMed

Sun, Mei-Na; Zhao, Xue-Jiao; Zhao, Han-Dong; Zhang, Wei-Guang; Li, Feng-Lan; Chen, Ming-Zi; Li, Hui; Li, Guangchao

2013-06-01

The aim of the present study was to investigate the effects of recombinant Escherichia coli (E. coli) Trx-jingzhaotoxin (JZTX)-III on cell growth in the mouse hepatocellular carcinoma (HCC) cell line Hepa1-6. The JZTX-III gene sequence was synthesized and cloned into the pET-32a(+) vector to construct the recombinant fusion protein Trx-JZTX-III, which was subsequently purified. Hepa1-6 cells were treated with 0 to 1,000-µg/ml concentrations of Trx-JZTX-III; this was demonstrated to affect cell viability, as determined by the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The expression of the proliferating cell nuclear antigen (PCNA) protein was investigated using western blot analysis. A colony formation assay was used to determine Hepa1-6 cell proliferation, and the migration ability of cells was determined using a wound‑healing assay. Additionally, flow cytometry was employed to observe changes in the cell cycle. The MTT assay and quantification of PCNA expression indicated that recombinant E. coli Trx-JZTX-III significantly repressed the proliferation of Hepa1-6 cells. Colony formation and the migration of malignant cells was inhibited following treatment with recombinant E. coli Trx-JZTX-III. Flow cytometry showed that recombinant E. coli Trx-JZTX-III induced G0/G1 cell cycle arrest. In conclusion, recombinant E. coli Trx-JZTX-III functions as a tumor suppressor drug in mouse HCC and its underlying mechanism may involve the induction of G0/G1 cell cycle arrest.

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

PubMed Central

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

1985-01-01

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

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

pRb phosphorylation regulates the proliferation of supporting cells in gentamicin-damaged neonatal avian utricle.

PubMed

Wu, Jingfang; Sun, Shan; Li, Wenyan; Chen, Yan; Li, Huawei

2014-10-01

The ability of nonmammalian vertebrates to regenerate hair cells (HCs) after damage-induced HC loss has stimulated and inspired research in the field of HC regeneration. The protein pRb encoded by retinoblastoma gene Rb1 forces sensory progenitor cells to exit cell cycle and maintain differentiated HCs and supporting cells (SCs) in a quiescent state. pRb function is regulated by phosphorylation through the MEK/ERK or the pRb/Raf-1 signaling pathway. In our previous study, we have shown that pRb phosphorylation is crucial for progenitor cell proliferation and survival during the early embryonic stage of avian otocyst sensory epithelium development. However, in damaged avian utricle, the role of pRb in regulating the cell cycling of SCs or HCs regeneration still remains unclear. To further elucidate the function of pRb phosphorylation on SCs re-entering the cell cycle triggered by gentamycin-induced HCs damage, we isolated neonatal chicken utricles and treated them with the MEK inhibitor U0126 or the pRb/Raf-1 inhibitor RRD-251, respectively in vitro. We found that after gentamycin-induced HCs damage, pRb phosphorylation is important for the quiescent SCs re-entering the cell cycle in the neonatal chicken utricle. In addition, the proliferation of SCs decreased in a dose-dependent manner in response to both U0126 and RRD-251, which indicates that both the MEK/ERK and the pRb/Raf-1 signaling pathway play important roles in pRb phosphorylation in damaged neonatal chicken utricle. Together, these findings on the function of pRb in damaged neonatal chicken utricle improve our understanding of the regulation of the cell cycle of SCs after HCs loss and may shed light on the mammalian HC regeneration from SCs in damaged organs.

Toxicology and cellular effect of manufactured nanomaterials

DOEpatents

Chen, Fanqing

2014-07-22

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Herein are described methods and assays to predict and evaluate the cellular effects of nanomaterial exposure. Exposing cells to nanomaterials at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis, activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. Certain nanomaterials induce genes indicative of a strong immune and inflammatory response within skin fibroblasts. Furthermore, the described multiwall carbon nanoonions (MWCNOs) can be used as a therapeutic in the treatment of cancer due to its cytotoxicity.

The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

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

Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com; Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522; Tooyama, Ikuo

2012-04-13

Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor ofmore » p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.« less

Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin.

PubMed

Einbond, Linda Saxe; Wu, Hsan-Au; Kashiwazaki, Ryota; He, Kan; Roller, Marc; Su, Tao; Wang, Xiaomei; Goldsberry, Sarah

2012-10-01

Studies indicate that extracts and purified components, including carnosic acid, from the herb rosemary display significant growth inhibitory activity on a variety of cancers. This paper examines the ability of rosemary/carnosic acid to inhibit the growth of human breast cancer cells and to synergize with curcumin. To do this, we treated human breast cancer cells with rosemary/carnosic acid and assessed effects on cell proliferation, cell cycle distribution, gene expression patterns, activity of the purified Na/K ATPase and combinations with curcumin. Rosemary/carnosic acid potently inhibits proliferation of ER-negative human breast cancer cells and induces G1 cell cycle arrest. Further, carnosic acid is selective for MCF7 cells transfected for Her2, indicating that Her2 may function in its action. To reveal primary effects, we treated ER-negative breast cancer cells with carnosic acid for 6h. At a low dose, 5 μg/ml (15 μM), carnosic acid activated the expression of 3 genes, induced through the presence of antioxidant response elements, including genes involved in glutathione biosynthesis (CYP4F3, GCLC) and transport (SLC7A11). At a higher dose, 20 μg/ml, carnosic acid activated the expression of antioxidant (AKR1C2, TNXRD1, HMOX1) and apoptosis (GDF15, PHLDA1, DDIT3) genes and suppressed the expression of inhibitor of transcription (ID3) and cell cycle (CDKN2C) genes. Carnosic acid exhibits synergy with turmeric/curcumin. These compounds inhibited the activity of the purified Na-K-ATPase which may contribute to this synergy. Rosemary/carnosic acid, alone or combined with curcumin, may be useful to prevent and treat ER-negative breast cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

P63 EXPRESSION LEVELS IN SIDE POPULATION AND LOW LIGHT SCATTERING OCULAR SURFACE EPITHELIAL CELLS

PubMed Central

Epstein, Seth P; Wolosin, J. Mario; Asbell, Penny A

2005-01-01

Purpose Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell–free regions, we examined p63 expression in these stem cell–associated cohorts compared with their controls. Methods Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. Results All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. Conclusions Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health. PMID:17057802

Cycle training modulates satellite cell and transcriptional responses to a bout of resistance exercise.

PubMed

Murach, Kevin A; Walton, R Grace; Fry, Christopher S; Michaelis, Sami L; Groshong, Jason S; Finlin, Brian S; Kern, Philip A; Peterson, Charlotte A

2016-09-01

This investigation evaluated whether moderate-intensity cycle ergometer training affects satellite cell and molecular responses to acute maximal concentric/eccentric resistance exercise in middle-aged women. Baseline and 72 h postresistance exercise vastus lateralis biopsies were obtained from seven healthy middle-aged women (56 ± 5 years, BMI 26 ± 1, VO2max 27 ± 4) before and after 12 weeks of cycle training. Myosin heavy chain (MyHC) I- and II-associated satellite cell density and cross-sectional area was determined via immunohistochemistry. Expression of 93 genes representative of the muscle-remodeling environment was also measured via NanoString. Overall fiber size increased ~20% with cycle training (P = 0.052). MyHC I satellite cell density increased 29% in response to acute resistance exercise before endurance training and 50% with endurance training (P < 0.05). Following endurance training, MyHC I satellite cell density decreased by 13% in response to acute resistance exercise (acute resistance × training interaction, P < 0.05). Genes with an interaction effect tracked with satellite cell behavior, increasing in the untrained state and decreasing in the endurance trained state in response to resistance exercise. Similar satellite cell and gene expression response patterns indicate coordinated regulation of the muscle environment to promote adaptation. Moderate-intensity endurance cycle training modulates the response to acute resistance exercise, potentially conditioning the muscle for more intense concentric/eccentric activity. These results suggest that cycle training is an effective endurance exercise modality for promoting growth in middle-aged women, who are susceptible to muscle mass loss with progressing age. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Hybrid cars now, fuel cell cars later.

PubMed

Demirdöven, Nurettin; Deutch, John

2004-08-13

We compare the energy efficiency of hybrid and fuel cell vehicles as well as conventional internal combustion engines. Our analysis indicates that fuel cell vehicles using hydrogen from fossil fuels offer no significant energy efficiency advantage over hybrid vehicles operating in an urban drive cycle. We conclude that priority should be placed on hybrid vehicles by industry and government.

Electron and photon degradation in aluminum, gallium and boron doped float zone silicon solar cells

NASA Technical Reports Server (NTRS)

Rahilly, W. P.; Scott-Monck, J.; Anspaugh, B.; Locker, D.

1976-01-01

Solar cells fabricated from Al, Ga and B doped Lopex silicon over a range of resistivities were tested under varying conditions of 1 MeV electron fluence, light exposures and thermal cycling. Results indicate that Al and Ga can replace B as a P type dopant to yield improved solar cell performance.

Hybrid Cars Now, Fuel Cell Cars Later

NASA Astrophysics Data System (ADS)

Demirdöven, Nurettin; Deutch, John

2004-08-01

We compare the energy efficiency of hybrid and fuel cell vehicles as well as conventional internal combustion engines. Our analysis indicates that fuel cell vehicles using hydrogen from fossil fuels offer no significant energy efficiency advantage over hybrid vehicles operating in an urban drive cycle. We conclude that priority should be placed on hybrid vehicles by industry and government.

Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells.

PubMed

Lin, Shuw-Yuan; Lai, Wan-Wen; Chou, Chi-Chung; Kuo, Hsiu-Maan; Li, Te-Mao; Chung, Jing-Gung; Yang, Jen-Hung

2006-12-01

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.

Inhibition of Aurora A Kinase by Alisertib Induces Autophagy and Cell Cycle Arrest and Increases Chemosensitivity in Human Hepatocellular Carcinoma HepG2 Cells.

PubMed

Zhu, Qiaohua; Yu, Xinfa; Zhou, Zhi-Wei; Zhou, Chengyu; Chen, Xiao-Wu; Zhou, Shu-Feng

2017-01-01

Aurora A kinase represent a feasible target in cancer therapy. To evaluate the proteomic response of human liver carcinoma cells to alisertib (ALS) and identify the molecular targets of ALS, we examined the effects of ALS on the proliferation, cell cycle, autophagy, apoptosis, and chemosensitivity in HepG2 cells. The stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomic study was performed to evaluate the proteomic response to ALS. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. Our SILAC proteomic study showed that ALS regulated the expression of 914 proteins, with 407 molecules being up-regulated and 507 molecules being down-regulated in HepG2 cells. Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 146 and 32 signaling pathways were regulated by ALS, respectively, which were associated with cell survival, programmed cell death, and nutrition-energy metabolism. Subsequently, the verification experiments showed that ALS remarkably arrested HepG2 cells in G2/M phase and led to an accumulation of aneuploidy via regulating the expression of key cell cycle regulators. ALS induced a marked autophagy in a concentration- and time-dependent manner via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Autophagy inhibition promoted the pro-apoptotic effect of ALS, indicating a cyto-protective role of ALS-induced autophagy. ALS increased the chemosensitivity of HepG2 cells to cisplatin and doxorubicin. Taken together, ALS induces autophagy and cell cycle arrest in HepG2 cells via PI3K/Akt/mTOR-mediated pathway. Autophagy inhibition may promote the anticancer effect of ALS and sensitize the chemotherapy in HepG2 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line.

PubMed

Bakshi, Hamid; Sam, Smitha; Rozati, Roya; Sultan, Phalisteen; Islam, Tajamul; Rathore, Babita; Lone, Zahoor; Sharma, Manik; Triphati, Jagrati; Saxena, Ramesh Chand

2010-01-01

Apoptosis, a widely important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus in different cancer types. The present study was designed to elucidate apoptosis induction by crocin, a main component of Crocus sativus in a human pancreatic cancer cell line (BxPC-3). Cell viability was measured by MTT assay, Hoechest33258 staining was used to detect the chromatin condensation characteristic of apoptosis, and DNA fragmentation was assessed by gel electrophoresis and cell cycle analysis by flow cytometry. Crocin induced apoptosis and G1-phase cell cycle arrest of BxPC-3 cells, while decreasing cell viability in a dose dependent and time dependent manner. Cells treated with 10μg/L crocin exhibited apoptotic morphology (brightly blue-fluorescent condensed nuclei on Hoechst 33258 staining) and reduction of volume. DNA analysis revealed typical ladders as early as 12 hours after treatment indicative of apoptosis. Our preclinical study demonstrated a pancreatic cancer cell line to be highly sensitive to crocin-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of crocin action are not yet clearly understood, it appears to have potential as a therapeutic agent.

lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth of colon cancer cells via apoptosis and sub G1 cell cycle arrest.

PubMed

Karthikeyan, Chandrabose; Amawi, Haneen; Viana, Arabela Guedes; Sanglard, Leticia; Hussein, Noor; Saddler, Maria; Ashby, Charles R; Moorthy, N S Hari Narayana; Trivedi, Piyush; Tiwari, Amit K

2018-07-15

A series of lH-pyrazolo[3,4-b]quinolin-3-amine derivatives were synthesized and evaluated for anticancer efficacy in a panel of ten cancer cell lines, including breast (MDAMB-231 and MCF-7), colon (HCT-116, HCT-15, HT-29 and LOVO), prostate (DU-145 and PC3), brain (LN-229), ovarian (A2780), and human embryonic kidney (HEK293) cells, a non-cancerous cell line. Among the eight derivatives screened, compound QTZ05 had the most potent and selective antitumor efficacy in the four colon cancer cell lines, with IC 50 values ranging from 2.3 to 10.2 µM. Furthermore, QTZ05 inhibited colony formation in HCT-116 cells in a concentration-dependent manner. Cell cycle analysis data indicated that QTZ05 caused an arrest in the sub G1 cell cycle in HCT-116 cells. QTZ05 induced apoptosis in HCT-116 cells in a concentration-dependent manner that was characterized by chromatin condensation and increase in the fluorescence of fluorochrome-conjugated Annexin V. The findings from our study suggest that QTZ05 may be a valuable prototype for the development of chemotherapeutics targeting apoptotic pathways in colorectal cancer cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Radiosensitivity of Mammalian Cells

PubMed Central

Walters, R. A.; Petersen, D. F.

1968-01-01

Radiation effects on macromolecular synthesis essential for the Chinese hamster cell to traverse the life cycle and to divide have been investigated. Life-cycle analysis techniques employing inhibitors of macromolecular synthesis were used in determining the kinetics of cell growth for specific segments of the population following spontaneous recovery from radiation-induced division delay. The results indicated that recovery does not occur in the absence of functional protein synthesis. Under conditions which inhibit normal RNA and DNA synthesis, irradiated cells can recover the capacity to traverse the life cycle and to divide. The stability of mRNA species coding for proteins essential for division in irradiated cells was also measured. The mean functional lifetime of these mRNA species was 1 hr. The data demonstrate the existence of a specific segment of the population consisting of cells which have completed transcription related to division but not concomitant translation and which can recover from the radiation injury without synthesis of additional RNA. Thus, initial recovery of the ability to divide has an obligate requirement for protein synthesis but no corresponding requirement for nucleic acid synthesis during the period when original messenger remains intact. PMID:5753224

Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest.

PubMed

Gudarzi, Hoda; Salimi, Mona; Irian, Saeed; Amanzadeh, Amir; Mostafapour Kandelous, Hirsa; Azadmanesh, Keyhan; Salimi, Misha

2015-01-01

Ferula gummosa Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of F. gummosa as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC50 value at 72 h (0.001 ± 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ± 0.037, 0.01 ± 2.51 mg/g of dry powder). Generally, the results obtained indicate that F. gummosa ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent.

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

Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle.

PubMed

Picazo, R A; García Ruiz, J P; Santiago Moreno, J; González de Bulnes, A; Muñoz, J; Silván, G; Lorenzo, P L; Illera, J C

2004-11-01

The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle.

Repair of oxidative DNA damage, cell-cycle regulation and neuronal death may influence the clinical manifestation of Alzheimer's disease.

PubMed

Silva, Aderbal R T; Santos, Ana Cecília Feio; Farfel, Jose M; Grinberg, Lea T; Ferretti, Renata E L; Campos, Antonio Hugo Jose Froes Marques; Cunha, Isabela Werneck; Begnami, Maria Dirlei; Rocha, Rafael M; Carraro, Dirce M; de Bragança Pereira, Carlos Alberto; Jacob-Filho, Wilson; Brentani, Helena

2014-01-01

Alzheimer's disease (AD) is characterized by progressive cognitive decline associated with a featured neuropathology (neuritic plaques and neurofibrillary tangles). Several studies have implicated oxidative damage to DNA, DNA repair, and altered cell-cycle regulation in addition to cell death in AD post-mitotic neurons. However, there is a lack of studies that systematically assess those biological processes in patients with AD neuropathology but with no evidence of cognitive impairment. We evaluated markers of oxidative DNA damage (8-OHdG, H2AX), DNA repair (p53, BRCA1, PTEN), and cell-cycle (Cdk1, Cdk4, Cdk5, Cyclin B1, Cyclin D1, p27Kip1, phospho-Rb and E2F1) through immunohistochemistry and cell death through TUNEL in autopsy hippocampal tissue samples arrayed in a tissue microarray (TMA) composed of three groups: I) "clinical-pathological AD" (CP-AD)--subjects with neuropathological AD (Braak ≥ IV and CERAD = B or C) and clinical dementia (CDR ≥ 2, IQCODE>3.8); II) "pathological AD" (P-AD)--subjects with neuropathological AD (Braak ≥ IV and CERAD = B or C) and without cognitive impairment (CDR 0, IQCODE<3.2); and III) "normal aging" (N)--subjects without neuropathological AD (Braak ≤ II and CERAD 0 or A) and with normal cognitive function (CDR 0, IQCODE<3.2). Our results show that high levels of oxidative DNA damage are present in all groups. However, significant reductions in DNA repair and cell-cycle inhibition markers and increases in cell-cycle progression and cell death markers in subjects with CP-AD were detected when compared to both P-AD and N groups, whereas there were no significant differences in the studied markers between P-AD individuals and N subjects. This study indicates that, even in the setting of pathological AD, healthy cognition may be associated with a preserved repair to DNA damage, cell-cycle regulation, and cell death in post-mitotic neurons.

Physangulidine A, a withanolide from Physalis angulata, perturbs the cell cycle and induces cell death by apoptosis in prostate cancer cells.

PubMed

Reyes-Reyes, E Merit; Jin, Zhuang; Vaisberg, Abraham J; Hammond, Gerald B; Bates, Paula J

2013-01-25

Recently, our group reported the discovery of three new withanolides, physangulidines A-C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G(2)/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.

Development of nickel-metal hydride cell: An update

NASA Technical Reports Server (NTRS)

Kuwajima, S.; Kusawake, Hiroaki; Nakatani, Kensuke; Yano, Y.

1994-01-01

This paper presents in viewgraph format an overview of NASDA's evaluation of commercial nickel metal-hydride (Ni-MH) cells and the development and testing of Ni-MH cells for use in space. The commercial cells are concluded to be feasible and suitable for use in LEO; for GEO, the durability for overcharge is needed because long-term charge retention is required. For the aerospace Ni-MH cell design, two activation procedures are applied to evaluate the effect of the difference in the amount of overcharge protection and precharge. Specific energy of the Ni-MH cell is nearly accomplished at 50 Wh/kg. Initial characteristics indicate the effect derived from precharge. Thirty-five amp-hour class Ni-MH cells have good performance for LEO cycle of 25 and 40 percent DOD up to 3000 cycles as similar to commercial cells. The effect of the difference in the amount of overcharge protection will appear in life test.

Moderate temperature rechargeable sodium batteries

NASA Technical Reports Server (NTRS)

Abraham, K. M.; Rupich, M. W.; Pitts, L.; Elliott, J. E.

1983-01-01

Cells utilizing the organic electrolyte, NaI in triglyme, operated at approx. 130 C with Na(+) - intercalating cathodes. However, their rate and stability were inadequate. NaAlCl4 was found to be a highly useful electrolyte for cell operation at 165-190 C. Na(+) intercalating chalcogenides reacted with NaAlCl4 during cycling to form stable phases. Thus, VS2 became essentially VS2Cl, with reversible capacity of approx 2.8 e(-)/V, and a mid-discharge voltage of approx 2.5V and 100 deep discharge cycles were readily achieved. A positive electrode consisting of VCl3 and S plus NaAlCl4 was subjected to deep-discharge cycles 300 times and it demonstrated identity with the in-situ-formed BSxCly cathode. NiS2 and NiS which are not Na(+)-intercalating structures formed highly reversible electrodes in NaAlCl4. The indicated discharge mechanism implies a theoretical capacity 4e(-)/Ni for NiS2 and 2e(-)/Ni for NiS. The mid-discharge potentials are, respectively, 2.4V and 2.1V. A Na/NiS2 cell cycling at a C/5 rate has exceeded 500 deep discharge cycles with 2.5e(-)/Ni average utilization. A 4 A-hr nominal capacity prototype Na/NiS2 cell was tested at 190 C. It was voluntarily terminated after 80 cycles. Further development, particularly of cathode structure and hardware should produce a battery capable of at least 50-W-hr/lb and more than 1000 cycles.

CFS-1686 causes cell cycle arrest at intra-S phase by interference of interaction of topoisomerase 1 with DNA.

PubMed

Lin, Ru-Wei; Yang, Chia-Ning; Ku, ShengYu; Ho, Cheng-Jung; Huang, Shih-Bo; Yang, Min-Chi; Chang, Hsin-Wen; Lin, Chun-Mao; Hwang, Jaulang; Chen, Yeh-Long; Tzeng, Cherg-Chyi; Wang, Chihuei

2014-01-01

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.

The MAPK Signaling Cascade is a Central Hub in the Regulation of Cell Cycle, Apoptosis and Cytoskeleton Remodeling by Tripeptidyl-Peptidase II

PubMed Central

Sompallae, Ramakrishna; Stavropoulou, Vaia; Houde, Mathieu; Masucci, Maria G.

2008-01-01

Tripeptidyl-peptidase II (TPPII) is a serine peptidase highly expressed in malignant Burkitt’s lymphoma cells (BL). We have previously shown that overexpression of TPPII correlates with chromosomal instability, centrosomal and mitotic spindle abnormalities and resistance to apoptosis induced by spindle poisons. Furthermore, TPPII knockdown by RNAi was associated with endoreplication and the accumulation of polynucleated cells that failed to complete cell division, indicating a role of TPPII in the cell cycle. Here we have applied a global approach of gene expression analysis to gain insights on the mechanism by which TPPII regulates this phenotype. mRNA profiling of control and TPPII knockdown BL cells identified one hundred and eighty five differentially expressed genes. Functional categorization of these genes highlighted major physiological functions such as apoptosis, cell cycle progression, cytoskeleton remodeling, proteolysis, and signal transduction. Pathways and protein interactome analysis revealed a significant enrichment in components of MAP kinases signaling. These findings suggest that TPPII influences a wide network of signaling pathways that are regulated by MAPKs and exerts thereby a pleiotropic effect on biological processes associated with cell survival, proliferation and genomic instability. PMID:19787088

Analysis of cardiomyocyte movement in the developing murine heart

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

Hashimoto, Hisayuki; Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp; Tabata, Hidenori

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cellmore » cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.« less

Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

PubMed

Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

2014-10-17

Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300μL/L citral and 100μL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the complexity of the mechanisms of bacterial inactivation among EO constituents. Advancements in the description of these mechanisms will help in extending and improving the use of these compounds as natural antimicrobials. Copyright © 2014 Elsevier B.V. All rights reserved.

Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells

PubMed Central

Wang, Feng; Wang, Qi; Zhou, Zhi-Wei; Yu, Song-Ning; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Yin-Xue; Yang, Tianxing; Sun, Tao; Li, Min; Qiu, Jia-Xuan; Zhou, Shu-Feng

2015-01-01

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways. PMID:25632222

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

PubMed Central

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

2007-01-01

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

Advances in rechargeable lithium molybdenum disulfide batteries

NASA Technical Reports Server (NTRS)

Brandt, K.; Stiles, J. A. R.

1985-01-01

The lithium molybdenum disulfide system as demonstrated in a C size cell, offers performance characteristics for applications where light weight and low volume are important. A gravimetric energy density of 90 watt hours per kilogram can be achieved in a C size cell package. The combination of charge retention capabilities, high energy density and a state of charge indicator in a rechargeable cell provides power package for a wide range of devices. The system overcomes the memory effect in Nicads where the full capacity of the battery cannot be utilized unless it was utilized on previous cycles. The development of cells with an advanced electrolyte formulation led to an improved rate capability especially at low temperatures and to a significantly improved life cycle.

CCS52A2/FZR1, a cell cycle regulator, is an essential factor for shoot apical meristem maintenance in Arabidopsis thaliana.

PubMed

Liu, Yajie; Ye, Wei; Li, Beibei; Zhou, Xiaojing; Cui, Yuhai; Running, Mark P; Liu, Kede

2012-08-08

Cell division and cell fate decisions regulate organ formation and function in plant growth and development. It is still unclear how specific meristematic regulatory networks operate with the cell cycle machinery to translate stem cell identity and maintenance into cellular behavior. In this study, we address these questions by analysis of a shoot apex defective mutant, namely xcm9. Phenotypic analysis of the xcm9 mutant reveals concomitant premature termination of floral shoots with frequent bifurcation of the shoot apices, stems, and flowers. Microscopic observations show irregular cell organization in shoot apical meristems of xcm9. Positional cloning revealed that xcm9 is a loss of function allele of the CCS52A2/FZR1 gene, which has previously been implicated in root development. Expression analysis demonstrated that CCS52A2 maintains a higher transcriptional expression level in actively dividing tissue. Genetic studies indicated that the CCS52A2 gene functions together with WUSCHEL (WUS) and CLAVATA3 (CLV3) in regulating the development of the shoot meristem, and also contributes to this regulation together with the chromatin remodeling pathway. In addition, fewer xcm9 cells express CYCLIN B1:1, showing that cell cycle progression is disrupted in the mutant. We propose that the CCS52A2 gene is a mediator that functions together with meristematic genes to regulate meristem organization, and cross-functions with chromatin regulators in cell cycle progression during shoot apical meristem development.

Sevoflurane suppresses proliferation by upregulating microRNA-203 in breast cancer cells.

PubMed

Liu, Jiaying; Yang, Longqiu; Guo, Xia; Jin, Guangli; Wang, Qimin; Lv, Dongdong; Liu, Junli; Chen, Qiu; Song, Qiong; Li, Baolin

2018-05-03

Rapid proliferation is one of the critical characteristics of breast cancer. However, the underlying regulatory mechanism of breast cancer cell proliferation is largely unclear. The present study indicated that sevoflurane, one of inhalational anesthetics, could significantly suppress breast cancer cell proliferation by arresting cell cycle at G1 phase. Notably, the rescue experiment indicated that miR-203 was upregulated by sevoflurane and mediated the function of sevoflurane on suppressing the breast cancer cell proliferation. The present study indicated the function of the sevoflurane/miR-203 signaling pathway on regulating breast cancer cell proliferation. These results provide mechanistic insight into how the sevoflurane/miR-203 signaling pathway supresses proliferation of breast cancer cells, suggesting the sevoflurane/miR-203 pathway may be a potential target in the treatment of breast cancer.

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

Asymmetric Distribution of Primary Cilia Allocates Satellite Cells for Self-Renewal.

PubMed

Jaafar Marican, Nur Hayati; Cruz-Migoni, Sara B; Borycki, Anne-Gaëlle

2016-06-14

Regeneration of vertebrate skeletal muscles requires satellite cells, a population of stem cells that are quiescent in normal conditions and divide, differentiate, and self-renew upon activation triggered by exercise, injury, and degenerative diseases. Satellite cell self-renewal is essential for long-term tissue homeostasis, and previous work has identified a number of external cues that control this process. However, little is known of the possible intrinsic control mechanisms of satellite cell self-renewal. Here, we show that quiescent satellite cells harbor a primary cilium, which is rapidly disassembled upon entry into the cell cycle. Contrasting with a commonly accepted belief, cilia reassembly does not occur uniformly in cells exiting the cell cycle. We found that primary cilia reassemble preferentially in cells committed to self-renew, and disruption of cilia reassembly causes a specific deficit in self-renewing satellite cells. These observations indicate that primary cilia provide an intrinsic cue essential for satellite cell self-renewal. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2014-04-01

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

Relationships between host and symbiont cell cycles in sea anemones and their symbiotic dinoflagellates.

PubMed

Dimond, James L; Pineda, Rea R; Ramos-Ascherl, Zullaylee; Bingham, Brian L

2013-10-01

The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbiotic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastrodermal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across species and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

PubMed Central

Róna, Gergely; Borsos, Máté; Ellis, Jonathan J; Mehdi, Ahmed M; Christie, Mary; Környei, Zsuzsanna; Neubrandt, Máté; Tóth, Judit; Bozóky, Zoltán; Buday, László; Madarász, Emília; Bodén, Mikael; Kobe, Bostjan; Vértessy, Beáta G

2014-01-01

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle. PMID:25483092

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.

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isotype-selective inhibition

PubMed Central

Torbett, Neil E; Luna, Antonio; Knight, Zachary A.; Houk, Andrew; Moasser, Mark; Weiss, William; Shokat, Kevan M.; Stokoe, David

2011-01-01

Synopsis The Phosphoinositide-3-kinase (PI3K) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we demonstrate that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110α inhibitors were generally effective in inhibiting the phosphorylation of Akt and S6, two downstream components of PI3K signaling, in most cell lines examined. In contrast, 110β selective inhibitors only reduced Akt phosphorylation in PTEN mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing a cell cycle arrest in the G1 phase of the cell cycle, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signaling pathways. Taken together our data indicates that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts. PMID:18498248

Thermal modelling of Li-ion polymer battery for electric vehicle drive cycles

NASA Astrophysics Data System (ADS)

Chacko, Salvio; Chung, Yongmann M.

2012-09-01

Time-dependent, thermal behaviour of a lithium-ion (Li-ion) polymer cell has been modelled for electric vehicle (EV) drive cycles with a view to developing an effective battery thermal management system. The fully coupled, three-dimensional transient electro-thermal model has been implemented based on a finite volume method. To support the numerical study, a high energy density Li-ion polymer pouch cell was tested in a climatic chamber for electric load cycles consisting of various charge and discharge rates, and a good agreement was found between the model predictions and the experimental data. The cell-level thermal behaviour under stressful conditions such as high power draw and high ambient temperature was predicted with the model. A significant temperature increase was observed in the stressful condition, corresponding to a repeated acceleration and deceleration, indicating that an effective battery thermal management system would be required to maintain the optimal cell performance and also to achieve a full battery lifesapn.

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

PubMed

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

2012-12-01

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

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

PubMed

Burgos-Barragan, Guillermo; Wit, Niek; Meiser, Johannes; Dingler, Felix A; Pietzke, Matthias; Mulderrig, Lee; Pontel, Lucas B; Rosado, Ivan V; Brewer, Thomas F; Cordell, Rebecca L; Monks, Paul S; Chang, Christopher J; Vazquez, Alexei; Patel, Ketan J

2017-08-31

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Examining live cell cultures during apoptosis by digital holographic phase imaging and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Khmaladze, Alexander

2017-11-01

Cellular apoptosis is a unique, organized series of events, leading to programmed cell death. In this work, we present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy measurements of live cell cultures yield information about cell shape and volume, changes to which are indicative of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopic measurements provide complementary information about cells, such as protein, lipid and nucleic acid content, and the spectral signatures associated with structural changes in molecules. Our work indicates that the chemical changes in proteins, which were detected by Raman measurements, preceded morphological changes, which were seen with digital holographic microscopy.

Amarogentin secoiridoid inhibits in vivo cancer cell growth in xenograft mice model and induces apoptosis in human gastric cancer cells (SNU-16) through G2/M cell cycle arrest and PI3K/Akt signalling pathway.

PubMed

Zhao, Jian-Guo; Zhang, Ling; Xiang, Xiao-Jun; Yu, Feng; Ye, Wan-Li; Wu, Dong-Ping; Wang, Jian-Fang; Xiong, Jian-Ping

2016-01-01

To investigate the in vitro and in vivo antitumor effects of amarogentin in SNU-16 human gastric cancer cells as well as in nude mice xenograft model. The effects of this compound on cell apoptosis, cell cycle phase distribution and PI3K/Akt and m-TOR signalling pathways were also studied in detail. MTT assay was used to study the effect of amarogentin on SNU-16 cell viability while clonogenic assay indicated the effect of the compound on colony formation tendency of these cells. Phase contrast microscopy revealed the effect on cellular morphology while flow cytometry was engaged to study the effects on cell apoptosis and cell cycle arrest. SNU-16 cancer cells were subcutaneously inoculated into nude mice to investigate the in vivo antitumor effects of amarogentin. Amarogentin induced potent, dose-dependent as well as time-dependent cytotoxic effects on the growth of SNU-16 human gastric cancer cells. Amarogentin also inhibited the colony forming capability of these tumor cells and its treatment led to morphological alterations in these cells in which the cells became withered and rounded, detached from one another and adopted irregular shapes while floating freely in the culture medium. In comparison to untreated control cells, the amarogentin treated cells with 10, 50 and 75 μM exhibited 32.5, 45.2 and 57.1 % apoptotic cells, respectively. Amarogentin induced potent and dose-dependent G2/M cell cycle arrest in these cells and led to downregulation of m-TOR, p-PI3K, PI3K, p-Akt and Akt and upregulation of cyclin D1 and cyclin E protein expressions. The tumor tissues obtained from the amarogentin-treated mice were much smaller than the tumor tissues derived from the control group. Amarogentin exerts potent in vitro and in vivo antitumor effects in SNU-16 cell model as well as in nude mice xenograft model. These antitumor effects were found to be mediated through apoptosis induction, G2/M cell cycle arrest and downregulation of PI3K/Akt/m-TOR signalling pathways.

p205, a potential tumor suppressor, inhibits cell proliferation via multiple pathways of cell cycle regulation.

PubMed

Asefa, Benyam; Dermott, Jonathan M; Kaldis, Philipp; Stefanisko, Karen; Garfinkel, David J; Keller, Jonathan R

2006-02-20

p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.

Raman spectral observation of a new phase observed in nickel electrodes cycled to failure

NASA Technical Reports Server (NTRS)

Loyselle, Patricia L.; Shan, X.; Cornilsen, B. C.; Reid, Margaret A.

1991-01-01

A new phase is reported in nickel electrodes from Ni/H2 boilerplate cells which were cycled to failure in electrolyte of variable KOH concentration (21 to 36 percent). Raman spectra clearly show the presence of this phase, and these spectra have been used to estimate the amounts present on these electrodes. Ten of 12 electrodes examined contain this new phase. The cycle life at higher KOH concentrations (31 and 36 percent) was greatly reduced, and nickel electrodes from these cells exhibited extensive amounts of this new phase. The presence of this new phase correlates with cell failure defined by low end of discharge voltages. It is proposed that the lowered capacity and failure of these electrodes was caused by loss of active mass and formation of a phase with reduced electrochemical activity. These results indicate that formation of the new phase is accelerated at higher KOH concentrations.

Cytotoxic effect of achatinin(H) (lectin) from Achatina fulica against a human mammary carcinoma cell line (MCF7).

PubMed

Dharmu, Indra; Ramamurty, N; Kannan, Ramalingam; Babu, Mary

2007-01-01

The hemolymph-derived achatinin(H) (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF7, a human mammary carcinoma cell line. IC(50) values as measured by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay for achatinin(H) ranged from 6 to 10 microg/ml in the MCF7 cells. MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed after 48 h of treatment with 8 microg/ml when compared to untreated cells. Alterations in the tumor marker enzymes, as well as in antioxidant enzymes, were observed after achatinin(H) treatment. The specificity and purity of the achatinin(H) was confirmed by the Western blot assay. Achatinin(H) binding to MCF7 cells was detected by anti-achatinin(H), and visualization of the achatinin(H) binding sites on confluent MCF7 cells was confirmed by flourescein isothiocyanate conjugated secondary antibody. MCF7-treated cells fluoresced, indicating the presence of achatinin(H) binding sites. Fluorescence-activated cell sorting analysis of the cell cycle showed a significant increase in S-phase in MCF7 cells after 48 h of achatinin(H) treatment. The cells were arrested in G(2)/M phase of the cell cycle after 48 h with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis.

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

PubMed

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

2006-05-01

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

Study on the Mechanism of Cell Cycle Checkpoint Kinase 2 (CHEK2) Gene Dysfunction in Chemotherapeutic Drug Resistance of Triple Negative Breast Cancer Cells.

PubMed

Luo, Li; Gao, Wei; Wang, Jinghui; Wang, Dingxue; Peng, Xiaobo; Jia, Zhaoyang; Jiang, Ye; Li, Gongzhuo; Tang, Dongxin; Wang, Yajie

2018-05-15

BACKGROUND This study aimed to investigate the mechanism of CHEK2 gene dysfunction in drug resistance of triple negative breast cancer (TNBC) cells. MATERIAL AND METHODS To perform our study, a stable CHEK2 wild type (CHEK2 WT) or CHEK2 Y390C mutation (CHEK2 Y390C) expressed MDA-MB-231 cell line was established. MTT assay, cell apoptosis assay and cell cycle assay were carried out to analyze the cell viability, apoptosis, and cell cycle respectively. Western blotting and qRT-PCR were applied for related protein and gene expression detection. RESULTS We found that the IC50 value of DDP (Cisplatin) to CHEK2 Y390C expressed MDA-MB-231 cells was significantly higher than that of the CHEK2 WT expressed cells and the control cells. After treatment with DDP for 48 h, cells expressing CHEK2 WT showed lower cell viability than that of the CHEK2 Y390C expressed cells and the control cells; compared with the CHEK2 Y390C expressed cells and the control cells, cells expressing CHEK2 WT showed significant G1/S arrest. Meanwhile, we found that compared with the CHEK2 Y390C expressed cells and the control cells, cell apoptosis was significantly increased in CHEK2 WT expressed cells. Moreover, our results suggested that cells expressing CHEK2 WT showed higher level of p-CDC25A, p-p53, p21, Bax, PUMA, and Noxa than that of the CHEK2 Y390C expressed cells and the control cells. CONCLUSIONS Our findings indicated that CHEK2 Y390C mutation induced the drug resistance of TNBC cells to chemotherapeutic drugs through administrating cell apoptosis and cell cycle arrest via regulating p53 activation and CHEK2-p53 apoptosis pathway.

Tri-ortho-cresyl phosphate induces autophagy of rat spermatogonial stem cells.

PubMed

Liu, Meng-Ling; Wang, Jing-Lei; Wei, Jie; Xu, Lin-Lin; Yu, Mei; Liu, Xiao-Mei; Ruan, Wen-Li; Chen, Jia-Xiang

2015-02-01

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame retardants in industry and reported to have a deleterious effect on the male reproductive system in animals besides delayed neurotoxicity. Our preliminary results found that TOCP could disrupt the seminiferous epithelium in the testis and inhibit spermatogenesis, but the precise mechanism is yet to be elucidated. This study shows that TOCP inhibited viability of rat spermatogonial stem cells in a dose-dependent manner. TOCP could not lead to cell cycle arrest in the cells; the mRNA levels of p21, p27, p53, and cyclin D1 in the cells were also not affected by TOCP. Meanwhile, TOCP did not induce apoptosis of rat spermatogonial stem cells. After treatment with TOCP, however, both LC3-II and the ratio of LC3-II/LC3-I were markedly increased; autophagy proteins ATG5 and beclin 1 were also increased after treatment with TOCP, indicating that TOCP could induce autophagy in the cells. Ultrastructural observation under the transmission electron microscopy indicated that autophagic vesicles in the cytoplasm containing extensively degraded organelles such as mitochondria and endoplasmic reticulum increased significantly after the cells were treated with TOCP. In summary, we have shown that TOCP can inhibit viability of rat spermatogonial stem cells and induce autophagy of the cells, without affecting cell cycle and apoptosis. © 2015 Society for Reproduction and Fertility.

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

PubMed Central

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

2009-01-01

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

HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

PubMed

Das, Mitali; Prasad, Shyam Babu; Yadav, Suresh Singh; Modi, Arusha; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

2015-12-01

Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2-7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4-/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells.

Turning the Table: Plants Consume Microbes as a Source of Nutrients

PubMed Central

Paungfoo-Lonhienne, Chanyarat; Rentsch, Doris; Robatzek, Silke; Webb, Richard I.; Sagulenko, Evgeny; Näsholm, Torgny

2010-01-01

Interactions between plants and microbes in soil, the final frontier of ecology, determine the availability of nutrients to plants and thereby primary production of terrestrial ecosystems. Nutrient cycling in soils is considered a battle between autotrophs and heterotrophs in which the latter usually outcompete the former, although recent studies have questioned the unconditional reign of microbes on nutrient cycles and the plants' dependence on microbes for breakdown of organic matter. Here we present evidence indicative of a more active role of plants in nutrient cycling than currently considered. Using fluorescent-labeled non-pathogenic and non-symbiotic strains of a bacterium and a fungus (Escherichia coli and Saccharomyces cerevisiae, respectively), we demonstrate that microbes enter root cells and are subsequently digested to release nitrogen that is used in shoots. Extensive modifications of root cell walls, as substantiated by cell wall outgrowth and induction of genes encoding cell wall synthesizing, loosening and degrading enzymes, may facilitate the uptake of microbes into root cells. Our study provides further evidence that the autotrophy of plants has a heterotrophic constituent which could explain the presence of root-inhabiting microbes of unknown ecological function. Our discovery has implications for soil ecology and applications including future sustainable agriculture with efficient nutrient cycles. PMID:20689833

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage

PubMed Central

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein–protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage. PMID:24675884

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

PubMed

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response

PubMed Central

Malzer, Elke; Daly, Marie-Louise; Moloney, Aileen; Sendall, Timothy J.; Thomas, Sally E.; Ryder, Edward; Ryoo, Hyung Don; Crowther, Damian C.; Lomas, David A.; Marciniak, Stefan J.

2010-01-01

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2α phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest. PMID:20682638

An accelerated calendar and cycle life study of Li-ion cells.

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

Bloom, I.; Cole, B. W.; Sohn, J. J.

2001-10-15

The accelerated calendar and cycle life of lithium-ion cells was studied. Useful cell life was strongly affected by temperature, time, state-of-charge (SOC) and change in state-of-charge ({Delta}SOC). In calendar life experiments, useful cell life was strongly affected by temperature and time. Temperature accelerated cell performance degradation. The rates of area specific impedance (ASI) increase and power fade followed simple laws based on a power of time and Arrhenius kinetics. The data have been modeled using these two concepts and the calculated data agree well with the experimental values. The calendar life ASI increase and power fade data follow (time){sup 1/2}more » kinetics. This behavior may be due to solid electrolyte interface layer growth. From the cycle life experiments, the ASI increase data follow (time){sup 1/2} kinetics also, but there is an apparent change in overall power fade mechanism when going from 3 to 6% {Delta}SOC. Here, the power of time drops to below 1/2, which indicates that the power fade mechanism is more complex than layer growth.« less

Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias).

PubMed

McClusky, L M

2005-01-01

To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at the mitosis-meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12-13 require 9-10 months to reach the mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4-5-fold less than the number that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed. Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related. PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited to spermatogonia and increased stage-dependently in the PrM region (L-PrM = mid-stage PrM >E-PrM >GZ), correlating with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are stage- and process-specific.

Disrupted cell cycle arrest and reduced proliferation in corneal fibroblasts from GCD2 patients: A potential role for altered autophagy flux

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

Choi, Seung-il; Dadakhujaev, Shorafidinkhuja; Maeng, Yong-Sun

Highlights: • Reduced cell proliferation in granular corneal dystrophy type 2. • Abnormal cell cycle arrest by defective autophagy. • Decreased Cyclin A1, B1, and D1 in Atg7 gene knockout cells. • Increase in p16 and p27 expressions were observed in Atg7 gene knockout cells. - Abstract: This study investigates the role of impaired proliferation, altered cell cycle arrest, and defective autophagy flux of corneal fibroblasts in granular corneal dystrophy type 2 (GCD2) pathogenesis. The proliferation rates of homozygous (HO) GCD2 corneal fibroblasts at 72 h, 96 h, and 120 h were significantly lower (1.102 ± 0.027, 1.397 ± 0.039,more » and 1.527 ± 0.056, respectively) than those observed for the wild-type (WT) controls (1.441 ± 0.029, 1.758 ± 0.043, and 2.003 ± 0.046, respectively). Flow cytometry indicated a decreased G{sub 1} cell cycle progression and the accumulation of cells in the S and G{sub 2}/M phases in GCD2 cells. These accumulations were associated with decreased levels of Cyclin A1, B1, and E1, and increased expression of p16 and p27. p21 and p53 expression was also significantly lower in GCD2 cells compared to the WT. Interestingly, treatment with the autophagy flux inhibitor, bafilomycin A{sub 1}, resulted in similarly decreased Cyclin A1, B1, D1, and p53 expression in WT fibroblasts. Furthermore, similar findings, including a decrease in Cyclin A1, B1, and D1 and an increase in p16 and p27 expression were observed in autophagy-related 7 (Atg7; known to be essential for autophagy) gene knockout cells. These data provide new insight concerning the role of autophagy in cell cycle arrest and cellular proliferation, uncovering a number of novel therapeutic possibilities for GCD2 treatment.« less

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.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway.

PubMed

Pande, Hari Om; Tesfaye, Dawit; Hoelker, Michael; Gebremedhn, Samuel; Held, Eva; Neuhoff, Christiane; Tholen, Ernst; Schellander, Karl; Wondim, Dessie Salilew

2018-05-01

The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches. The role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling pathway, granulosa cells were treated with activin A. Activin A treatment increased cell proliferation and downregulation of both miRNA-424/503 members and its target gene, indicated the presence of negative feedback loop between activin A and the expression of miRNA-424/503. This study suggests that the miRNA-424/503 cluster members are involved in regulating bovine granulosa cell proliferation and cell cycle progression. Further, miRNA-424/503 cluster members target the SMAD7 and ACVR2A genes which are involved in the activin signalling pathway.

Expression of Progesterone Receptor Membrane Component-2 Within the Immature Rat Ovary and Its Role in Regulating Mitosis and Apoptosis of Spontaneously Immortalized Granulosa Cells1

PubMed Central

Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K.; Peluso, John J.

2014-01-01

ABSTRACT Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular 3H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development. PMID:24990806

Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis*

PubMed Central

Wong, Ming-Kin; Guan, Daogang; Ng, Kaoru Hon Chun; Ho, Vincy Wing Sze; An, Xiaomeng; Li, Runsheng; Ren, Xiaoliang

2016-01-01

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development. PMID:27056332

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

PubMed

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

2010-11-01

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

The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells.

PubMed

Mayr, Christian; Wagner, Andrej; Neureiter, Daniel; Pichler, Martin; Jakab, Martin; Illig, Romana; Berr, Frieder; Kiesslich, Tobias

2015-06-23

The green tea catechin epigallocatechin gallate (EGCG) was shown to effectively inhibit tumor growth in various types of cancer including biliary tract cancer (BTC). For most BTC patients only palliative therapy is possible, leading to a median survival of about one year. Chemoresistance is a major problem that contributes to the high mortality rates of BTC. The aim of this study was to investigate the cytotoxic effect of EGCG alone or in combination with cisplatin on eight BTC cell lines and to investigate the cellular anti-cancer mechanisms of EGCG. The effect of EGCG treatment alone or in combination with the standard chemotherapeutic cisplatin on cell viability was analyzed in eight BTC cell lines. Additionally, we analyzed the effects of EGCG on caspase activity, cell cycle distribution and gene expression in the BTC cell line TFK-1. EGCG significantly reduced cell viability in all eight BTC cell lines (p < 0.05 or p < 0.01, respectively, for most cell lines and EGCG concentrations > 5 μM). Combined EGCG and cisplatin treatment showed a synergistic cytotoxic effect in five cell lines and an antagonistic effect in two cell lines. Furthermore, EGCG reduced the mRNA levels of various cell cycle-related genes, while increasing the expression of the cell cycle inhibitor p21 and the apoptosis-related death receptor 5 (p < 0.05). This observation was accompanied by an increase in caspase activity and cells in the sub-G1 phase of the cell cycle, indicating induction of apoptosis. EGCG also induced a down-regulation of expression of stem cell-related genes and genes that are associated with an aggressive clinical character of the tumor, such as cd133 and abcg2. EGCG shows various anti-cancer effects in BTC cell lines and might therefore be a potential anticancer drug for future studies in BTC. Additionally, EGCG displays a synergistic cytotoxic effect with cisplatin in most tested BTC cell lines. Graphical abstract Summary illustration.

Effects of demethoxycurcumin on the viability and apoptosis of skin cancer cells.

PubMed

Wu, Yaoqun; Zhang, Pei; Yang, Hongyun; Ge, Yong; Xin, Yong

2017-07-01

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.

Effect of heat-assisted pulsed electric fields and bacteriophage on enterohemorrhagic Escherichia coli O157:H7.

PubMed

Walkling-Ribeiro, Markus; Anany, Hany; Griffiths, Mansel W

2015-01-01

Pulsed electric fields (PEF), heat-assisted PEF (H-PEF), and virulent bacteriophage (VP) are non-thermal techniques for pathogen inactivation in liquids that were investigated individually, and in combination (PEF/VP, H-PEF/VP) to control enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Luria-Bertani broth (LBB) and Ringer's solution (RS). Treated cells were subsequently incubated at refrigeration (4°C) and temperature-abuse conditions (12°C) for 5 days. When EHEC cells grown in LBB were subjected to non-thermal processing and subsequently stored at 12°C for 5 days, reductions in count of between 0.1 and 0.6 log cycles were observed and following storage at 4°C the decrease in counts varied between 0.2 and 1.1 log10 . For bacteria cells suspended in RS values ranged from 0.1 to ≥3.9 log cycles at both storage temperatures. The most effective treatments were H-PEF and H-PEF/VP, both producing a >3.4 log cycle reduction of cells suspended in non-nutrient RS. Analysis of EHEC recovery on selective and non-selective media indicated no occurrence of sub-lethal damage for VP, PEF/VP, and H-PEF/VP-treated cells. The findings indicate that combining PEF and lytic phage may represent a suitable alternative to conventional fluid decontamination following further process optimization. © 2014 American Institute of Chemical Engineers.

Human transcription factor hTAF(II)150 (CIF150) is involved in transcriptional regulation of cell cycle progression.

PubMed

Martin, J; Halenbeck, R; Kaufmann, J

1999-08-01

Here we present evidence that CIF150 (hTAF(II)150), the human homolog of Drosophila TAF(II)150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAF(II)150) seems to be less tightly associated with human transcription factor IID than hTAF(II)130 is associated with hTAF(II)250. The transient functional knockout of CIF150 (hTAF(II)150) protein led to cell cycle arrest at the G(2)/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAF(II)150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAF(II)150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAF(II)150) function. We defined a CIF150 (hTAF(II)150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAF(II)150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression.

Human Transcription Factor hTAFII150 (CIF150) Is Involved in Transcriptional Regulation of Cell Cycle Progression

PubMed Central

Martin, Jay; Halenbeck, Robert; Kaufmann, Jörg

1999-01-01

Here we present evidence that CIF150 (hTAFII150), the human homolog of Drosophila TAFII150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAFII150) seems to be less tightly associated with human transcription factor IID than hTAFII130 is associated with hTAFII250. The transient functional knockout of CIF150 (hTAFII150) protein led to cell cycle arrest at the G2/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAFII150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAFII150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAFII150) function. We defined a CIF150 (hTAFII150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAFII150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression. PMID:10409744

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

PubMed

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

2004-06-18

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

Lamprey immune protein-1 (LIP-1) from Lampetra japonica induces cell cycle arrest and cell death in HeLa cells.

PubMed

Chi, Xiaoyuan; Su, Peng; Bi, Dan; Tai, Zhao; Li, Yingying; Pang, Yue; Li, Qingwei

2018-04-01

The lamprey (Lampetra japonica), a representative of the jawless vertebrates, is the oldest extant species in the world. LIP-1, which has a jacalin-like domain and an aerolysin pore-forming domain, has previously been identified in Lampetra japonica. However, the structure and function of the LIP-1 protein have not been described. In this study, the LIP-1 gene was overexpressed in HeLa cells and H293T cells. The results showed that the overexpression of LIP-1 in HeLa cells significantly elevated LDH release (P < 0.05), phosphatidylserine exposure and ROS accumulation. The overexpression of LIP-1 also had remarkable effects on the organelles in HeLa cells, while it had no effect on H293T cell organelles. Array data indicated that overexpression of LIP-1 primarily upregulated P53 signaling pathways in HeLa cells. Cell cycle assay results confirmed that LIP-1 caused arrest in the G 2 /M phase of the cell cycle in HeLa cells. In summary, our findings provide insights into the function and characterization of LIP-1 genes in vertebrates and establish the foundation for further research into the biological function of LIP-1. Our observations suggest that this lamprey protein has the potential for use in new applications in the medical field. Copyright © 2018. Published by Elsevier Ltd.

[Immunohistochemical analysis of cell cycle-regulating protein (p21, p27 and Ki67) expression in endoscopic biopsy samples from patients with gastroesophageal reflux disease].

PubMed

Koyama, Shigeki; Nishiyama, Yorihiro; Ishizuka, Izumi

2007-05-01

We performed an immunohistochemical analysis of cell cycle-regulating protein (p21, p27 and Ki67) expression in endoscopic biopsy samples from the patients with gastroesophageal reflux disease (GERD) using angled -biopsy forceps. Inflammatory cell accumulation into the lamina propria was detected even in patients with modified Los Angeles (LA) system grades N or M. In grade N or M patients with no changes in the epithelium, the area of p21, p27 and Ki67 positive cells was expanded compared to normal mucosa. The area of p21, p27 and Ki67 positive cells tended to expand upward in the epithelium with GERD severity based on the LA classification grading. These indicate that inflammatory cell infiltration into the lamina propria is initial histological change of GERD.

Novel functions for the transcription factor E2F4 in development and disease

PubMed Central

Sage, Julien

2016-01-01

ABSTRACT The E2F family of transcription factors is a key determinant of cell proliferation in response to extra- and intra-cellular signals. Within this family, E2F4 is a transcriptional repressor whose activity is critical to engage and maintain cell cycle arrest in G0/G1 in conjunction with members of the retinoblastoma (RB) family. However, recent observations challenge this paradigm and indicate that E2F4 has a multitude of functions in cells besides this cell cycle regulatory role, including in embryonic and adult stem cells, during regenerative processes, and in cancer. Some of these new functions are independent of the RB family and involve direct activation of target genes. Here we review the canonical functions of E2F4 and discuss recent evidence expanding the role of this transcription factor, with a focus on cell fate decisions in tissue homeostasis and regeneration. PMID:27753528

β-Sitosterol enhances cellular glutathione redox cycling by reactive oxygen species generated from mitochondrial respiration: protection against oxidant injury in H9c2 cells and rat hearts.

PubMed

Wong, Hoi Shan; Chen, Na; Leong, Pou Kuan; Ko, Kam Ming

2014-07-01

Herba Cistanches (Cistanche deserticola Y. C. Ma) is a 'Yang-invigorating' tonic herb in Chinese medicine. Preliminary chemical analysis indicated that β-sitosterol (BS) is one of the chemical constituents in an active fraction of Herba Cistanches. To investigate whether BS is an active ingredient of Herba Cistanches, the effects of BS on H9c2 cells and rat hearts were examined. The results indicated that BS stimulated the mitochondrial ATP generation capacity in H9c2 cells, which was associated with the increased production of mitochondrial reactive oxygen species. BS also stimulated mitochondrial state 3 and state 4 respiration, with the resultant decrease in coupling efficiency. BS produced an up-regulation of cellular glutathione redox cycling and protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. However, the protective effect of BS against myocardial ischemia/reperfusion injury was seen in female but not male rats ex vivo. The cardioprotection afforded by BS was likely mediated by an up-regulation of mitochondrial glutathione redox cycling in female rat hearts. In conclusion, the ensemble of results suggests that BS is an active ingredient of Herba Cistanches. The gender-dependent effect of BS on myocardial protection will further be investigated. Copyright © 2013 John Wiley & Sons, Ltd.

An authentic imaging probe to track cell fate from beginning to end.

PubMed

Lee, Seung Koo; Mortensen, Luke J; Lin, Charles P; Tung, Ching-Hsuan

2014-10-17

Accurate tracing of cell viability is critical for optimizing delivery methods and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. On incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently and the first colour is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second colour appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively.

Cytoskeleton disorder and cell cycle arrest may be associated with the alteration of protein CEP135 by microgravity

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Liu, Zhiyuan

In the past decades, alterations in the morphology, cytoskeleton and cell cycle have been observed in cells in vitro under microgravity conditions. But the underlying mechanisms are not absolutely identified yet. Our previous study on proteomic and microRNA expression profiles of zebrafish embryos exposed to simulated-microgravity has demonstrated a serial of microgravity-sensitive molecules. Centrosomal protein of 135 kDa (CEP135) was found down-regulated, but the mRNA expression level of it was up-regulated in zebrafish embryos after simulated-microgravity. However, the functional study on CEP135 is very limited and it has not been cloned in zebrafish till now. In this study, we try to determine whether the cytoskeleton disorder and cell cycle arrest is associated with the alteration of CEP135 by microgravity. Full-length cDNA of cep135 gene was firstly cloned from mitosis phase of ZF4. The sequence was analyzed and the phylogenetic tree was constructed based on the similarity to other species. Zebrafish embryonic cell line ZF4 were exposed to simulated microgravity for 24 and 48 hours, using a rotary cell culture system (RCCS) designed by NASA. Quantitative analysis by western blot showed that CEP135 expression level was significantly decreased two times after 24 hour simulated microgravity. Cell cycle detection by flow cytometer indicated ZF4 cells were blocked in G1 phase after 24 and 48 hour simulated microgravity. Moreover, double immunostained ZF4 cells with anti-tubulin and anti-CEP135antibodies demonstrated simulated microgravity could lead to cytoskeleton disorder and CEP135 abnormality. Further investigations are currently being carried out to determine whether knockdown and over-expression of CEP135 will modulate cytoskeleton and cell cycle. In vitro data in combination within vivo results might, at least in part, explain the dramatic effects of microgravity. Key Words: microgravity; CEP135; Cytoskeleton disorder; G1 arrest; ZF4 cell line

Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

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

Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon

Voltage-dependent K{sup +} channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G{sub 1}-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletalmore » muscle cell proliferation.« less

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

PubMed Central

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

2016-01-01

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

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression.

PubMed

Christopher, R A; Judge, S R; Vincent, P A; Higgins, P J; McKeown-Longo, P J

1999-10-01

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

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

PubMed Central

Rouse, Michael; Rao, Roshni; Nagarkatti, Mitzi

2014-01-01

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

Formononetin induces cell cycle arrest of human breast cancer cells via IGF1/PI3K/Akt pathways in vitro and in vivo.

PubMed

Chen, J; Zeng, J; Xin, M; Huang, W; Chen, X

2011-09-01

Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study further investigated that formononetin inactivated IGF1/IGF1R-PI3K/Akt pathways and decreased cyclin D1 mRNA and protein expression in human breast cancer cells in vitro and in vivo. MCF-7 cells were treated with different concentrations of formononetin. The proliferation of the cells treated with formononetin was tested by MTT assay. The cell cycle in the treated cells was examined by flow cytometry. The levels of p-IGF-1 R, p-Akt, and cyclin D1 protein expression and cyclin D1 mRNA expression in the treated cells were determined by Western blot and RT-PCR, respectively. In addition, the antitumor activity of formononetin was evaluated in nude mice bearing orthotopic tumor implants. Compared with the control, formononetin inhibited the proliferation of MCF-7 cells and effectively induced cell cycle arrest. The levels of p-IGF-1 R, p-Akt, cyclin D1 protein expression, and cyclin D1 mRNA expression were also downregulated. On the other hand, formononetin also prevented the tumor growth of human breast cancer cells in nude mouse xenografts. These results show that formononetin causes cell cycle arrest at the G0/G1 phase by inactivating IGF1/IGF1R-PI3K/Akt pathways and decreasing cyclin D1 mRNA and protein expression, indicating the use of formononetin in the prevention of breast cancer carcinogenesis. Georg Thieme Verlag KG Stuttgart · New York.

Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway

PubMed Central

Xie, You-Li; Tao, Wen-Hui; Yang, Ti-Xiong; Qiao, Jian-Guo

2016-01-01

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma. PMID:27698776

15-PGDH/15-KETE plays a role in hypoxia-induced pulmonary vascular remodeling through ERK1/2-dependent PAR-2 pathway.

PubMed

Wei, Liuping; Yu, Xiufeng; Shi, Hengyuan; Zhang, Bo; Lian, Mingming; Li, Jing; Shen, Tingting; Xing, Yan; Zhu, Daling

2014-07-01

We have established that 15-hydroxyeicosatetraenoic acid is an important factor in regulation of pulmonary vascular remodeling (PVR) associated with hypoxia-induced pulmonary hypertension (PH), which is further metabolized by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to form 15-ketoeicosatetraenoic acid (15-KETE). However, the role of 15-PGDH and 15-KETE on PH has not been identified. The purpose of this study was to investigate whether 15-PGDH/15-KETE pathway regulates hypoxia-induced PVR in PH and to characterize the underlying mechanisms. To accomplish this, Immunohistochemistry, Ultra Performance Liquid Chromatography, Western blot, bromodeoxyuridine incorporation and cell cycle analysis were preformed. Our results showed that the levels of 15-PGDH expression and endogenous 15-KETE were drastically elevated in the lungs of humans with PH and hypoxic PH rats. Hypoxia stimulated pulmonary arterial smooth muscle cell (PASMC) proliferation, which seemed to be due to the increased 15-PGDH/15-KETE. 15-PGDH/15-KETE pathway was also capable of stimulating the cell cycle progression and promoting the cell cycle-related protein expression. Furthermore, 15-KETE-promoted cell cycle progression and proliferation in PASMCs depended on protease-activated receptor 2 (PAR-2). ERK1/2 signaling was likely required for 15-PGDH/15-KETE-induced PAR-2 expression under hypoxia. Our study indicates that 15-PGDH/15-KETE stimulates the cell cycle progression and proliferation of PASMCs involving ERK1/2-mediated PAR-2 expression, and contributes to hypoxia-induced PVR. Copyright © 2014 Elsevier Inc. All rights reserved.

Three-volt lithium-ion battery with Li[Ni 1/2Mn 3/2]O 4 and the zero-strain insertion material of Li[Li 1/3Ti 5/3]O 4

NASA Astrophysics Data System (ADS)

Ariyoshi, Kingo; Yamamoto, Satoshi; Ohzuku, Tsutomu

A 3 V lithium-ion cell with Li[Ni 1/2Mn 3/2]O 4 ( Fd 3¯m ; a=8.17 Å) and the zero-strain insertion material of Li[Li 1/3Ti 5/3]O 4 ( Fd 3¯m ; a=8.36 Å) was examined with an emphasis on rate-capability and cycle life. This cell showed a quite flat operating voltage of 3.2 V with excellent cycleability. Accelerated cycle tests indicated that 83% of the initial capacity was delivered and stored even after 1100 cycles. Although the calculated energy density of a Li[Li 1/3Ti 5/3]O 4/Li[Ni 1/2Mn 3/2]O 4 cell was about 250 Wh kg -1 or 1000 Wh dm -3 based on the active material weight or volume, the 3 V lithium-ion battery exhibited positive characteristic features, such as flatness in operating voltage, high rate capability, and cycle life.

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.

Doping of carbon foams for use in energy storage devices

DOEpatents

Mayer, Steven T.; Pekala, Richard W.; Morrison, Robert L.; Kaschmitter, James L.

1994-01-01

A polymeric foam precursor, wetted with phosphoric acid, is pyrolyzed in an inert atmosphere to produce an open-cell doped carbon foam, which is utilized as a lithium intercalation anode in a secondary, organic electrolyte battery. Tests were conducted in a cell containing an organic electrolyte and using lithium metal counter and reference electrodes, with the anode located therebetween. Results after charge and discharge cycling, for a total of 6 cycles, indicated a substantial increase in the energy storage capability of the phosphorus doped carbon foam relative to the undoped carbon foam, when used as a rechargeable lithium ion battery.

Life Cycle Assessment of Titania Perovskite Solar Cell Technology for Sustainable Design and Manufacturing.

PubMed

Zhang, Jingyi; Gao, Xianfeng; Deng, Yelin; Li, Bingbing; Yuan, Chris

2015-11-01

Perovskite solar cells have attracted enormous attention in recent years due to their low cost and superior technical performance. However, the use of toxic metals, such as lead, in the perovskite dye and toxic chemicals in perovskite solar cell manufacturing causes grave concerns for its environmental performance. To understand and facilitate the sustainable development of perovskite solar cell technology from its design to manufacturing, a comprehensive environmental impact assessment has been conducted on titanium dioxide nanotube based perovskite solar cells by using an attributional life cycle assessment approach, from cradle to gate, with manufacturing data from our laboratory-scale experiments and upstream data collected from professional databases and the literature. The results indicate that the perovskite dye is the primary source of environmental impact, associated with 64.77% total embodied energy and 31.38% embodied materials consumption, contributing to more than 50% of the life cycle impact in almost all impact categories, although lead used in the perovskite dye only contributes to about 1.14% of the human toxicity potential. A comparison of perovskite solar cells with commercial silicon and cadmium-tellurium solar cells reveals that perovskite solar cells could be a promising alternative technology for future large-scale industrial applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells

PubMed Central

Kobayashi, Yuki; Kanesaki, Yu; Tanaka, Ayumi; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Tanaka, Kan

2009-01-01

Eukaryotic cells arose from an ancient endosymbiotic association of prokaryotes, with plant cells harboring 3 genomes as the remnants of such evolution. In plant cells, plastid and mitochondrial DNA replication [organelle DNA replication (ODR)] occurs in advance of the subsequent cell cycles composed of nuclear DNA replication (NDR) and cell division. However, the mechanism by which replication of these genomes with different origins is coordinated is largely unknown. Here, we show that NDR is regulated by a tetrapyrrole signal in plant cells, which has been suggested as an organelle-to-nucleus retrograde signal. In synchronized cultures of the primitive red alga Cyanidioschyzon merolae, specific inhibition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cell cycle. In contrast, inhibition of ODR by nalidixic acid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirement of ODR for NDR was bypassed by addition of the tetrapyrrole intermediates protoporphyrin IX (ProtoIX) or Mg-ProtoIX, both of which activated CDKA without inducing ODR. This scheme was also observed in cultured tobacco cells (BY-2), where inhibition of ODR by nalidixic acid prevented CDKA activation and NDR, and these inhibitions were circumvented by Mg-ProtoIX without inducing ODR. We thus show that tetrapyrrole-mediated organelle–nucleus replicational coupling is an evolutionary conserved process among plant cells. PMID:19141634

HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

PubMed

Evans, Edward L; Becker, Jordan T; Fricke, Stephanie L; Patel, Kishan; Sherer, Nathan M

2018-04-01

Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production, reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors cyclin T1 (CCNT1) and exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's postintegration stages, we studied HIV-1 NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1-compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines, which we mapped to the viral protein Vif and its apparent species-specific capacity to induce G 2 /M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that Vif NL4-3 's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G 2 /M cell cycle arrest in other nonhuman cell types, including cells derived from nonhuman primates, leading us to propose that one or more human-specific cofactors underpin Vif's ability to modulate the cell cycle. IMPORTANCE Cells derived from mice and other rodents exhibit profound blocks to HIV-1 replication, thus hindering the development of a low-cost small-animal model for studying HIV/AIDS. Here, we engineered otherwise-nonpermissive mouse cells to express HIV-1-compatible versions of two species-specific host dependency factors, cyclin T1 (CCNT1) and exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1 particle production but, unexpectedly, are completely resistant to virus-induced cytopathic effects. We mapped these effects to the viral accessory protein Vif, which induces a prolonged G 2 /M cell cycle arrest followed by apoptosis in human cells. Combined, our results indicate that one or more additional human-specific cofactors govern HIV-1's capacity to modulate the cell cycle, with potential relevance to viral pathogenesis in people and existing animal models. Copyright © 2018 American Society for Microbiology.

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

PubMed Central

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-01-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588] PMID:26277981

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

PubMed

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-10-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function.

Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

PubMed Central

Wilson, Korey A.; Elefanty, Andrew G.; Stanley, Edouard G.; Gilbert, David M.

2016-01-01

ABSTRACT Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification. PMID:27433885

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

PubMed

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

2018-01-01

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

Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

PubMed

Bácsi, A; Aranyosi, J; Beck, Z; Ebbesen, P; Andirkó, I; Szabó, J; Lampé, L; Kiss, J; Gergely, L; Tóth, F D

1999-10-01

Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-β-d-glucan in human lymphocytes.

PubMed

Malini, Maressa; Souza, Marilesia Ferreira de; Oliveira, Marcelo Tempesta de; Antunes, Lusânia Maria Greggi; Figueiredo, Suely Gomes de; Barbosa, Aneli M; Dekker, Robert F H; Cólus, Ilce Mara de Syllos

2015-01-01

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

RBP-J-interacting and tubulin-associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7 pathway

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

Wang, Haihe; Yang, Zhanchun; Liu, Chunbo

2014-11-07

Highlights: • RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. • RITA can significantly inhibit the in vitro growth of SMMC7721 and HepG2 cells. • RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC. - Abstract: Aberrant Notch signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell growth. However, the role of Notch signaling in HCC and its underlying mechanism remain elusive.more » RBP-J-interacting and tubulin-associated (RITA) mediates the nuclear export of RBP-J to tubulin fibers and downregulates Notch-mediated transcription. In this study, we found that RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Our findings indicate that RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC.« less

Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

PubMed Central

Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

2015-01-01

Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

PubMed Central

Shimizu-Sato, Sae; Ike, Yoko

2007-01-01

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds. PMID:18034314

Intrinsic, nondeterministic circadian rhythm generation in identified mammalian neurons.

PubMed

Webb, Alexis B; Angelo, Nikhil; Huettner, James E; Herzog, Erik D

2009-09-22

Circadian rhythms are modeled as reliable and self-sustained oscillations generated by single cells. The mammalian suprachiasmatic nucleus (SCN) keeps near 24-h time in vivo and in vitro, but the identity of the individual cellular pacemakers is unknown. We tested the hypothesis that circadian cycling is intrinsic to a unique class of SCN neurons by measuring firing rate or Period2 gene expression in single neurons. We found that fully isolated SCN neurons can sustain circadian cycling for at least 1 week. Plating SCN neurons at <100 cells/mm(2) eliminated synaptic inputs and revealed circadian neurons that contained arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) or neither. Surprisingly, arrhythmic neurons (nearly 80% of recorded neurons) also expressed these neuropeptides. Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation. In SCN explants treated with tetrodotoxin to block spike-dependent signaling, neurons gained or lost circadian cycling over many days. The rate of PERIOD2 protein accumulation on the previous cycle reliably predicted the spontaneous onset of arrhythmicity. We conclude that individual SCN neurons can generate circadian oscillations; however, there is no evidence for a specialized or anatomically localized class of cell-autonomous pacemakers. Instead, these results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.

Effect of nickel chloride on cell proliferation.

PubMed

D'Antò, Vincenzo; Valletta, Rosa; Amato, Massimo; Schweikl, Helmut; Simeone, Michele; Paduano, Sergio; Rengo, Sandro; Spagnuolo, Gianrico

2012-01-01

Metal alloys used in dentistry and in other biomedical fields may release nickel ions in the oral environment. The release of nickel might influence the normal biological and physiological processes, including tissue wound healing, cell growth and proliferation. The aim of this study was to evaluate in vitro the effects of nickel ions on cell cycle, viability and proliferation. Human osteosarcoma cells (U2OS) and human keratinocytes (HaCat) were exposed to different nickel chloride (NiCl(2)) concentrations (0 - 5mM) for various periods exposure. The viability of cultured cells was estimated by flow cytometry using Annexin V-FITC and Propidium Iodide (PI). Cell proliferation was evaluated by using carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometry. Finally, the effects of NiCl(2) on cell cycle were assessed and quantified by flow cytometry. Statistical analysis was performed by means of ANOVA followed by Tukey's test. NiCl(2) induced a dose and time dependent decrease in cell viability. After 24h, 1mM NiCl(2) caused a similar and significant reduction of viability in U2OS and HaCat cells, while higher NiCl(2) concentrations and longer exposure times showed a reduced cytotoxic effect in HaCat as compared to U2OS cells. Exposure to NiCl(2) caused a dose- and time-dependent inhibition of cell proliferation in both cell lines tested, with a prominent effect on U2OS cells. Furthermore, both cell lines exposed to NiCl(2) exhibited significant changes in cell cycle distribution after 24h exposure 2mM NiCl2, as compared to untreated cells (p<0.05). Our results indicate that release of nickel ions may affect cell proliferation. The inhibition of cell growth by NiCl2 is mediated by both cell cycle arrest and by induction of cell death.

Effect of Nickel Chloride on Cell Proliferation

PubMed Central

D’Antò, Vincenzo; Valletta, Rosa; Amato, Massimo; Schweikl, Helmut; Simeone, Michele; Paduano, Sergio; Rengo, Sandro; Spagnuolo, Gianrico

2012-01-01

Objective: Metal alloys used in dentistry and in other biomedical fields may release nickel ions in the oral environment. The release of nickel might influence the normal biological and physiological processes, including tissue wound healing, cell growth and proliferation. The aim of this study was to evaluate in vitro the effects of nickel ions on cell cycle, viability and proliferation. Materials and Methods: Human osteosarcoma cells (U2OS) and human keratinocytes (HaCat) were exposed to different nickel chloride (NiCl2) concentrations (0 - 5mM) for various periods exposure. The viability of cultured cells was estimated by flow cytometry using Annexin V-FITC and Propidium Iodide (PI). Cell proliferation was evaluated by using carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometry. Finally, the effects of NiCl2 on cell cycle were assessed and quantified by flow cytometry. Statistical analysis was performed by means of ANOVA followed by Tukey’s test. Results: NiCl2 induced a dose and time dependent decrease in cell viability. After 24h, 1mM NiCl2 caused a similar and significant reduction of viability in U2OS and HaCat cells, while higher NiCl2 concentrations and longer exposure times showed a reduced cytotoxic effect in HaCat as compared to U2OS cells. Exposure to NiCl2 caused a dose- and time-dependent inhibition of cell proliferation in both cell lines tested, with a prominent effect on U2OS cells. Furthermore, both cell lines exposed to NiCl2 exhibited significant changes in cell cycle distribution after 24h exposure 2mM NiCl2, as compared to untreated cells (p<0.05). Conclusion: Our results indicate that release of nickel ions may affect cell proliferation. The inhibition of cell growth by NiCl2 is mediated by both cell cycle arrest and by induction of cell death. PMID:23198004

Human Papillomavirus Promotes Epstein-Barr Virus Maintenance and Lytic Reactivation in Immortalized Oral Keratinocytes

PubMed Central

Makielski, Kathleen R.; Lee, Denis; Lorenz, Laurel D.; Nawandar, Dhananjay M.; Chiu, Ya- Fang; Kenney, Shannon C.; Lambert, Paul F.

2016-01-01

Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other’s life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity. PMID:27179345

MicroRNA-155 acts as a tumor suppressor in colorectal cancer by targeting CTHRC1 in vitro.

PubMed

Liu, Jingtian; Chen, Zongyou; Xiang, Jianbin; Gu, Xiaodong

2018-04-01

Colorectal cancer is one of the most common malignancies. Aberrant expressed microRNAs (miRNAs) have been demonstrated to have strong associations with colorectal cancer by repressing their targets. Therefore, miRNAs are thought to have significant promise in the diagnosis and prognosis of colorectal cancer. Previous studies indicated that miR-155 and collagen triple helix repeat containing 1 (CTHRC1) were both involved in pathogenesis of colorectal cancer, but the underlying mechanisms of miR-155 and CTHRC1 are still unknown. The present study aimed to investigate the biological functions of miR-155 and CTHRC1 in colorectal cancer. Reverse transcription-quantitative polymerase chain reaction was used to examine miR-155 and CTHRC1 expression levels. A dual-luciferase reporter assay was applied to verify the target interaction between miR-155 and CTHRC1. Proliferation, cell cycle, apoptosis, cell migration and invasion were measured using the MTT assay, flow cytometry and Transwell assays, respectively. Results showed that miR-155 expression was decreased, but CTHRC1 expression was increased in colorectal cancer tissue and cell lines. Furthermore, it was demonstrated that miR-155 negatively regulated CTHRC1. Additionally, miR-155 overexpression suppressed cell proliferation, induced cell cycle arrest and promoted cell apoptosis, while an inhibitor of miR-155 facilitated cell proliferation and cell cycle and repressed apoptosis. Transwell experiments indicated that miR-155 inhibited the cell migratory and invasive abilities of HT-29 cells, but miR-155 inhibitor enhanced these abilities of HT-29 cells. These results suggested that miR-155 prevented colorectal cancer progression and metastasis via silencing CTHRC1 in vitro , which provides evidence for miR-155 and CTHRC1 as a novel anti-onco molecular target for the treatment of colorectal cancer in the future.

Quantification of Intracellular Ice Formation and Recrystallization During Freeze-Thaw Cycles and Their Relationship with the Viability of Pig Iliac Endothelium Cells.

PubMed

Liu, Xiaoli; Zhao, Gang; Shu, Zhiquan; Niu, Dan; Zhang, Zhiguo; Zhou, Ping; Cao, Yunxia; Gao, Dayong

2016-12-01

Quantitative evaluation of the inherent correlation between cell cryoinjuries and intracellular ice formation (IIF) together with recrystallization (IIR) is of primary importance for both optimization of biopreservation and cryotherapy. The objective of this study is to thoroughly explore the roles of IIF on cell viability by using pig iliac endothelium cells (PIECs) as model cells during freezing and thawing. The experimental results indicated that both the probabilities of IIF (PIF) and IIR (PIR) increased along with the increase of cooling rates (p < 0.05) during the freeze-thaw cycles at cooling rates of 40, 60, 80, 100, and 150°C/min and the same warming rates of 100°C/min in phosphate-buffered saline-based solutions with or without 1 M DMSO. Viability evaluation with Hoechst 33342/propidium iodide double staining showed that most of the cells were killed (viability <20%) by the abovementioned freeze-thaw cycles, which indicated that the cooling rates investigated were all too rapid since large amounts of IIF and IIR were introduced. Another interesting phenomenon is that the presence of a low concentration of DMSO (1 M) tends to improve cell viability while increasing the PIF and PIR during freezing/thawing, contrary to the common belief that larger PIF corresponds to greater cryoinjury. This may be attributed to the intrinsic protection effect of DMSO by reduction of solution injury or other potential injuries. These findings may be of potential application value for both cryopreservation and cryosurgery by providing helpful additions to the existing studies on investigation of cryoinjuries of PIECs.

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

Neuroblastoma cells depend on HDAC11 for mitotic cell cycle progression and survival

PubMed Central

Thole, Theresa M; Lodrini, Marco; Fabian, Johannes; Wuenschel, Jasmin; Pfeil, Sebastian; Hielscher, Thomas; Kopp-Schneider, Annette; Heinicke, Ulrike; Fulda, Simone; Witt, Olaf; Eggert, Angelika; Fischer, Matthias; Deubzer, Hedwig E

2017-01-01

The number of long-term survivors of high-risk neuroblastoma remains discouraging, with 10-year survival as low as 20%, despite decades of considerable international efforts to improve outcome. Major obstacles remain and include managing resistance to induction therapy, which causes tumor progression and early death in high-risk patients, and managing chemotherapy-resistant relapses, which can occur years after the initial diagnosis. Identifying and validating novel therapeutic targets is essential to improve treatment. Delineating and deciphering specific functions of single histone deacetylases in neuroblastoma may support development of targeted acetylome-modifying therapeutics for patients with molecularly defined high-risk neuroblastoma profiles. We show here that HDAC11 depletion in MYCN-driven neuroblastoma cell lines strongly induces cell death, mostly mediated by apoptotic programs. Genes necessary for mitotic cell cycle progression and cell division were most prominently enriched in at least two of three time points in whole-genome expression data combined from two cell systems, and all nine genes in these functional categories were strongly repressed, including CENPA, KIF14, KIF23 and RACGAP1. Enforced expression of one selected candidate, RACGAP1, partially rescued the induction of apoptosis caused by HDAC11 depletion. High-level expression of all nine genes in primary neuroblastomas significantly correlated with unfavorable overall and event-free survival in patients, suggesting a role in mediating the more aggressive biological and clinical phenotype of these tumors. Our study identified a group of cell cycle-promoting genes regulated by HDAC11, being both predictors of unfavorable patient outcome and essential for tumor cell viability. The data indicate a significant role of HDAC11 for mitotic cell cycle progression and survival of MYCN-amplified neuroblastoma cells, and suggests that HDAC11 could be a valuable drug target. PMID:28252645

Tunicamycin inhibits progression of glioma cells through downregulation of the MEG-3-regulated wnt/β-catenin signaling pathway.

PubMed

Li, Xin; Xue, Lei; Peng, Qin

2018-06-01

Glioma is derived from the oncogenic transformation of brain and spinal cord glial cells, and is one of the most common primary brain tumors. Tunicamycin (TUN) can significantly inhibit glioma growth and aggressiveness by promoting apoptosis in glioma cells. The purpose of the present study was to investigate the effects of TUN on growth of glioma cells and examine the TUN-mediated signaling pathway. The inhibitory effects of TUN on apoptosis, growth, aggressiveness and cell cycle arrest of glioma tumor cells were determined by western blotting, reverse transcription-quantitative polymerase chain reaction, apoptotic assays and immunofluorescence. The results demonstrated that treatment with TUN suppressed growth, migration and invasion of glioma carcinoma cells. In addition, TUN treatment induced apoptosis of glioma cells through downregulation of Bcl-2 and P53 expression levels. Findings also indicated that TUN suppressed proliferation and arrested the glioma cells in the S phase of the cell cycle. Further analysis of the mechanisms of TUN demonstrated that TUN treatment upregulated the expression levels of maternally expressed gene (MEG)-3, wnt and β-catenin in glioma cells. Furthermore, knockdown of MEG-3 expression reversed the TUN-decreased wnt/β-catenin signaling pathway, which subsequently also reversed the TUN-inhibited growth and aggressiveness of glioma cells. In conclusion, the findings in the present study indicated that TUN treatment inhibited growth and aggressiveness through MEG-3-mediated wnt/β-catenin signaling, suggesting that TUN may be an efficient anticancer agent for the treatment of glioma.

[Mechanism research on the lupeol treatment on MCF-7 breast cancer cells based on cell metabonomics].

PubMed

Shi, Dongdong; Kuang, Yuanyuan; Wang, Guiming; Peng, Zhangxiao; Wang, Yan; Yan, Chao

2014-03-01

The objective of this research is to investigate the suppressive effects of lupeol on MCF-7 breast cancer cells, and explore its mechanism on inhibiting the proliferation of MCF-7 cells based on cell metabonomics and cell cycle. Gas chromatography-mass spectrometry (GC-MS) was used in the cell metabonomics assay to identify metabolites of MCF-7 cells and MCF-7 cells treated with lupeol. Then, orthogonal partial least squares discriminant analysis (OPLS-DA) was used to process the metabolic data and model parameters of OPLS-DA were as follows: R2Ycum = 0.988, Q2Ycum = 0.964, which indicated that these two groups could be distinguished clearly. The metabolites (VIP (variable importance in the projection) > 1) were analyzed by t-test, and finally, metabolites (t < 0.05) were identified to be biomarkers. Eleven metabolites such as butanedioic acid, phosphoric acid, L-leucine and isoleucine which had a significant contribution to classification were selected and preliminarily identified due to the accurate mass. Cell cycle assay was analyzed by FACSCalibur. Since the cells in the phase of G1 were increased significantly after the treatment of lupeol, we speculated that lupeol has a blocking effect on the generation of succinyl-CoA and the reaction of substrate phosphorylation of tricarboxylic acid cycle of MCF-7 cells. This study provided a novel approach to the mechanism research on the lupeol treatment on MCF-7 breast cancer cells based on cell metabonomics.

Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell.

PubMed

Kavitha, Nowroji; Ein Oon, Chern; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

2017-04-06

Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. MDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G 0 /G 1 and G 2 /M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆ Ψm ) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G 0 /G 1 and G 2 /M-phases cell cycle arrest by p53-mediated mechanism. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Cytogenetic effects of space-relevant hze-particles in human blood lymphocytes

NASA Astrophysics Data System (ADS)

Lee, R.; Nasonova, E.; Ritter, S.

The analysis of aberrations in human lymphocytes collected 48 h after exposure is used since the 1960s to estimate the radiation risk. However, evidence is increasing that this protocol is not reliable in the case of high LET exposure, because particle induced cell cycle delays influence the aberration yield. To contribute to this issue lymphocytes obtained from a healthy donor were irradiated with Fe-ions (200 MeV/n, 440 keV/μ m), iron-like particles (˜ 4 MeV/n Ni- and Cr-ions, ˜ 4000 keV/μ m) and X-rays. Directly after irradiation PHA and BrdU was added to the cell culture medium. Aberrations were measured in first mitoses collected at 48, 60 and 72 h post-irradiation following colcemid treatment and in prematurely condensed G2-cells (PCCs) at 48 h using calyculin A. Samples were stained with the FPG-technique to allow cell cycle discrimination. Additionally, the mitotic index, the BrdU-labelling index and the number of apoptotic cells were determined at several time-points. Analysis of the BrdU-labelling indices and the mitotic indices revealed a dose- and LET-dependent delay in the cell cycle progression. Cells that reached the first mitosis 48 h after high LET exposure carried only a few aberrations. However, cells that entered the first mitosis 60 to 72 h after high LET exposure carried at least five times more aberrations than those collected at 48 h. The analysis of chromosomal damage in G2-PCCs showed that the delayed entry of severely damaged cells into mitosis results from a prolonged arrest in G2. Conversely, after X-ray exposure a stable aberration-yield was observed in lymphocytes collected at different time-points post-irradiation and the number of aberrations measured in G2-PCCs was only slightly higher than in metaphase cells. Furthermore, only in samples exposed to stopping heavy charged particles a high frequency of apoptotic cells was detected indicating that under this exposure conditions a large proportion of heavily damaged cells is rapidly removed from the cell population. In summary, our data demonstrate that the genetic risk of high energy Fe-ions will be considerably underestimated, when metaphase cells are only analysed at 48 h post-treatment, because of a selective delay of heavily damaged lymphocytes. Similarly, stopping heavy particles affect the time-course of aberrations. However, due to the low mitotic indices and the high apoptotic rates observed in samples exposed to stopping particles, it is reasonable to assume that most aberrant cells are rapidly eliminated from the cell population. Thus, the neoplastic risk of low energy, high LET particles is expected to be low. Supported by BMBF (Bonn), grant 02S8203.

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

6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells.

PubMed

Li, Ting-Yi; Chiang, Been-Huang

2017-09-01

6-shogaol is a phytochemical of dietary ginger, we found that 6-shogaol could induced both autophagic and apoptotic death in human colon adenocarcinoma (HT-29) cells. Results of this study showed that 6-shogal induced cell cycle arrest, autophagy, and apoptosis in HT-29 cells in a time sequence. After 6h, 6-shogal induced apparent G2/M arrest, then the HT-29 cells formed numerous autophagosomes in each phase of the cell cycle. After 18h, increases in acidic vesicles and LAMP-1 (Lysosome-associated membrane proteins 1) showed that 6-shogaol had caused autophagic cell death. After 24h, cell shrinkage and Caspase-3/7 activities rising, suggesting that apoptotic cell death had increased. And after 48h, the result of TUNEL assay indicated the highest occurrence of apoptosis upon 6-shogaol treatment. It appeared that apoptosis is triggered by autophagy in 6-shogaol treated HT-29 cells, the damage of autophagic cell death initiated apoptosis program. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

SU-E-J-65: Evaluation of a Radiation-Induced Cell Proliferation Probability Formula Using Monte Carlo Simulation

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

Watanabe, Y; Dahlman, E

2014-06-01

Purpose: To evaluate the analytic formula of the cell death probability after single fraction dose. Methods: Cancer cells endlessly divide, but radiation causes the cancer cells to die. Not all cells die right away after irradiation. Instead, they continue dividing for next few cell cycles before they stop dividing and die. At the end of every cell cycle, the cell decides if it undertakes the mitotic process with a certain probability, Pdiv, which is altered by the radiation. Previously, by using a simple analytic model of radiobiology experiments, we obtained a formula of Pdeath (= 1 − Pdiv). A questionmore » is if the proposed probability can reproduce the well-known survival data of the LQ model. In this study, we evaluated the formula by doing a Monte Carlo simulation of the cell proliferation process. Starting with Ns seed cells, the cell proliferation process was simulated for N generations or until all cells die. We counted the number of living cells at the end. Assuming that the cell colony survived when more than Nc cells were still alive, the surviving fraction S was estimated. We compared the S vs. dose, or S-D curve, with the LQ model. Results: The results indicated that our formula does not reproduce the experimentally observed S-D curve without selecting appropriate α and α/β. With parameter optimization, there was a fair agreement between the MC result and the LQ curve of dose lower than 20Gy. However, the survival fraction of MC decreased much faster in comparison to the LQ data for doses higher than 20 Gy. Conclusion: This study showed that the previously derived probability of cell death per cell cycle is not sufficiently accurate to replicate common radiobiological experiments. The formula must be modified by considering its cell cycle dependence and some other unknown effects.« less

Ca{sup 2+}-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

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

Stenovec, Matjaz; Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana; Kreft, Marko

2007-11-01

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca{sup 2+}-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowlymore » between the plasma membrane and the cytoplasm. When the cytosolic Ca{sup 2+} level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca{sup 2+}-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.« less

Gene Expression of Pneumocystis murina after Treatment with Anidulafungin Results in Strong Signals for Sexual Reproduction, Cell Wall Integrity, and Cell Cycle Arrest, Indicating a Requirement for Ascus Formation for Proliferation.

PubMed

Cushion, Melanie T; Ashbaugh, Alan; Hendrix, Keeley; Linke, Michael J; Tisdale, Nikeya; Sayson, Steven G; Porollo, Aleksey

2018-05-01

The echinocandins are a class of antifungal agents that target β-1,3-d-glucan (BG) biosynthesis. In the ascigerous Pneumocystis species, treatment with these drugs depletes the ascus life cycle stage, which contains BG, but large numbers of forms which do not express BG remain in the infected lungs. In the present study, the gene expression profiles of Pneumocystis murina were compared between infected, untreated mice and mice treated with anidulafungin for 2 weeks to understand the metabolism of the persisting forms. Almost 80 genes were significantly up- or downregulated. Like other fungi exposed to echinocandins, genes associated with sexual replication, cell wall integrity, cell cycle arrest, and stress comprised the strongest upregulated signals in P. murina from the treated mice. The upregulation of the P. murina β-1,3-d-glucan endohydrolase and endo-1,3-glucanase was notable and may explain the disappearance of the existing asci in the lungs of treated mice since both enzymes can degrade BG. The biochemical measurement of BG in the lungs of treated mice and fluorescence microscopy with an anti-BG antibody supported the loss of BG. Downregulated signals included genes involved in cell replication, genome stability, and ribosomal biogenesis and function and the Pneumocystis -specific genes encoding the major surface glycoproteins (Msg). These studies suggest that P. murina attempted to undergo sexual replication in response to a stressed environment and was halted in any type of proliferative cycle, likely due to a lack of BG. Asci appear to be a required part of the life cycle stage of Pneumocystis , and BG may be needed to facilitate progression through the life cycle via sexual replication. Copyright © 2018 Cushion et al.

Agonistic activity of ICI 182 780 on activation of GSK 3β/AKT pathway in the rat uterus during the estrous cycle.

PubMed

Baranda-Avila, Noemi; Mendoza-Rodríguez, C Adriana; Morimoto, Sumiko; Camacho-Arroyo, Ignacio; Guerra-Araiza, Christian; Langley, Elizabeth; Cerbón, Marco

2013-07-01

We examined the ability of ICI 182,780 (ICI) to block uterine cell proliferation via protein kinase b/AKT pathway in the uterus of the rat during the estrous cycle. Intact rats, with regular estrous cycles, received a subcutaneous (s.c.) injection of either vehicle or ICI at 08:00 h on the day of proestrus or at 00:00 h on the day of estrus and sacrificed at 13:00 h of metaestrus. Estradiol (E₂) and progesterone (P₄) plasma levels were measured by radioimmunoassay. Both ICI treatments, induced a significant decrease (p<0.01) in uterine estrogen receptor alpha (ERα) content, had no effect on uterine progesterone receptor (PR) protein expression and caused marked nuclear localization of cyclin D1, in both luminal and glandular uterine epithelium, as compared to vehicle-treated animals. Furthermore, we detected that ICI treatment induced glycogen synthase kinase (Gsk3-β) Ser 9 phosphorylation, which correlates with cyclin D1 nuclear localization. However, some differences were observed between the two different time schedules of administration. We observed that the administration of ICI at 08:00 h on proestrus day produced a 15% inhibition of luminal epithelial cell proliferation, reduced uterine wet weight by 21% and caused reduction of Akt phosphorylation at Ser 473 as compared to vehicle-treated animals, whereas ICI treatment at 00:00 h on estrus day had no effect on these parameters. The overall results indicate that ICI may exert agonistic and antagonistic effects on uterine cell proliferation through differential activation of the Akt pathway depending on the administration period during the estrous cycle, and indicates that the mechanism of cell proliferation during the physiological conditions of the estrous cycle, is under a different and more complex regulation than in the ovariectomized + E₂ animal model. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

Antitumor effects of vitamins K1, K2 and K3 on hepatocellular carcinoma in vitro and in vivo.

PubMed

Hitomi, Misuzu; Yokoyama, Fumi; Kita, Yuko; Nonomura, Takako; Masaki, Tsutomu; Yoshiji, Hitoshi; Inoue, Hideyuki; Kinekawa, Fumihiko; Kurokohchi, Kazutaka; Uchida, Naohito; Watanabe, Seishiro; Kuriyama, Shigeki

2005-03-01

A number of studies have shown that various K vitamins, specifically vitamins K2 and K3, possess antitumor activity on various types of rodent- and human-derived neoplastic cell lines. In the present study, we examined the antitumor effects of vitamins K1, K2 and K3 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of these vitamins in vitro and in vivo. Although vitamin K1 did not inhibit proliferation of PLC/PRF/5 cells at a 90-microM concentration (the highest tested), vitamins K2 and K3 suppressed proliferation of the cells at concentrations of 90 and 9 microM, respectively. By flow cytometric analysis, it was shown that not only vitamin K1, but also vitamin K2 did not induce apoptosis or cell cycle arrest on PLC/PRF/5 cells. In contrast, vitamin K3 induced G1 arrest, but not apoptosis on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that both vitamins K2 and K3 markedly suppressed the growth of HCC tumors to similar extent. Protein expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4), but not p16INK4a Cdk inhibitor in the tumor was significantly reduced by vitamin K2 or K3 treatment, indicating that vitamins K2 and K3 may induce G1 arrest of cell cycle on PLC/PRF/5 cells in vivo. Taken collectively, vitamins K2 and K3 were able to induce potent antitumor effects on HCC in vitro and in vivo, at least in part, by inducing G1 arrest of the cell cycle. The results indicate that vitamins K2 and K3 may be useful agents for the treatment of patients with HCC.

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.

Germinal zones in the developing cerebral cortex of ferret: ontogeny, cell cycle kinetics, and diversity of progenitors.

PubMed

Reillo, Isabel; Borrell, Víctor

2012-09-01

Expansion and folding of the cerebral cortex are landmark features of mammalian brain evolution. This is recapitulated during embryonic development, and specialized progenitor cell populations known as intermediate radial glia cells (IRGCs) are believed to play central roles. Because developmental mechanisms involved in cortical expansion and folding are likely conserved across phylogeny, it is crucial to identify features specific for gyrencephaly from those unique to primate brain development. Here, we studied multiple features of cortical development in ferret, a gyrencephalic carnivore, in comparison with primates. Analyzing the combinatorial expression of transcription factors, cytoskeletal proteins, and cell cycle parameters, we identified a combination of traits that distinguish in ferret similar germinal layers as in primates. Transcription factor analysis indicated that inner subventricular zone (ISVZ) and outer subventricular zone (OSVZ) may contain an identical mixture of progenitor cell subpopulations in ferret. However, we found that these layers emerge at different time points, differ in IRGC abundance, and progenitors have different cell cycle kinetics and self-renewal dynamics. Thus, ISVZ and OSVZ are likely distinguished by genetic differences regulating progenitor cell behavior and dynamics. Our findings demonstrate that some, but not all, features of primate cortical development are shared by the ferret, suggesting a conserved role in the evolutionary emergence of gyrencephaly.

Effects of Bauhinia championii (Benth.) Benth. polysaccharides on the proliferation and cell cycle of chondrocytes.

PubMed

Cai, Liangliang; Ye, Hongzhi; Yu, Fangrong; Li, Huiting; Chen, Jiashou; Liu, Xianxiang

2013-05-01

It has been recently shown that polysaccharides isolated from plants exhibit a number of beneficial therapeutic properties. Bauhinia championii (Benth.) Benth. has been widely used for the clinical treatment of knee osteoarthritis (OA) in China. However, the underlying molecular mechanisms of knee OA treatment have yet to be elucidated. In the present study, we investigated the effects of Bauhinia championii (Benth.) Benth. polysaccharides (BCBPs) on the proliferation and cell cycle of chondrocytes on 4-week-old male Sprague Dawley rats. Immunohistochemical staining was used to identify chondrocytes and an MTT assay was used to evaluate cell viability. Flow cytometry was used for cell cycle analysis. The mRNA and protein expression levels of cyclin D1, CDK4 and CDK6 in chondrocytes were detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The data demonstrate that BCBP treatment increased the viability of chondrocytes. In addition, BCBP treatment reduced the cell population in the G0/G1 phase, whereas the cell population was increased in the S phase. Furthermore, BCBP treatment enhanced the expression of cyclin D1, CDK4 and CDK6. These results indicate that BCBP treatment promotes cell proliferation by accelerating the G1/S transition.

Adrenomedullin is a key Protein Mediating Rotary Cell Culture System that Induces the Effects of Simulated Microgravity on Human Breast Cancer Cells

NASA Astrophysics Data System (ADS)

Chen, Li; Yang, Xi; Cui, Xiang; Jiang, Minmin; Gui, Yu; Zhang, Yanni; Luo, Xiangdong

2015-11-01

Microgravity or simulated microgravity promotes stem cell proliferation and inhibits differentiation. But, researchers have not yet been able to understand the underlying mechanism through which microgravity or simulated microgravity brings about stem cell proliferation and inhibition of differentiation. In this study, we investigated the effect of simulated microgravity (SMG) on MDA-MB-231 and MCF-7 human breast cancer cells using rotary cell culture system (RCCS). SMG induced a significant accumulation of these cancer cells in S phase of the cell cycle. But, compared with the static group, there was no effect on the overall growth rate of cells in the RCCS group. Furthermore, the expression of cyclin D1 was inhibited in the RCCS group, indicating that RCCS induced cell cycle arrest. In addition, RCCS also induced glycolytic metabolism by increasing the expression of adrenomedullin (ADM), but not HIF1 a. The addition of ADM further enhanced the effects of SMG, which was induced by RCCS. But, the addition of adrenomedullin antagonist (AMA) reversed these effects of SMG. Finally, our results proved that RCCS, which induced cells cycle arrest of breast cancer cells, enhanced glycolysis and upregulated the expression of ADM. But, this did not lead to an increase in hypoxia-inducible factor-1 a (HIF1 a) expression. Thus, we have uncovered a new mechanism for understanding the Warburg effect in breast cancer cells, this mechanism is not the same as hypoxia induced glycolysis.

KPU-300, a Novel Benzophenone–Diketopiperazine–Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase

PubMed Central

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300. PMID:26716455

KPU-300, a Novel Benzophenone-Diketopiperazine-Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase.

PubMed

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

Free fatty acids block glucose-induced β-cell proliferation in mice by inducing cell cycle inhibitors p16 and p18.

PubMed

Pascoe, Jordan; Hollern, Douglas; Stamateris, Rachel; Abbasi, Munira; Romano, Lia C; Zou, Baobo; O'Donnell, Christopher P; Garcia-Ocana, Adolfo; Alonso, Laura C

2012-03-01

Pancreatic β-cell proliferation is infrequent in adult humans and is not increased in type 2 diabetes despite obesity and insulin resistance, suggesting the existence of inhibitory factors. Free fatty acids (FFAs) may influence proliferation. In order to test whether FFAs restrict β-cell proliferation in vivo, mice were intravenously infused with saline, Liposyn II, glucose, or both, continuously for 4 days. Lipid infusion did not alter basal β-cell proliferation, but blocked glucose-stimulated proliferation, without inducing excess β-cell death. In vitro exposure to FFAs inhibited proliferation in both primary mouse β-cells and in rat insulinoma (INS-1) cells, indicating a direct effect on β-cells. Two of the fatty acids present in Liposyn II, linoleic acid and palmitic acid, both reduced proliferation. FFAs did not interfere with cyclin D2 induction or nuclear localization by glucose, but increased expression of inhibitor of cyclin dependent kinase 4 (INK4) family cell cycle inhibitors p16 and p18. Knockdown of either p16 or p18 rescued the antiproliferative effect of FFAs. These data provide evidence for a novel antiproliferative form of β-cell glucolipotoxicity: FFAs restrain glucose-stimulated β-cell proliferation in vivo and in vitro through cell cycle inhibitors p16 and p18. If FFAs reduce proliferation induced by obesity and insulin resistance, targeting this pathway may lead to new treatment approaches to prevent diabetes.

Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier.

PubMed

Massodi, Iqbal; Moktan, Shama; Rawat, Aruna; Bidwell, Gene L; Raucher, Drazen

2010-01-15

Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37 degrees C) but aggregates when the temperature is raised above 39 degrees C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37 degrees C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42 degrees C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.

Studying the rapid bioconversion of lignocellulosic sugars into ethanol using high cell density fermentations with cell recycle

PubMed Central

2014-01-01

Background The Rapid Bioconversion with Integrated recycle Technology (RaBIT) process reduces capital costs, processing times, and biocatalyst cost for biochemical conversion of cellulosic biomass to biofuels by reducing total bioprocessing time (enzymatic hydrolysis plus fermentation) to 48 h, increasing biofuel productivity (g/L/h) twofold, and recycling biocatalysts (enzymes and microbes) to the next cycle. To achieve these results, RaBIT utilizes 24-h high cell density fermentations along with cell recycling to solve the slow/incomplete xylose fermentation issue, which is critical for lignocellulosic biofuel fermentations. Previous studies utilizing similar fermentation conditions showed a decrease in xylose consumption when recycling cells into the next fermentation cycle. Eliminating this decrease is critical for RaBIT process effectiveness for high cycle counts. Results Nine different engineered microbial strains (including Saccharomyces cerevisiae strains, Scheffersomyces (Pichia) stipitis strains, Zymomonas mobilis 8b, and Escherichia coli KO11) were tested under RaBIT platform fermentations to determine their suitability for this platform. Fermentation conditions were then optimized for S. cerevisiae GLBRCY128. Three different nutrient sources (corn steep liquor, yeast extract, and wheat germ) were evaluated to improve xylose consumption by recycled cells. Capacitance readings were used to accurately measure viable cell mass profiles over five cycles. Conclusion The results showed that not all strains are capable of effectively performing the RaBIT process. Acceptable performance is largely correlated to the specific xylose consumption rate. Corn steep liquor was found to reduce the deleterious impacts of cell recycle and improve specific xylose consumption rates. The viable cell mass profiles indicated that reduction in specific xylose consumption rate, not a drop in viable cell mass, was the main cause for decreasing xylose consumption. PMID:24847379

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

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells.

PubMed

Li, Xiao-Li; Wang, Chong-Zhi; Mehendale, Sangeeta R; Sun, Shi; Wang, Qi; Yuan, Chun-Su

2009-11-01

Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20-100 microM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 microM) for 24 and 48 h. Panaxadiol (25 microM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 microM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.

γ-Tocotrienol Inhibits Proliferation and Induces Apoptosis Via the Mitochondrial Pathway in Human Cervical Cancer HeLa Cells.

PubMed

Xu, Weili; Mi, Yaqing; He, Pan; He, Shenghua; Niu, Lingling

2017-08-04

γ-Tocotrienol, a kind of isoprenoid phytochemical, has antitumor activity. However, there is limited evidence that it has an effect on cervical cancer. In this study, the capacity to inhibit proliferation and induce apoptosis in human cervical cancer HeLa cells and the mechanism underlying these effects were examined. The results indicated that a γ-tocotrienol concentration over 30 μM inhibited the growth of HeLa cells with a 50% inhibitory concentration (IC 50 ) of 46.90 ± 3.50 μM at 24 h, and significantly down-regulated the expression of proliferative cell nuclear antigen (PCNA) and Ki-67. DNA flow cytometric analysis indicated that γ-tocotrienol arrested the cell cycle at G0/G1 phase and reduced the S phase in HeLa cells. γ-tocotrienol induced apoptosis of HeLa cells in a time- and dose-dependent manner. γ-tocotrienol-induced apoptosis in HeLa cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, release of cytochrome from mitochondria, activation of caspase-9 and caspase-3, and subsequent poly (ADP-ribose) polymerase (PARP) cleavage. These results suggested that γ-tocotrienol could significantly inhibit cell proliferation through G0/G1 cell cycle arrest, and induce apoptosis via the mitochondrial apoptotic pathway in human cervical cancer HeLa cells. Thus, our findings revealed that γ-tocotrienol may be considered as a potential agent for cervical cancer therapy.

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.

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

Low-dose cisplatin protects human neuroblastoma SH-SY5Y cells from paclitaxel-induced apoptosis.

PubMed

Villa, Daniela; Miloso, Mariarosaria; Nicolini, Gabriella; Rigolio, Roberta; Villa, Antonello; Cavaletti, Guido; Tredici, Giovanni

2005-09-01

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin alpha, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis.

Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

PubMed

Jiao, Fei; Wang, Juan; Dong, Zhao-Lun; Wu, Min-Juan; Zhao, Ting-Bao; Li, Dan-Dan; Wang, Xin

2012-08-01

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

Electronic waste leachate-mediated DNA fragmentation and cell death by apoptosis in mouse fibroblast (NIH/3T3) cell line.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Filippin-Monteiro, Fabíola B; Sierra, Jelver A; Creczynski-Pasa, Tânia B

2013-08-01

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N-acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N-acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Upregulation of long noncoding RNA HOXA-AS3 promotes tumor progression and predicts poor prognosis in glioma

PubMed Central

Wu, Fan; Zhang, Chuanbao; Cai, Jinquan; Yang, Fan; Liang, Tingyu; Yan, Xiaoyan; Wang, Haoyuan; Wang, Wen; Chen, Jing; Jiang, Tao

2017-01-01

Long noncoding RNAs (lncRNAs) have recently emerged as new potentially promising therapeutic targets in many cancers. However, their prognostic value and biological functions associated with glioma remain to be elucidated. Here, High-throughput RNAseq was performed to detect the expression profiles of lncRNAs in 325 human glioma tissues. It was shown that a novel lncRNA HOXA-AS3 was one of the most significantly upregulated lncRNAs in glioma tissues. Quantitative PCR further verified the increased expression of HOXA-AS3 in patient samples and glioma cell lines. Uni and Multivariate Cox regression analysis revealed that HOXA-AS3 was an independent prognostic factor in glioma patients. Gene set enrichment analysis indicated that the gene sets correlated with HOXA-AS3 expression were involved in cell cycle progression and E2F targets. Functionally, HOXA-AS3 silencing resulted in proliferation arrest by altering cell cycle progression and promoting cell apoptosis, and impaired cell migration in glioma cells. Furthermore, the growth-inhibiting effect of HOXA-AS3 knockdown was also demonstrated in Xenograft mouse model. Our results highlight the important role of HOXA-AS3 in glioma progression, and indicate that HOXA-AS3 may be served as a valuable prognostic biomarker for glioma. PMID:28881797

The ROCK/GGTase Pathway Are Essential to the Proliferation and Differentiation of Neural Stem Cells Mediated by Simvastatin.

PubMed

Zhang, Chan; Wu, Jian-Min; Liao, Min; Wang, Jun-Ling; Xu, Chao-Jin

2016-12-01

Simvastatin, a lipophilic and fermentation-derived natural statin, is reported to treat neurological disorders, such as traumatic brain injury, Parkinson's disease (PD), Alzheimer disease (AD), etc. Recently, research also indicated that simvastatin could promote regeneration in the dentate gyrus of adult mice by Wnt/β-catenin signaling (Robin et al. in Stem Cell Reports 2:9-17, 2014). However, the effect and mechanisms by which simvastatin may affect the neural stem cells (NSCs; from the embryonic day 14.5 (E14.5) SD rat brain) are not fully understood. Here, we investigated the effects of different doses of simvastatin on the survival, proliferation, differentiation, migration, and cell cycle of NSCs as well as underlying intracellular signaling pathways. The results showed that simvastatin not only inhibits the proliferation of NSCs but also enhances the βIII-tubulin + neuron differentiation rate. Additionally, we find that simvastatin could also promote NSC migration and induce cell cycle arrest at M2 phrase. All these effects of simvastatin on NSCs were mimicked with an inhibitor of Rho kinase (ROCK) and a specific inhibitor of geranylgeranyl transferase (GGTase). In conclusion, these data indicate that simvastatin could promote neurogenesis of neural stem cells, and these effects were mediated through the ROCK/GGTase pathway.

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage.

PubMed Central

Early, Anne; Drury, Lucy S; Diffley, John F X

2004-01-01

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start. PMID:15065654

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

Cdc2-like kinase 2 is a key regulator of the cell cycle via FOXO3a/p27 in glioblastoma.

PubMed

Park, Soon Young; Piao, Yuji; Thomas, Craig; Fuller, Gregory N; de Groot, John F

2016-05-03

Cdc2-like kinase 2 (CLK2) is known as a regulator of RNA splicing that ultimately controls multiple physiological processes. However, the function of CLK2 in glioblastoma progression has not been described. Reverse-phase protein array (RPPA) was performed to identify proteins differentially expressed in CLK2 knockdown cells compared to controls. The RPPA results indicated that CLK2 knockdown influenced the expression of survival-, proliferation-, and cell cycle-related proteins in GSCs. Thus, knockdown of CLK2 expression arrested the cell cycle at the G1 and S checkpoints in multiple GSC lines. Depletion of CLK2 regulated the dephosphorylation of AKT and decreased phosphorylation of Forkhead box O3a (FOXO3a), which not only translocated to the nucleus but also increased p27 expression. In two glioblastoma xenograft models, the survival duration of mice with CLK2-knockdown GSCs was significantly longer than mice with control tumors. Additionally, tumor volumes were significantly smaller in CLK2-knockdown mice than in controls. Knockdown of CLK2 expression reduced the phosphorylation of FOXO3a and decreased Ki-67 in vivo. Finally, high expression of CLK2 protien was significantly associated with worse patient survival. These findings suggest that CLK2 plays a critical role in controlling the cell cycle and survival of glioblastoma via FOXO3a/p27.

Cdc2-like kinase 2 is a key regulator of the cell cycle via FOXO3a/p27 in glioblastoma

PubMed Central

Thomas, Craig; Fuller, Gregory N.; de Groot, John F.

2016-01-01

Cdc2-like kinase 2 (CLK2) is known as a regulator of RNA splicing that ultimately controls multiple physiological processes. However, the function of CLK2 in glioblastoma progression has not been described. Reverse-phase protein array (RPPA) was performed to identify proteins differentially expressed in CLK2 knockdown cells compared to controls. The RPPA results indicated that CLK2 knockdown influenced the expression of survival-, proliferation-, and cell cycle-related proteins in GSCs. Thus, knockdown of CLK2 expression arrested the cell cycle at the G1 and S checkpoints in multiple GSC lines. Depletion of CLK2 regulated the dephosphorylation of AKT and decreased phosphorylation of Forkhead box O3a (FOXO3a), which not only translocated to the nucleus but also increased p27 expression. In two glioblastoma xenograft models, the survival duration of mice with CLK2-knockdown GSCs was significantly longer than mice with control tumors. Additionally, tumor volumes were significantly smaller in CLK2-knockdown mice than in controls. Knockdown of CLK2 expression reduced the phosphorylation of FOXO3a and decreased Ki-67 in vivo. Finally, high expression of CLK2 protien was significantly associated with worse patient survival. These findings suggest that CLK2 plays a critical role in controlling the cell cycle and survival of glioblastoma via FOXO3a/p27. PMID:27050366

13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes.

PubMed

Nelson, Amanda M; Gilliland, Kathryn L; Cong, Zhaoyuan; Thiboutot, Diane M

2006-10-01

Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.

Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

PubMed Central

Illescas-Montes, Rebeca; Melguizo-Rodríguez, Lucía; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción

2017-01-01

Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2) using different transmission modes (continuous or pulsed). The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing. PMID:28773152

Degradation diagnosis of aged Li4Ti5O12/LiFePO4 batteries

NASA Astrophysics Data System (ADS)

Castaing, Rémi; Reynier, Yvan; Dupré, Nicolas; Schleich, Donald; Jouanneau Si Larbi, Séverine; Guyomard, Dominique; Moreau, Philippe

2014-12-01

Li4Ti5O12/LiFePO4 cells are cycled under 4 different conditions of discharge profile (galvanostatic or driving-based) and cycling rates (C/8 or 1C) during 4-5 months. All the cells exhibit capacity fade whose extent is not correlated with the aging condition. In order to understand aging phenomena, cells are disassembled at the end of cycle life and the recovered electrodes are analyzed using electrochemistry, electron microscopy, XRD and MAS-NMR. Positive and negative electrodes show no loss in active material and no change in electrochemical activity, active material structure and composite electrode structure. This rules out any irreversible electrode degradation. Lithium stoichiometry estimated by both XRD and electrochemistry is unexpectedly low in the positive electrode when the aging is stopped at full discharge. That indicates a loss of cyclable lithium or electrons leading to cell balancing evolution. That loss may have been caused by parasitic reactions occurring at both electrodes, in accordance with their rich surface chemistry as evidenced by MAS-NMR.

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

PubMed Central

Stoop, JMH.; Pharr, D. M.

1993-01-01

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

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells*

PubMed Central

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

2013-01-01

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

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

Magnolol suppresses the proliferation and invasion of cholangiocarcinoma cells via inhibiting the NF-κB signaling pathway.

PubMed

Zhang, Fu-Hui; Ren, Hong-Yue; Shen, Jin-Xing; Zhang, Xiao-Yun; Ye, Hui-Ming; Shen, Dong-Yan

2017-10-01

Magnolol has shown the potential anticancer properties against a variety of cancers. However, the role of magnolol in cholangiocarcinoma (CCA) cells is unknown. In this study, we assessed the effect of magnolol on the CCA cells. CCA cells were treated with magnolol in the absence or presence of TNFα, the activator for NF-κB. After co-incubation with magnolol, cell proliferation and growth were examined by MTT, colony formation and xenograft tumors; cell cycle was analyzed by flow cytometry; cell migration and invasion were detected by wound healing and transwell assays; the expression of PCNA, Ki67, CyclinD1, MMP-2, MMP-7 and MMP-9 and NF-κB pathway were evaluated by using Western blot. Magnolol inhibited the abilities of CCA cell growth, migration and invasion accompanying with a decreased expression of PCNA, Ki67, MMP-2, MMP-7 and MMP-9 (all P<0.05). with magnolol induced cell cycle arrest in G1 phase with a downregulation of cell cycle protein CyclinD1 (all P<0.05). In addition, magnolol suppressed the expression of p-IκBα and p-P65 and the effect of magnolol on CCA cells could be inhibited by TNFα. Magnolol could inhibit the growth, migration and invasion of CCA cells through regulation of NF-κB pathway, and these data indicate that magnolol is a potential candidate for treating of CCA. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Pak3 promotes cell cycle exit and differentiation of β-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.

PubMed

Piccand, Julie; Meunier, Aline; Merle, Carole; Jia, Zhengping; Barnier, Jean-Vianney; Gradwohl, Gérard

2014-01-01

The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase Pak3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that Pak3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In Pak3-deficient embryos, the proliferation of Ngn3(+) progenitors and β-cells is transiently increased concomitantly with an upregulation of Ccnd1. β-Cell differentiation is impaired at E15.5 but resumes at later stages. Pak3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, Pak3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that Pak3 is a novel regulator of β-cell differentiation and function. Pak3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, Pak3 is required for the proper control of glucose homeostasis under challenging HFD.

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

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

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

1986-03-01

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

Cycling hypoxia: A key feature of the tumor microenvironment.

PubMed

Michiels, Carine; Tellier, Céline; Feron, Olivier

2016-08-01

A compelling body of evidence indicates that most human solid tumors contain hypoxic areas. Hypoxia is the consequence not only of the chaotic proliferation of cancer cells that places them at distance from the nearest capillary but also of the abnormal structure of the new vasculature network resulting in transient blood flow. Hence two types of hypoxia are observed in tumors: chronic and cycling (intermittent) hypoxia. Most of the current work aims at understanding the role of chronic hypoxia in tumor growth, response to treatment and metastasis. Only recently, cycling hypoxia, with spatial and temporal fluctuations in oxygen levels, has emerged as another key feature of the tumor environment that triggers different responses in comparison to chronic hypoxia. Either type of hypoxia is associated with distinct effects not only in cancer cells but also in stromal cells. In particular, cycling hypoxia has been demonstrated to favor, to a higher extent than chronic hypoxia, angiogenesis, resistance to anti-cancer treatments, intratumoral inflammation and tumor metastasis. These review details these effects as well as the signaling pathway it triggers to switch on specific transcriptomic programs. Understanding the signaling pathways through which cycling hypoxia induces these processes that support the development of an aggressive cancer could convey to the emergence of promising new cancer treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.

PubMed

Silver, R B

1989-01-01

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

PubMed

Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

2015-12-01

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

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.

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.

In vitro short-term exposure to air pollution PM{sub 2.5-0.3} induced cell cycle alterations and genetic instability in a human lung cell coculture model

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

Abbas, Imane; EA4492-UCEIV, Université du Littoral-Côte d’Opale, Dunkerque; Lebanese Atomic Energy Commission – CNRS, Beirut

Although its adverse health effects of air pollution particulate matter (PM2.5) are well-documented and often related to oxidative stress and pro-inflammatory response, recent evidence support the role of the remodeling of the airway epithelium involving the regulation of cell death processes. Hence, the overarching goals of the present study were to use an in vitro coculture model, based on human AM and L132 cells to study the possible alteration of TP53-RB gene signaling pathways (i.e. cell cycle phases, gene expression of TP53, BCL2, BAX, P21, CCND1, and RB, and protein concentrations of their active forms), and genetic instability (i.e. LOHmore » and/or MSI) in the PM{sub 2.5-0.3}-exposed coculture model. PM{sub 2.5-0.3} exposure of human AM from the coculture model induced marked cell cycle alterations after 24 h, as shown by increased numbers of L132 cells in subG1 and S+G2 cell cycle phases, indicating apoptosis and proliferation. Accordingly, activation of the TP53-RB gene signaling pathways after the coculture model exposure to PM{sub 2.5-0.3} was reported in the L132 cells. Exposure of human AM from the coculture model to PM{sub 2.5-0.3} resulted in MS alterations in 3p chromosome multiple critical regions in L132 cell population. Hence, in vitro short-term exposure of the coculture model to PM{sub 2.5-0.3} induced cell cycle alterations relying on the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation and genetic instability. - Highlights: • Better knowledge on health adverse effects of air pollution PM{sub 2.5}. • Human alveolar macrophage and normal human epithelial lung cell coculture. • Molecular abnormalities from TP53-RB gene signaling pathway. • Loss of heterozygosity and microsatellite instability. • Pathologic changes in morphology and number of cells in relation to airway remodeling.« less

Aqueous extract from pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell show activity against breast cancer cell line MCF-7 and Ehrlich ascites tumor in Balb-C mice.

PubMed

Hilbig, Josiane; Policarpi, Priscila de Britto; Grinevicius, Valdelúcia Maria Alves de Souza; Mota, Nádia Sandrine Ramos Santos; Toaldo, Isabela Maia; Luiz, Marilde Terezinha Bordignon; Pedrosa, Rozangela Curi; Block, Jane Mara

2018-01-30

In Brazil many health disorders are treated with the consumption of different varieties of tea. Shell extracts of pecan nut (Carya illinoinensis), which have significant amounts of phenolic compounds in their composition, are popularly taken as tea to prevent diverse pathologies. Phenolic compounds from pecan nut shell extract have been associated with diverse biological effects but the effect on tumor cells has not been reported yet. The aim of the current work was to evaluate the relationship between DNA fragmentation, cell cycle arrest and apoptosis induced by pecan nut shell extract and its antitumor activity. Cytotoxicity, proliferation, cell death and cell cycle were evaluated in MCF-7 cells by MTT, colony assay, differential coloring and flow cytometry assays, respectively. DNA damage effects were evaluated through intercalation into CT-DNA and plasmid DNA cleavage. Tumor growth inhibition, survival time increase, apoptosis and cell cycle arrest were assessed in Ehrlich ascites tumor in Balb/C mice. The cytotoxic effect of pecan nut shell extracts, the induction of cell death by apoptosis and also the cell cycle arrest in MCF-7 cells have been demonstrated. The survival time in mice with Ehrlich ascites tumor increased by 67%. DNA damage was observed in the CT-DNA, plasmid DNA and comet assays. The mechanism involved in the antitumor effect of pecan nut shell extracts may be related to the activation of key proteins involved in apoptosis cell death (Bcl-XL, Bax and p53) and on the cell cycle regulation (cyclin A, cyclin B and CDK2). These results were attributed to the phenolic profile of the extract, which presented compounds such as gallic, 4-hydroxybenzoic, chlorogenic, vanillic, caffeic and ellagic acid, and catechin, epicatechin, epigallocatechin and epicatechin gallate. The results indicated that pecan nut shell extracts are effective against tumor cells growth and may be considered as an alternative to the treatment of cancer. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Stimulatory effect of vascular endothelial growth factor on progesterone production and survivability of cultured bubaline luteal cells.

PubMed

Chouhan, V S; Dangi, S S; Gupta, M; Babitha, V; Khan, F A; Panda, R P; Yadav, V P; Singh, G; Sarkar, M

2014-08-01

The objectives of the present study were to investigate the effects of vascular endothelial growth factor (VEGF) on progesterone (P4) synthesis in cultured luteal cells from different stages of the estrous cycle and on expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side chain cleavage (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B), antiapoptotic gene PCNA, and proapoptotic gene BAX in luteal cells obtained from mid-luteal phase (MLP) of estrous cycle in buffalo. Corpus luteum samples from the early luteal phase (ELP; day 1st-4th; n=4), MLP (day 5th-10th; n=4), and the late luteal phase (LLP; day 11th-16th; n=4) of oestrous cycle were obtained from a slaughterhouse. Luteal cell cultures were treated with VEGF (0, 1, 10 and 100 ng/ml) for 24, 48 and 72h. Progesterone was assessed by RIA, while mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Results indicated a dose- and time-dependent stimulatory effect of VEGF on P4 synthesis and expression of steroidogenic enzymes. Moreover, VEGF treatment led to an increase in PCNA expression and decrease in BAX expression. In summary, these findings suggest that VEGF acts locally in the bubaline CL to modulate steroid hormone synthesis and cell survivability, which indicates that this factor has an important role as a regulator of CL development and function in buffalo. Copyright © 2014 Elsevier B.V. All rights reserved.

Apigenin inhibits renal cell carcinoma cell proliferation.

PubMed

Meng, Shuai; Zhu, Yi; Li, Jiang-Feng; Wang, Xiao; Liang, Zhen; Li, Shi-Qi; Xu, Xin; Chen, Hong; Liu, Ben; Zheng, Xiang-Yi; Xie, Li-Ping

2017-03-21

Apigenin, a natural flavonoid found in vegetables and fruits, has antitumor activity in several cancer types. The present study evaluated the effects and mechanism of action of apigenin in renal cell carcinoma (RCC) cells. We found that apigenin suppressed ACHN, 786-0, and Caki-1 RCC cell proliferation in a dose- and time-dependent manner. A comet assay suggested that apigenin caused DNA damage in ACHN cells, especially at higher doses, and induced G2/M phase cell cycle arrest through ATM signal modulation. Small interfering RNA (siRNA)-mediated p53 knockdown showed that apigenin-induced apoptosis was likely p53 dependent. Apigenin anti-proliferative effects were confirmed in an ACHN cell xenograft mouse model. Apigenin treatment reduced tumor growth and volume in vivo, and immunohistochemical staining revealed lower Ki-67 indices in tumors derived from apigenin-treated mice. These findings suggest that apigenin exposure induces DNA damage, G2/M phase cell cycle arrest, p53 accumulation and apoptosis, which collectively suppress ACHN RCC cell proliferation in vitro and in vivo. Given its antitumor effects and low in vivo toxicity, apigenin is a highly promising agent for treatment of RCC.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

PubMed Central

Hematulin, Arunee; Meethang, Sutiwan; Utapom, Kitsana; Wongkham, Sopit; Sagan, Daniel

2018-01-01

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint. PMID:29541168

Changes of ploidy during the Azotobacter vinelandii growth cycle.

PubMed Central

Maldonado, R; Jiménez, J; Casadesús, J

1994-01-01

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory. Images PMID:8021173

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.

Ochratoxin A at nanomolar concentration perturbs the homeostasis of neural stem cells in highly differentiated but not in immature three-dimensional brain cell cultures.

PubMed

Zurich, Marie-Gabrielle; Honegger, Paul

2011-08-28

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A Role of hIPI3 in DNA Replication Licensing in Human Cells.

PubMed

Huang, Yining; Amin, Aftab; Qin, Yan; Wang, Ziyi; Jiang, Huadong; Liang, Lu; Shi, Linjing; Liang, Chun

2016-01-01

The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

PubMed

Wang, Juan; Huang, Shujuan; Xing, Lingxiao; Cui, Jinfeng; Tian, Ziqiang; Shen, Haitao; Jiang, Xiujuan; Yan, Xia; Wang, Junling; Zhang, Xianghong

2015-11-01

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53-p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.

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.

The role of cell size in density gradient electrophoretic separation of mouse leukemia cells according to position in the cell cycle

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

Cultured mouse leukemia cells line L5178Y were subjected to upward electrophoresis in a density gradient and the slower migrating cell populations were enriched in G2 cells. It is indicated that this cell line does not change electrophoretic mobility through the cell cycle. The possibility that increased sedimentation downward on the part of the larger G2 cells caused this separation was explored. Two different cell populations were investigated. The log phase population was found to migrate upward faster than the G2 population, and a similar difference between their velocities and calculated on the basis of a 1 um diameter difference between the two cell populations. The G2 and G1 enriched populations were isolated by Ficoll density gradient sedimentation. The bottom fraction was enriched in G2 cells and the top fraction was enriched with G1 cells, especially when compared with starting materials. The electrophoretic mobilities of these two cell populations did not differ significantly from one another. Cell diameter dependent migration curves were calculated and were found to be different. Families of migration curves that differ when cell size is considered as a parameter are predicted.

Mitotic spindle defects and chromosome mis-segregation induced by LDL/cholesterol-implications for Niemann-Pick C1, Alzheimer's disease, and atherosclerosis.

PubMed

Granic, Antoneta; Potter, Huntington

2013-01-01

Elevated low-density lipoprotein (LDL)-cholesterol is a risk factor for both Alzheimer's disease (AD) and Atherosclerosis (CVD), suggesting a common lipid-sensitive step in their pathogenesis. Previous results show that AD and CVD also share a cell cycle defect: chromosome instability and up to 30% aneuploidy-in neurons and other cells in AD and in smooth muscle cells in atherosclerotic plaques in CVD. Indeed, specific degeneration of aneuploid neurons accounts for 90% of neuronal loss in AD brain, indicating that aneuploidy underlies AD neurodegeneration. Cell/mouse models of AD develop similar aneuploidy through amyloid-beta (Aß) inhibition of specific microtubule motors and consequent disruption of mitotic spindles. Here we tested the hypothesis that, like upregulated Aß, elevated LDL/cholesterol and altered intracellular cholesterol homeostasis also causes chromosomal instability. Specifically we found that: 1) high dietary cholesterol induces aneuploidy in mice, satisfying the hypothesis' first prediction, 2) Niemann-Pick C1 patients accumulate aneuploid fibroblasts, neurons, and glia, demonstrating a similar aneugenic effect of intracellular cholesterol accumulation in humans 3) oxidized LDL, LDL, and cholesterol, but not high-density lipoprotein (HDL), induce chromosome mis-segregation and aneuploidy in cultured cells, including neuronal precursors, indicating that LDL/cholesterol directly affects the cell cycle, 4) LDL-induced aneuploidy requires the LDL receptor, but not Aß, showing that LDL works differently than Aß, with the same end result, 5) cholesterol treatment disrupts the structure of the mitotic spindle, providing a cell biological mechanism for its aneugenic activity, and 6) ethanol or calcium chelation attenuates lipoprotein-induced chromosome mis-segregation, providing molecular insights into cholesterol's aneugenic mechanism, specifically through its rigidifying effect on the cell membrane, and potentially explaining why ethanol consumption reduces the risk of developing atherosclerosis or AD. These results suggest a novel, cell cycle mechanism by which aberrant cholesterol homeostasis promotes neurodegeneration and atherosclerosis by disrupting chromosome segregation and potentially other aspects of microtubule physiology.

Mitotic Spindle Defects and Chromosome Mis-Segregation Induced by LDL/Cholesterol—Implications for Niemann-Pick C1, Alzheimer’s Disease, and Atherosclerosis

PubMed Central

Granic, Antoneta; Potter, Huntington

2013-01-01

Elevated low-density lipoprotein (LDL)-cholesterol is a risk factor for both Alzheimer’s disease (AD) and Atherosclerosis (CVD), suggesting a common lipid-sensitive step in their pathogenesis. Previous results show that AD and CVD also share a cell cycle defect: chromosome instability and up to 30% aneuploidy–in neurons and other cells in AD and in smooth muscle cells in atherosclerotic plaques in CVD. Indeed, specific degeneration of aneuploid neurons accounts for 90% of neuronal loss in AD brain, indicating that aneuploidy underlies AD neurodegeneration. Cell/mouse models of AD develop similar aneuploidy through amyloid-beta (Aß) inhibition of specific microtubule motors and consequent disruption of mitotic spindles. Here we tested the hypothesis that, like upregulated Aß, elevated LDL/cholesterol and altered intracellular cholesterol homeostasis also causes chromosomal instability. Specifically we found that: 1) high dietary cholesterol induces aneuploidy in mice, satisfying the hypothesis’ first prediction, 2) Niemann-Pick C1 patients accumulate aneuploid fibroblasts, neurons, and glia, demonstrating a similar aneugenic effect of intracellular cholesterol accumulation in humans 3) oxidized LDL, LDL, and cholesterol, but not high-density lipoprotein (HDL), induce chromosome mis-segregation and aneuploidy in cultured cells, including neuronal precursors, indicating that LDL/cholesterol directly affects the cell cycle, 4) LDL-induced aneuploidy requires the LDL receptor, but not Aß, showing that LDL works differently than Aß, with the same end result, 5) cholesterol treatment disrupts the structure of the mitotic spindle, providing a cell biological mechanism for its aneugenic activity, and 6) ethanol or calcium chelation attenuates lipoprotein-induced chromosome mis-segregation, providing molecular insights into cholesterol’s aneugenic mechanism, specifically through its rigidifying effect on the cell membrane, and potentially explaining why ethanol consumption reduces the risk of developing atherosclerosis or AD. These results suggest a novel, cell cycle mechanism by which aberrant cholesterol homeostasis promotes neurodegeneration and atherosclerosis by disrupting chromosome segregation and potentially other aspects of microtubule physiology. PMID:23593294

Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

PubMed Central

Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

2011-01-01

Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

PubMed Central

Peyton, Kelly J.; Shebib, Ahmad R.; Azam, Mohammad A.; Liu, Xiao-ming; Tulis, David A.; Durante, William

2012-01-01

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G2M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G0/G1 phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease. PMID:22470341

Centrioles: some self-assembly required.

PubMed

Song, Mi Hye; Miliaras, Nicholas B; Peel, Nina; O'Connell, Kevin F

2008-12-01

Centrioles play an important role in organizing microtubules and are precisely duplicated once per cell cycle. New (daughter) centrioles typically arise in association with existing (mother) centrioles (canonical assembly), suggesting that mother centrioles direct the formation of daughter centrioles. However, under certain circumstances, centrioles can also selfassemble free of an existing centriole (de novo assembly). Recent work indicates that the canonical and de novo pathways utilize a common mechanism and that a mother centriole spatially constrains the self-assembly process to occur within its immediate vicinity. Other recently identified mechanisms further regulate canonical assembly so that during each cell cycle, one and only one daughter centriole is assembled per mother centriole.

Doping of carbon foams for use in energy storage devices

DOEpatents

Mayer, S.T.; Pekala, R.W.; Morrison, R.L.; Kaschmitter, J.L.

1994-10-25

A polymeric foam precursor, wetted with phosphoric acid, is pyrolyzed in an inert atmosphere to produce an open-cell doped carbon foam, which is utilized as a lithium intercalation anode in a secondary, organic electrolyte battery. Tests were conducted in a cell containing an organic electrolyte and using lithium metal counter and reference electrodes, with the anode located there between. Results after charge and discharge cycling, for a total of 6 cycles, indicated a substantial increase in the energy storage capability of the phosphorus doped carbon foam relative to the undoped carbon foam, when used as a rechargeable lithium ion battery. 3 figs.

3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a novel combretastatin A-4 analog, induces G2/M arrest and apoptosis by disrupting tubulin polymerization in human cervical HeLa cells and fibrosarcoma HT-1080 cells.

PubMed

Zuo, Daiying; Guo, Dandan; Jiang, Xuewei; Guan, Qi; Qi, Huan; Xu, Jingwen; Li, Zengqiang; Yang, Fushan; Zhang, Weige; Wu, Yingliang

2015-02-05

Microtubule is a popular target for anticancer drugs. In this study, we describe the effect 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a newly synthesized analog of combretastatin A-4 (CA-4), showing a strong time- and dose-dependent anti-proliferative effect on human cervical cancer HeLa cells and human fibrosarcoma HT-1080 cells. We demonstrated that the growth inhibitory effects of G-1103 in HeLa and HT-1080 cells were associated with microtubule depolymerization and proved that G-1103 acted as microtubule destabilizing agent. Furthermore, cell cycle analysis revealed that G-1103 treatment resulted in cell cycle arrest at the G2/M phase in a time-dependent manner with subsequent apoptosis induction. Western blot analysis revealed that down-regulation of cdc25c and up-regulation of cyclin B1 was related with G2/M arrest in HeLa and HT-1080 cells treatment with G-1103. In addition, G-1103 induced HeLa cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8 expression, which indicated that G-1103 induced HeLa cell apoptosis was mainly associated with death receptor pathway. However, G-1103 induced HT-1080 cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8, Bax and cleaved caspase-9 expression and down-regulating anti-apoptotic protein Bcl-2 expression, which indicated that G-1103 induced HT-1080 cell apoptosis was associated with both mitochondrial and death receptor pathway. Taken together, all the data demonstrated that G-1103 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in HeLa and HT-1080 cells. Therefore, the novel compound G-1103 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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.

Lack of anodic capacitance causes power overshoot in microbial fuel cells.

PubMed

Peng, Xinhong; Yu, Han; Yu, Hongbing; Wang, Xin

2013-06-01

Power overshoot commonly makes the performance evaluation of microbial fuel cells (MFCs) inaccurate. Here, three types of carbon with different capacitance (ultracapacitor activated carbon (UAC), plain activated carbon (PAC) and carbon black (CB)) rolled on stainless steel mesh (SSM) as anodes to investigate the relationship between overshoot and anodic capacitance. It was not observed in all cycles of UAC-MFCs (from Cycle 2 to 4) due to the largest abiotic capacitance (Cm(abiotic)) of 2.1F/cm(2), while this phenomenon was eliminated in PAC-MFCs (Cm(abiotic)=1.6 F/cm(2)) from Cycle 3 and in CB-MFCs (Cm(abiotic)=0.5F/cm(2)) from Cycle 4, indicated that the Cm(abiotic) of the anode stored charges and functioned as electron shuttle to overcome the power overshoot. With bacterial colonization, the transient charge storage in biofilm resulted in a 0.1-0.4F/cm(2) increase in total capacitance for anodes, which was the possible reason for the elimination of power overshoot in PAC/CB-MFCs after multi cycle acclimation. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

PubMed Central

Bandara, L R; Buck, V M; Zamanian, M; Johnston, L H; La Thangue, N B

1993-01-01

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation. Images PMID:8223441

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

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

Colon Cancer-Upregulated Long Non-Coding RNA lincDUSP Regulates Cell Cycle Genes and Potentiates Resistance to Apoptosis.

PubMed

Forrest, Megan E; Saiakhova, Alina; Beard, Lydia; Buchner, David A; Scacheri, Peter C; LaFramboise, Thomas; Markowitz, Sanford; Khalil, Ahmad M

2018-05-09

Long non-coding RNAs (lncRNAs) are frequently dysregulated in many human cancers. We sought to identify candidate oncogenic lncRNAs in human colon tumors by utilizing RNA sequencing data from 22 colon tumors and 22 adjacent normal colon samples from The Cancer Genome Atlas (TCGA). The analysis led to the identification of ~200 differentially expressed lncRNAs. Validation in an independent cohort of normal colon and patient-derived colon cancer cell lines identified a novel lncRNA, lincDUSP, as a potential candidate oncogene. Knockdown of lincDUSP in patient-derived colon tumor cell lines resulted in significantly decreased cell proliferation and clonogenic potential, and increased susceptibility to apoptosis. The knockdown of lincDUSP affects the expression of ~800 genes, and NCI pathway analysis showed enrichment of DNA damage response and cell cycle control pathways. Further, identification of lincDUSP chromatin occupancy sites by ChIRP-Seq demonstrated association with genes involved in the replication-associated DNA damage response and cell cycle control. Consistent with these findings, lincDUSP knockdown in colon tumor cell lines increased both the accumulation of cells in early S-phase and γH2AX foci formation, indicating increased DNA damage response induction. Taken together, these results demonstrate a key role of lincDUSP in the regulation of important pathways in colon cancer.

Ailanthone Inhibits Huh7 Cancer Cell Growth via Cell Cycle Arrest and Apoptosis In Vitro and In Vivo

PubMed Central

Zhuo, Zhenjian; Hu, Jianyang; Yang, Xiaolin; Chen, Minfen; Lei, Xueping; Deng, Lijuan; Yao, Nan; Peng, Qunlong; Chen, Zhesheng; Ye, Wencai; Zhang, Dongmei

2015-01-01

While searching for natural anti-hepatocellular carcinoma (HCC) components in Ailanthus altissima, we discovered that ailanthone had potent antineoplastic activity against HCC. However, the molecular mechanisms underlying the antitumor effect of ailanthone on HCC have not been examined. In this study, the antitumor activity and the underlying mechanisms of ailanthone were evaluated in vitro and in vivo. Mechanistic studies showed that ailanthone induced G0/G1-phase cell cycle arrest, as indicated by decreased expression of cyclins and CDKs and increased expression of p21 and p27. Our results demonstrated that ailanthone triggered DNA damage characterized by activation of the ATM/ATR pathway. Moreover, ailanthone-induced cell death was associated with apoptosis, as evidenced by an increased ratio of cells in the subG1 phase and by PARP cleavage and caspase activation. Ailanthone-induced apoptosis was mitochondrion-mediated and involved the PI3K/AKT signaling pathway in Huh7 cells. In vivo studies demonstrated that ailanthone inhibited the growth and angiogenesis of tumor xenografts without significant secondary adverse effects, indicating its safety for treating HCC. In conclusion, our study is the first to report the efficacy of ailanthone against Huh7 cells and to elucidate its underlying molecular mechanisms. These findings suggest that ailanthone is a potential agent for the treatment of liver cancer. PMID:26525771

Human Mesenchymal Stem Cells Derived From Limb Bud Can Differentiate into All Three Embryonic Germ Layers Lineages

PubMed Central

Jiao, Fei; Wang, Juan; Dong, Zhao-lun; Wu, Min-juan; Zhao, Ting-bao; Li, Dan-dan

2012-01-01

Abstract Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them “human limb bud–derived mesenchymal stem cells” (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro. PMID:22775353

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.

Synthesis and biological evaluation of novel N-phenyl ureidobenzenesulfonate derivatives as potential anticancer agents. Part 2. Modulation of the ring B.

PubMed

Gagné-Boulet, Mathieu; Moussa, Hanane; Lacroix, Jacques; Côté, Marie-France; Masson, Jean-Yves; Fortin, Sébastien

2015-10-20

DNA double strand-breaks (DSBs) are the most deleterious lesions that can affect the genome of living beings and are lethal if not quickly and properly repaired. Recently, we discovered a new family of anticancer agents designated as N-phenyl ureidobenzenesulfonates (PUB-SOs) that are blocking the cells cycle progression in S-phase and inducing DNA DSBs. Previously, we have studied the effect of several modifications on the molecular scaffold of PUB-SOs on their cytocidal properties. However, the effect of the nature and the position of substituents on the aromatic ring B is still poorly studied. In this study, we report the preparation and the biological evaluation of 45 new PUB-SO derivatives substituted by alkyl, alkoxy, halogen and nitro groups at different positions on the aromatic ring B. All PUB-SOs were active in the submicromolar to low micromolar range (0.24-20 μM). The cell cycle progression analysis showed that PUB-SOs substituted at position 2 by alkyl, halogen or nitro groups or substituted at position 4 by a hydroxyl group arrest the cell cycle progression in S-phase. Interestingly, all others PUB-SOs substituted at positions 3 and 4 arrested the cell cycle in G2/M-phase. PUB-SOs arresting the cell cycle progression in S-phase also induced the phosphorylation of H2AX (γH2AX) which is indicating the generation of DNA DSBs. We evidenced that few modifications on the ring B of PUB-SOs scaffold lead to cytocidal derivatives arresting the cell cycle in S-phase and inducing γH2AX and DSBs. In addition, this study shows that these new anticancer agents are promising and could be used as alternative to circumvent some of the biopharmaceutical complications that might be encountered during the development of PUB-SOs. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Gene structure, expression, and DNA methylation characteristics of sea cucumber cyclin B gene during aestivation.

PubMed

Zhu, Aijun; Chen, Muyan; Zhang, Xiumei; Storey, Kenneth B

2016-12-05

The sea cucumber, Apostichopus japonicus, is a good model for studying environmentally-induced aestivation by a marine invertebrate. One of the central requirements of aestivation is the repression of energy-expensive cellular processes such as cell cycle progression. The present study identified the gene structure of the cell cycle regulator, cyclin B, and detected the expression levels of this gene over three stages of the annual aestivation-arousal cycle. Furthermore, the DNA methylation characteristics of cyclin B were analyzed in non-aestivation and deep-aestivation stages of sea cucumbers. We found that the cyclin B promoter contains a CpG island, three CCAAT-boxes and three cell cycle gene homology regions (CHRs). Application of qRT-PCR analysis showed significant downregulation of cyclin B transcript levels during deep-aestivation in comparison with non-aestivation in both intestine and longitudinal muscle, and these returned to basal levels after arousal from aestivation. Methylation analysis of the cyclin B core promoter revealed that its methylation level showed significant differences between non-aestivation and deep-aestivation stages (p<0.05) and interestingly, a positive correlation between Cyclin B transcripts expression and methylation levels of the core promoter was also observed. Our findings suggest that cell cycle progression may be reversibly arrested during aestivation as indicated by the changes in cyclin B expression levels and we propose that DNA methylation is one of the regulatory mechanisms involved in cyclin B transcriptional variation. Copyright © 2016 Elsevier B.V. All rights reserved.

3D chromatin conformation correlates with replication timing and is conserved in resting cells

PubMed Central

Moindrot, Benoit; Audit, Benjamin; Klous, Petra; Baker, Antoine; Thermes, Claude; de Laat, Wouter; Bouvet, Philippe; Mongelard, Fabien; Arneodo, Alain

2012-01-01

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts. PMID:22879376

Geraniol suppresses prostate cancer growth through down-regulation of E2F8.

PubMed

Lee, Sanghoon; Park, Yu Rang; Kim, Su-Hwa; Park, Eun-Jung; Kang, Min Ji; So, Insuk; Chun, Jung Nyeo; Jeon, Ju-Hong

2016-10-01

Geraniol, an acyclic dietary monoterpene, has been found to suppress cancer survival and growth. However, the molecular mechanism underlying the antitumor action of geraniol has not been investigated at the genome-wide level. In this study, we analyzed the microarray data obtained from geraniol-treated prostate cancer cells. Geraniol potently altered a gene expression profile and primarily down-regulated cell cycle-related gene signatures, compared to linalool, another structurally similar monoterpene that induces no apparent phenotypic changes. Master regulator analysis using the prostate cancer-specific regulatory interactome identified that the transcription factor E2F8 as a specific target molecule regulates geraniol-specific cell cycle signatures. Subsequent experiments confirmed that geraniol down-regulated E2F8 expression and the knockdown of E2F8 was sufficient to suppress cell growth by inducing G 2 /M arrest. Epidemiological analysis showed that E2F8 is up-regulated in metastatic prostate cancer and associated with poor prognosis. These results indicate that E2F8 is a crucial transcription regulator controlling cell cycle and survival in prostate cancer cells. Therefore, our study provides insight into the role of E2F8 in prostate cancer biology and therapeutics. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons 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.

Assessing the Role of Dissolved Organic Phosphate on Rates of Microbial Phosphorus Cycling

NASA Astrophysics Data System (ADS)

Gonzalez, A. C.; Popendorf, K. J.; Duhamel, S.

2016-02-01

Phosphorus (P) is an element crucial to life, and it is limiting in many parts of the ocean. In oligotrophic environments, the dissolved P pool is cycled rapidly through the activity of microbes, with turnover times of several hours or less. The overarching aim of this study was to assess the flux of P from picoplankton to the dissolved pool and the role this plays in fueling rapid P cycling. To determine if specific microbial groups are responsible for significant return of P to the dissolved pool during cell lifetime, we compared the rate of cellular P turnover (cell-Pτ, the rate of cellular P uptake divided by cellular P content) to the rate of cellular biomass turnover (cellτ). High rates of P return to the dissolved pool during cell lifetime (high cell-Pτ/cellτ) indicate significant P regeneration, fueling more rapid turnover of the dissolved P pool. We hypothesized that cell-Pτ/cellτ varies widely across picoplankton groups. One factor influencing this variation may be each microbial group's relative uptake of dissolved organic phosphorus (DOP) versus dissolved inorganic phosphorus (DIP). As extracellular hydrolysis is necessary for P incorporation from DOP, this process may return more P to the dissolved pool than DIP incorporation. This leads to the question: does a picoplankton's relative uptake of DOP (versus DIP) affect the rate at which it returns phosphorus to the dissolved pool? To address this question, we compared the rate of cellular P turnover based on uptake of DOP and uptake DIP using cultured representatives of three environmentally significant picoplankton groups: Prochlorococcus, Synechococcus, and heterotrophic bacteria. These different picoplankton groups are known to take up different ratios of DOP to DIP, and may in turn make significantly different contributions to the regeneration and cycling phosphorus. These findings have implications towards our understanding of the timeframes of biogeochemical cycling of phosphorus in the ocean.

Radon-induced reduced apoptosis in human bronchial epithelial cells with knock-down of mitochondria DNA

PubMed Central

Li, Bing-Yan; Sun, Jing; Wei, Hong; Cheng, Yu-Zhi; Xue, Lian; Cheng, Zhi-Hai; Wan, Jian-Mei; Wang, Ai-Qing; Hei, Tom K.; Tong, Jian

2012-01-01

Radon and radon progeny inhalation exposure are recognized to induce lung cancer. To explore the role of mitochondria in radon-induced carcinogenesis in humans, an in vitro partially depleted mitochondrial DNA (mtDNA) cell line (ρ−) was generated by treatment of human bronchial epithelial (HBE) cells (ρ+) with ethidium bromide (EB). The characterization of ρ− cells indicated the presence of dysfunctional mitochondria and might thus serve a reliable model to investigate the role of mitochondria. In a gas inhalation chamber, ρ− and ρ+ cells were exposed to radon gas produced by a radium source. Results showed that apoptosis was significantly increased both in ρ− and ρ+ cells irradiated by radon. Moreover, apoptosis in ρ− cells showed a lower level than in ρ+ cells. Radon was further found to depress mitochondrial membrane potential (MMP) of HBE cells with knock-down mtDNA. Production of reactive oxygen species (ROS) was markedly elevated both in ρ− and ρ+ cells exposed to radon. The distribution of phases of cell cycle was different in ρ− compared to ρ+ cells. Radon-irradiation induced a rise in G2/M and decrease in S phase in ρ+ cells. In ρ− cells, G1, G2/M and S populations remained similar to cells exposed to radon. In conclusion, radon-induced changes in ROS generation, MMP and cell cycle are all attributed to reduction of apoptosis which may trigger and promote cell transformation leading to carcinogenesis. Our study indicates that the use of the ρ− knock-down mtDNA HBE cells may serve as a reliable model to study the role played by mitochondria in carcinogenic diseases. PMID:22891884

Role of the ARF Tumor Suppressor in Prostate Cancer

DTIC Science & Technology

2006-10-01

also a paucity of rough endoplasmic reticulum compared to wild-type cells ( diamond headed arrows, upper panels) indicating a decrease in protein...cell cycle and cell growth (48). For example, the Tuberous Sclerosis Complex inherited cancer syndrome is caused by germline mutations in either the...J. M. Gardner , F. J. Tavner, D. A. Jans, B. R. Henderson, and T. J. Gonda. 2003. Myb-binding protein 1a is a nucleocytoplasmic shuttling protein that

Charge Control Investigation of Rechargeable Lithium Cells

NASA Technical Reports Server (NTRS)

Otzinger, B.; Somoano, R.

1984-01-01

An ambient temperature rechargeable Li-TiS2 cell was cycled under conditions which simulate aerospace applications. A novel charge/discharge state-of-charge control scheme was used, together with tapered current charging, to overcome deleterious effects associated with end-of-charge and end-of-discharge voltages. The study indicates that Li-TiS2 cells hold promise for eventual synchronous satellite-type applications. Problem areas associated with performance degradation and reconditioning effects are identified.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

PubMed

Oshikawa, Mio; Okada, Kei; Tabata, Hidenori; Nagata, Koh-Ichi; Ajioka, Itsuki

2017-09-15

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance. © 2017. Published by The Company of Biologists Ltd.

High Concentration of Melatonin Regulates Leaf Development by Suppressing Cell Proliferation and Endoreduplication in Arabidopsis.

PubMed

Wang, Qiannan; An, Bang; Shi, Haitao; Luo, Hongli; He, Chaozu

2017-05-05

N -acetyl-5-methoxytryptamine (Melatonin), as a crucial messenger in plants, functions in adjusting biological rhythms, stress tolerance, plant growth and development. Several studies have shown the retardation effect of exogenous melatonin treatment on plant growth and development. However, the in vivo role of melatonin in regulating plant leaf growth and the underlying mechanism are still unclear. In this study, we found that high concentration of melatonin suppressed leaf growth in Arabidopsis by reducing both cell size and cell number. Further kinetic analysis of the fifth leaves showed that melatonin remarkably inhibited cell division rate. Additionally, flow cytometic analysis indicated that melatonin negatively regulated endoreduplication during leaf development. Consistently, the expression analysis revealed that melatonin regulated the transcriptional levels of key genes of cell cycle and ribosome. Taken together, this study suggests that high concentration of melatonin negatively regulated the leaf growth and development in Arabidopsis , through modulation of endoreduplication and the transcripts of cell cycle and ribosomal key genes.

High Concentration of Melatonin Regulates Leaf Development by Suppressing Cell Proliferation and Endoreduplication in Arabidopsis

PubMed Central

Wang, Qiannan; An, Bang; Shi, Haitao; Luo, Hongli; He, Chaozu

2017-01-01

N-acetyl-5-methoxytryptamine (Melatonin), as a crucial messenger in plants, functions in adjusting biological rhythms, stress tolerance, plant growth and development. Several studies have shown the retardation effect of exogenous melatonin treatment on plant growth and development. However, the in vivo role of melatonin in regulating plant leaf growth and the underlying mechanism are still unclear. In this study, we found that high concentration of melatonin suppressed leaf growth in Arabidopsis by reducing both cell size and cell number. Further kinetic analysis of the fifth leaves showed that melatonin remarkably inhibited cell division rate. Additionally, flow cytometic analysis indicated that melatonin negatively regulated endoreduplication during leaf development. Consistently, the expression analysis revealed that melatonin regulated the transcriptional levels of key genes of cell cycle and ribosome. Taken together, this study suggests that high concentration of melatonin negatively regulated the leaf growth and development in Arabidopsis, through modulation of endoreduplication and the transcripts of cell cycle and ribosomal key genes. PMID:28475148

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.

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.

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.

PubMed

Ahmed, Emad A; Vélaz, Eukene; Rosemann, Michael; Gilbertz, Klaus-P; Scherthan, Harry

2017-03-01

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G 1 -like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

Extra-virgin olive oil phenols block cell cycle progression and modulate chemotherapeutic toxicity in bladder cancer cells

PubMed Central

Coccia, Andrea; Mosca, Luciana; Puca, Rosa; Mangino, Giorgio; Rossi, Alessandro; Lendaro, Eugenio

2016-01-01

Epidemiological data indicate that the daily consumption of extra-virgin olive oil (EVOO), a common dietary habit of the Mediterranean area, lowers the incidence of certain types of cancer, in particular bladder neoplasm. The aim of the present study was to evaluate the antiproliferative activity of polyphenols extracted from EVOO on bladder cancer (BCa), and to clarify the biological mechanisms that trigger cell death. Furthermore, we also evaluated the ability of low doses of extra-virgin olive oil extract (EVOOE) to modulate the in vitro activity of paclitaxel or mitomycin, two antineoplastic drugs used in the management of different types of cancer. Our results showed that EVOOE significantly inhibited the proliferation and clonogenic ability of T24 and 5637 BCa cells in a dose-dependent manner. Furthermore, cell cycle analysis after EVOOE treatment showed a marked growth arrest prior to mitosis in the G2/M phase for both cell lines, with the subsequent induction of apoptosis only in the T24 cells. Notably, simultaneous treatment of mitomycin C and EVOOE reduced the drug cytotoxicity due to inhibition of ROS production. Conversely, the co-treatment of T24 cells with paclitaxel and the polyphenol extract strongly increased the apoptotic cell death at each tested concentration compared to paclitaxel alone. Our results support the epidemiological evidence indicating that olive oil consumption exerts health benefits and may represent a starting point for the development of new anticancer strategies. PMID:27748855

Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

PubMed Central

Lai, Chin-Yu; Tsai, An-Chi; Chen, Mei-Chuan; Chang, Li-Hsun; Sun, Hui-Lung; Chang, Ya-Ling; Chen, Chien-Chih

2012-01-01

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (−/−) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity. PMID:22912688

The extent of clonal structure in different lymphoid organs

PubMed Central

1992-01-01

To gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near- physiological chimeras. We introduced RT7b fetal liver cells into nonirradiated congenic RT7a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to 20%. The numbers of cell clones simultaneously contributing to cell generation in a particular histological feature were deduced from the variance in donor cell distribution. In bone marrow and thymus, donor-derived lymphoid cells were found scattered among host cells, indicating a high mobility of cells. In bone marrow, donor cells were evenly distributed over the entire marrow, even at low chimerism. This indicates that leukopoiesis is maintained by the proliferation of many clones. In the thymus, the various lobules showed different quantities of donor-derived lymphoid cells. Mathematical analysis of these differences indicated that 17-18 cell division cycles occur in the cortex. In spleen, the distribution of donor-derived cells over the germinal centers indicated that 5 d after antigenic stimulation, germinal centers develop oligoclonally. The main conclusions of this work are that (a) bone marrow and thymus are highly polyclonal; (b) 17-18 divisions occur between prothymocyte and mature T cell; and (c) lymphoid cells disperse rapidly while proliferating and differentiating. PMID:1569396

Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine ▿ †

PubMed Central

Page, Martin A.; Shisler, Joanna L.; Mariñas, Benito J.

2010-01-01

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery. PMID:20305026

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

Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

PubMed

Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K; Peluso, John J

2014-08-01

Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development. © 2014 by the Society for the Study of Reproduction, Inc.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

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

Promoter methylation patterns in Richter syndrome affect stem-cell maintenance and cell cycle regulation and differ from de novo diffuse large B-cell lymphoma.

PubMed

Rinaldi, Andrea; Mensah, Afua Adjeiwaa; Kwee, Ivo; Forconi, Francesco; Orlandi, Ester M; Lucioni, Marco; Gattei, Valter; Marasca, Roberto; Berger, Françoise; Cogliatti, Sergio; Cavalli, Franco; Zucca, Emanuele; Gaidano, Gianluca; Rossi, Davide; Bertoni, Francesco

2013-10-01

In a fraction of patients, chronic lymphocytic leukaemia (CLL) can transform to Richter syndrome (RS), usually a diffuse large B-cell lymphoma (DLBCL). We studied genome-wide promoter DNA methylation in RS and clonally related CLL-phases of transformed patients, alongside de novo DLBCL (of non-germinal centre B type), untransformed-CLL and normal B-cells. The greatest differences in global DNA methylation levels were observed between RS and DLBCL, indicating that these two diseases, although histologically similar, are epigenetically distinct. RS was more highly methylated for genes involved in cell cycle regulation. When RS was compared to the preceding CLL-phase and with untransformed-CLL, RS presented a higher degree of methylation for genes possessing the H3K27me3 mark and PRC2 targets, as well as for gene targets of TP53 and RB1. Comparison of the methylation levels of individual genes revealed that OSM, a stem cell regulatory gene, exhibited significantly higher methylation levels in RS compared to CLL-phases. Its transcriptional repression by DNA methylation was confirmed by 5-aza-2'deoxycytidine treatment of DLBCL cells, determining an increased OSM expression. Our results showed that methylation patterns in RS are largely different from de novo DLBCL. Stem cell-related genes and cell cycle regulation genes are targets of DNA methylation in RS. © 2013 John Wiley & Sons Ltd.

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

PubMed

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

2014-05-15

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

Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.

PubMed

Zhao, Zhenjun; Johnson, Michael S; Chen, Biyi; Grace, Michael; Ukath, Jaysree; Lee, Vivienne S; McRobb, Lucinda S; Sedger, Lisa M; Stoodley, Marcus A

2016-06-01

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-07-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed Central

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-01-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity. PMID:9649516

Antioxidant and Renoprotective Effects of Mushroom Extract: Implication in Prevention of Nephrolithiasis

PubMed Central

Schulman, Ariel; Chaimowitz, Matthew; Choudhury, Muhammad; Eshghi, Majid; Konno, Sensuke

2016-01-01

Background The pathogenesis of nephrolithiasis (kidney stone) remains elusive, while several therapeutic options are available but not effective as we expected. Accumulating data yet suggest that oxidative stress (generation of oxygen free radicals) may play a primary role in its occurrence. Particularly, calcium oxalate (CaOx) is a key element in the most common form (> 75%) of kidney stones, and its crystal form known as CaOx monohydrate (COM) has been shown to exert oxidative stress, facilitating CaOx stone formation. Hence, diminishing oxidative stress with certain antioxidants could be a potential strategic approach. We are interested in a bioactive extract of Poria mushroom, PE, which has been shown to have antioxidant and renoprotective activities. Accordingly, we investigated if PE might have antioxidant activity that would have implication in prevention of kidney stone formation. Methods Renal epithelial LLC-PK1 cells were employed and exposed to COM or hydrogen peroxide (H2O2) as a positive control capable of exerting oxidative stress. Possible antioxidant and protective effects of PE against oxidative stress (exerted by COM or H2O2) were assessed by cell viability test and lipid peroxidation (LPO) assay. To explore its protective mechanism, two glycolytic parameters, hexokinase (HK) activity and ATP synthesis, were examined and cell cycle analysis was also performed. Results Both H2O2 and COM led to a significant (P < 0.05) reduction in cell viability, accompanied by severe oxidative stress assessed by LPO assay. Such oxidative stress also caused the significant decline in HK activity and cellular ATP level, indicating the inhibition of glycolysis. Cell cycle analysis further indicated that oxidative stress interfered with cell cycle, inducing a G1 cell cycle arrest that presumably results in the cessation of cell proliferation. However, PE was capable of significantly preventing or diminishing all these cellular effects mediated through oxidative stress (exerted by H2O2 and COM). Conclusions The present study shows that the mushroom extract PE appears to have antioxidant and renoprotective effects against oxidative stress exerted by COM in renal cells. Therefore, PE with antioxidant activity is considered a promising natural agent that may have clinical implications in prevention of nephrolithiasis primarily induced by oxidative stress. PMID:27829958