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Sample records for cell cycle proteins

  1. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  2. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  3. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  4. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

  5. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik

    2003-01-01

    Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell......-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1......) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas...

  6. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    International Nuclear Information System (INIS)

    Zhang, Heyu; Ma, Xi; Shi, Taiping; Song, Quansheng; Zhao, Hongshan; Ma, Dalong

    2010-01-01

    NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

  7. The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

    Science.gov (United States)

    Morsczeck, Christian; Hullmann, Markus; Reck, Anja; Reichert, Torsten E

    2018-02-01

    Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of β-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.

  8. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    International Nuclear Information System (INIS)

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-01-01

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence

  9. The bacterial cell cycle checkpoint protein Obg and its role in programmed cell death

    Directory of Open Access Journals (Sweden)

    Liselot Dewachter

    2016-03-01

    Full Text Available The phenomenon of programmed cell death (PCD, in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli. Importantly, the PCD pathway mediated by mutant Obg (Obg* differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway.

  10. Regulation of the vertebrate cell cycle by the cdc2 protein kinase

    International Nuclear Information System (INIS)

    Draetta, G.; Brizuela, L.; Moran, B.; Beach, D.

    1988-01-01

    A homolog of the cdc2/CDC28 protein kinase of yeast is found in all vertebrate species that have been investigated. Human cdc2 exists as a complex with a 13-kD protein that is homologous to the suc1 gene product of fission yeast. In both human and fission yeast cells, the protein kinase also exists in a complex with a 62-kD polypeptide that has not been identified genetically but acts as a substrate in vitro. The authors have studied the properties of the protein kinase in rat and human cells, as well as in Xenopus eggs. They find that in baby rat kidney (BRK) cells, which are quiescent in cell culture, the cdc2 protein is not synthesized. However, synthesis is rapidly induced in response to proliferative activation by infection with adenovirus. In human HeLa cells, the protein kinase is present continuously. It behaves as a cell-cycle oscillator that is inactive in G 1 but displays maximal enzymatic activity during mitotic metaphase. These observations indicate that in a wide variety of vertebrate cells, the cdc2 protein kinase is involved in regulating mitosis. The authors' approach taken toward study of the cdc2 protein kinase highlights the possibilities that now exist for combining the advantages of ascomycete genetics with the cell-free systems of Xenopus and the biochemical advantages of tissue culture cells to investigate fundamental problems of the cell cycle

  11. 14-3-3 proteins as signaling integration points for cell cycle control and apoptosis

    OpenAIRE

    Gardino, Alexandra K.; Yaffe, Michael B.

    2011-01-01

    14-3-3 proteins play critical roles in the regulation of cell fate through phospho-dependent binding to a large number of intracellular proteins that are targeted by various classes of protein kinases. 14-3-3 proteins play particularly important roles in coordinating progression of cells through the cell cycle, regulating their response to DNA damage, and influencing life-death decisions following internal injury or external cytokine-mediated cues. This review focuses on 14-3-3-dependent path...

  12. Genes adopt non-optimal codon usage to generate cell cycle-dependent oscillations in protein levels

    DEFF Research Database (Denmark)

    Frenkel-Morgenstern, Milana; Danon, Tamar; Christian, Thomas

    2012-01-01

    The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle-regulated genes with that of other genes, we discovered that there is a significant preference for non-optimal codons. Moreover, genes encoding proteins that cycle a...

  13. Phosphorylation Variation during the Cell Cycle Scales with Structural Propensities of Proteins

    DEFF Research Database (Denmark)

    Tyanova, S.; Frishman, D.; Cox, J.

    2013-01-01

    of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels...

  14. The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

    Science.gov (United States)

    Mizejewski, G J

    2016-09-01

    The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

  15. Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins

    International Nuclear Information System (INIS)

    Deng, Lin; Lu, Yuanyuan; Zhao, Xiaodi; Sun, Yi; Shi, Yongquan; Fan, Hongwei; Liu, Changhao; Zhou, Jinfeng; Nie, Yongzhan; Wu, Kaichun; Fan, Daiming; Guo, Xuegang

    2013-01-01

    Highlights: •Overexpression of Ran in pancreatic cancer was correlated with histological grade. •Downregulation of Ran could induce cell apoptosis and inhibit cell proliferation. •The effects were mediated by cell cycle proteins, Survivin and cleaved Caspase-3. -- Abstract: Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3

  16. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

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    Nicola Vitulo

    2007-01-01

    Full Text Available The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confi rming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals fi ve distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed.

  17. Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

    International Nuclear Information System (INIS)

    Jung, Th.; Streffer, C.

    1992-01-01

    To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G 2 block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G 2 block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author)

  18. Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Th. (AFRC Institute of Animal Physiology and Genetics Research, Babraham (United Kingdom)); Streffer, C. (Essen Univ (Germany). Inst. fuer Medizinische Strahlenbiolgie)

    1992-08-01

    To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G[sub 2] block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G[sub 2] block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author).

  19. A comparative study of cell cycle mediator protein expression patterns in anaplastic and papillary thyroid carcinoma.

    Science.gov (United States)

    Evans, Juanita J; Crist, Henry S; Durvesh, Saima; Bruggeman, Richard D; Goldenberg, David

    2012-07-01

    Anaplastic thyroid carcinoma (ATC) is an extremely aggressive and rapidly fatal neoplasm. The aim of this study was to identify a limited cell cycle associated protein expression pattern unique to ATC and to correlate that pattern with clinical outcome. This represents one of the largest tissue micro-array projects comparing the cell cycle protein expression data of ATC to other well-differentiated tumors in the literature. Tissue microarrays were created from 21 patients with ATC and an age and gender matched cohort of patients with papillary thyroid carcinoma (PTC). Expression of epidermal growth factor receptor, cyclin D1, cyclin E, p53, p21, p16, aurora kinase A, opioid growth factor (OGF), OGF-receptor, thyroglobulin and Ki-67 was evaluated in a semi-quantitative fashion. Differences in protein expression between the cohorts were evaluated using chi-square tests with Bonferroni adjustments. Survival time and presence of metastasis at presentation were collected. The ATC cohort showed a statistically significant decrease (p cycle with aberrant expression of multiple protein markers suggesting increased proliferative activity and loss of control of cell cycle progression to G₁ phase. These findings support the assertion that ATC may represent the furthest end of a continuum of thyroid carcinoma dedifferentiation.

  20. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

    Directory of Open Access Journals (Sweden)

    Thiel Cora S

    2012-01-01

    Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

  1. Immunohistochemical study of the expression of cell cycle regulating proteins at different stages of bladder cancer

    DEFF Research Database (Denmark)

    Primdahl, Hanne; von der Maase, Hans; Sørensen, Flemming Brandt

    2002-01-01

    PURPOSE: The cell cycle is known to be deregulated in cancer. We therefore analyzed the expression of the cell cycle related proteins p21, p27, p16, Rb, and L-myc by immunohistochemical staining of bladder tumors.METHODS: The tissue material consisted of bladder tumors from three groups of patients......; group 1, 23 patients with recurrent stage Ta (non-invasive) tumors; group 2, 22 patients presenting at their first admission with T2-4 (muscle invasive) tumors; group 3, 24 patients who experienced disease progression from Ta or T1 (invasive in connective tissue) to a higher stage...

  2. Effect of low dose radiation on cell cycle and expression of its related proteins of HCT-8 cells

    International Nuclear Information System (INIS)

    Xu Ying; Ma Kewei; Li Wei; Wang Guanjun

    2009-01-01

    Objective: To study the effects of low dose radiation (LDR) on cell cycle and the expression of its related proteins of HCT-8 cells and provide theoretical basis for clinical application of LDR. Methods: Human colon carcinoma cells (HCT-8) cultivated in vitro were divided into seven groups: sham radiation group (0 mGy), LDR groups (25, 50, 75, 100 and 200 mGy) and high dose radiation group (1000 mGy). The proliferation rate was detected with the method of cell count and MTT, the ratios of G 0 /G 1 , S, G 2 /M in cell cycle were determined with flow cytometry after LDR, The cell cycle and expressions of related signal proteins were analyzed with protein assay system. Results: The results of cell count and MTT showed that there were no significant differences of proliferation rate of HCT-8 cells between 25, 50, 75, 100, 200 mGy LDR groups and sham radiation group (P>0.05); compared with high dose radiation group, there were significant differences (P 0 /G 1 phase of HCT-8 cells increased (P>0.05), the ratio of S phase decreased significantly (P 2 /M phase increased obviously (P 0 /G 1 , S, and G 2 /M phases of HCT-8 cells 48 h after radiation compared with sham radiation group (P>0.05). The protein assay result indicated that the expressions of AKt, PCNA, p27, CDK2, cyclin E, EGFR, ERK1/2, p-ERK, p-GSK-32/β in HCT-8 cells after LDR decreased compared with sham radiation group. Conclusion: LDR has no stimulating effect on HCT-8 cells. However, to some extent LDR suppress the expressions of some proteins related to proliferation and cell cycle. (authors)

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

    Science.gov (United States)

    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.

  4. Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells.

    Science.gov (United States)

    Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

    2011-10-01

    Gastric cancer is a leading cause of cancer-related deaths, worldwide being second only to lung cancer as a cause of death. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms of arctigenin for anti-tumor effect on gastric cancer have not been examined. This study examined the biological effects of arctigenin on the human gastric cancer cell line SNU-1 and AGS. Cell proliferation was determined by MTT assay. In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by arctigenin in a time and dose dependent manner, as compared with SNU-1 and AGS cells cultured in the absence of arctigenin. Inhibition of cell proliferation by arctigenin was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bcl-2 to Bax by arctigenin. Also, arctigenin blocked cell cycle arrest from G(1) to S phase by regulating the expression of cell cycle regulatory proteins such as Rb, cyclin D1, cyclin E, CDK4, CDK2, p21Waf1/Cip1 and p15 INK4b. The antiproliferative effect of arctigenin on SNU-1 and AGS gastric cancer cells revealed in this study suggests that arctigenin has intriguing potential as a chemopreventive or chemotherapeutic agent. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  5. A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells

    Directory of Open Access Journals (Sweden)

    Stephanie Chrysanthou

    2018-04-01

    Full Text Available Summary: The ten-eleven translocation (TET proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage. : TET proteins are well known for their role in pluripotency. Here, Hemberger and colleagues show that TET1 and TET2 are also critical for maintaining the epithelial integrity of trophoblast stem cells. TET1/2 ensure mitotic cell-cycle progression by stabilizing cyclin B1 and by regulating centrosome organization. These insights reveal the importance of TET proteins beyond their role in epigenome remodeling. Keywords: TET proteins, trophoblast stem cells, cell cycle, endoreduplication, self-renewal, mitosis, trophoblast giant cells, differentiation

  6. Late phase cell cycle proteins in Alzheimer’s disease: a possible target for therapy?

    KAUST Repository

    Bajic, Vladan

    2017-02-22

    Alzheimer’s disease (AD) is represented by neuronal loss and this loss is correlated to a constant state of neuronal instability induced by intrinsic and extrinsic factors. In this paper data is presented regarding the possible roles of late phase cell cycle proteins in normal and affected neurons with the goal that understanding the mechanisms involved in the regulation of these proteins may represent a novel strategy for AD treatment. The results demonstrate a relative differential pattern of expression of certain proteins (APC/C, Mad1 and Mad2, Bub R1, Bub1, CDK 11, cohesin subunit Rad 21 and astrin) in the AD brain versus age matched controls, and it is suggested that targeting these proteins might translate into potential treatments for AD. Although the data presented here is of some interest, the ability to translate such information into clinical applications is often a challenge.

  7. Late phase cell cycle proteins in Alzheimer’s disease: a possible target for therapy?

    KAUST Repository

    Bajic, Vladan; B. Bajic, Vladimir; Zivkovic, Lada; Arendt, Thomas; Perry, George; Spremo-Potparevic, Biljana

    2017-01-01

    Alzheimer’s disease (AD) is represented by neuronal loss and this loss is correlated to a constant state of neuronal instability induced by intrinsic and extrinsic factors. In this paper data is presented regarding the possible roles of late phase cell cycle proteins in normal and affected neurons with the goal that understanding the mechanisms involved in the regulation of these proteins may represent a novel strategy for AD treatment. The results demonstrate a relative differential pattern of expression of certain proteins (APC/C, Mad1 and Mad2, Bub R1, Bub1, CDK 11, cohesin subunit Rad 21 and astrin) in the AD brain versus age matched controls, and it is suggested that targeting these proteins might translate into potential treatments for AD. Although the data presented here is of some interest, the ability to translate such information into clinical applications is often a challenge.

  8. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins

    International Nuclear Information System (INIS)

    Duchrow, M.; Schlueter, C.; Key, G.; Kubbutat, H.G.; Wohlenberg, C.; Flad, H.D.; Gerdes

    1995-01-01

    A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the 'Ki-67 proteins') has made it abundantly clear that this structure is strictly associated with human cell proliferation and the expression of this protein can be used to access the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ('Ki-67 repeats'), each containing a highly conserved new motif of 66 bp ('Ki-67 motif'). The deduced peptide sequence of this central exon possesses 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3 H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner. (author). 30 refs, 2 figs

  9. Cell cycle deregulation by the HBx protein of hepatitis B virus

    Directory of Open Access Journals (Sweden)

    Vijay Kumar

    2007-02-01

    Full Text Available

    Cell cycle control by oncogenic viruses usually involves disruption of the normal restraints on cellular proliferation via abnormal proteolytic degradation and malignant transformation of cells. The cell cycle regulatory molecules viz. cyclins, cyclin-dependent kinases (cdks and inhibitors of cdks as well as the transcriptional targets of signaling pathways induce cells to move through the cell cycle checkpoints. These check points are often found deregulated in tumor cells and in the cells afflicted with DNA tumor viruses predisposing them towards transformation. The X protein or HBx of hepatitis B virus is a promiscuous transactivator that has been implicated in the development of hepatocellular carcinoma in humans. However, the exact role of HBx in establishing a permissive environment for hepatocarcinogenesis is not fully understood. HBx activates the Ras-Raf-MAP kinase signaling cascade, through which it activates transcription factors AP-1 and NFkappa B, and stimulates cell DNA synthesis. HBx shows a profound effect on cell cycle progression even in the absence of serum. It can override the replicative senescence of cells in G0 phase by binding to p55sen. It stimulates the G0 cells to transit through G1 phase by activating Src kinases and the cyclin A-cyclin-dependent kinase 2 complexes, that in turn induces the cyclin A promoter. There is an early and sustained level of cyclin-cdk2 complex in the presence of HBx during the cell cycle which is coupled with an increased protein kinase activity of cdk2 suggesting an early appearance of S phase. The interaction between cyclin-cdk2 complex and HBx occurs through its carboxyterminal region (amino acids 85-119 and requires a constitutive Src kinase activity. The increased cdk2 activity is associated with stabilization of cyclin E as well as proteasomal degradation of cdk inhibitor p27Kip1. Notably, the HBx mutant

  10. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    International Nuclear Information System (INIS)

    Sun, Bin; Cai, Yingyue; Li, Yongshu; Li, Jingjing; Liu, Kaiyu; Li, Yi; Yang, Yongbo

    2013-01-01

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization

  11. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Bin; Cai, Yingyue; Li, Yongshu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Jingjing [College of Life Science, Hubei Normal University, Huangshi 435002, Hubei (China); Liu, Kaiyu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Yi, E-mail: johnli2668@hotmail.com [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Bioengineering Department, Wuhan Bioengineering Institute, Wuhan 430415, Hubei (China); Yang, Yongbo, E-mail: yongboyang@mail.ccnu.edu.cn [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China)

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  12. Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

    Science.gov (United States)

    Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

    2010-01-01

    The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

  13. A Novel Family of Cell Wall-Related Proteins Regulated Differently during the Yeast Life Cycle

    Science.gov (United States)

    Rodríguez-Peña, José Manuel; Cid, Víctor J.; Arroyo, Javier; Nombela, César

    2000-01-01

    The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial β-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Δ crh2Δ strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1–green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle. PMID:10757808

  14. Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

    DEFF Research Database (Denmark)

    Hagen, Lars; Kavli, Bodil; Sousa, Mirta M L

    2008-01-01

    -catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA......) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23...

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

    Science.gov (United States)

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

    2006-12-01

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

  16. The Prognostic Impact of Some Cell Cycle Regulatory Proteins in Egyptian Breast Cancer Patients

    International Nuclear Information System (INIS)

    KAMEL, A.; Mokhtar, N.; Elshaknkiry, N.; Yassin, D.; Elnahass, Y.; Zakarya, O.; Elbasmy, A.; Elmetenawy, W.

    2006-01-01

    Purpose: The particular goal of this work is to study some cell cycle regulatory proteins and their potential impact on prognosis of breast cancer; p53, cyclin D 1 and p27 are potential effectors being the major contributors to the control of the restriction (R) check point of the cell cycle. We also aimed to evaluate different techniques used to detect these cell cycle proteins. Material and Methods: Forty five breast cancer patients as well as 10 controls with non malignant pathology were assessed for cell cycle regulators each by 2 different techniques; p53 was assessed by enzyme immunoassay (EJA) and immunohistochemistry (lHC), cyclin D1 by Western Blotting (WB) and IHC and p27 by WB and me. The cut-off was calculated as the mean of the normal controls +2 SD. Patients were followed up for 4 years and their laboratory data were correlated with different clinical parameters and with other studied regulators. Results: Eighty seven percent of cases (39/45) were positive for p53 by EIA with a range from 20 to 4300, and a mean of 464±97 I pg/mg protein. By mc, 80% (24/30) of the cases showed varying degrees of positivity. Using WB, cyclin D 1 showed high expression levels above cut off values in 69% of patients (31/45) and in 67% (20/30) by me. The corresponding positive figures for p27 were 82% (37/45) and 73% (22/30) using the two techniques, respectively. No significant association was found between p53, cyclin 01 and p27 on one side and different clinical parameters as lymph node status, tumor size or presence of distant metastases on the other side. Survival was poor in patients with high p53 expression. Cyclin D1 positive cases showed comparable survival with negative cases, whereas high p27 levels favored a longer disease free survival. Conclusions: Techniques more suitable for assessment of each of these markers in our consideration were EIA for p53, WB for cyclin D1 and IHC for p27. Moreover, this study demonstrated that these markers were relevant to the

  17. Multiparameter Cell Cycle Analysis.

    Science.gov (United States)

    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.

  18. Effect of irradiation on cell cycle, cell death and expression of its related proteins in normal human oral keratinocytes

    International Nuclear Information System (INIS)

    Kang, Mi Ae; Heo, Min Suk; Lee, Sam Sun; Oh, Sung Ook; Choi, Soon Chul; Park, Tae Won; Lee, Sul Mi; Jeon, In Seong

    2003-01-01

    To investigate the radiosensitivity of the normal human oral keratinocytes (NHOK), and the effect of irradiation on cell cycle and protein expression. To evaluate the radiosensitivity of NHOK, the number of colonies and cells were counted after irradiation and the SF2 (survival fraction as 2 Gy) value, and the cell survival curve fitted on a linear-quadratic model were obtained. LDH analysis was carried out to evaluate the necrosis of NHOK at 1, 2,3, and 4 days after 2, 10, and 20 Gy irradiation. Cell cycle arrest and the induction of apoptosis were analyzed using flow cytometry at 1, 2, 3, and 4 days after 2, 10, and 20 Gy irradiation. Finally, proteins related cell cycle arrest and apoptosis were analysed by Western blot. The number of survival cell was significantly decreased in a dose-dependent manner. The cell survival curve showed SF2, α, and β values to be 0.568, 0.209, and 0.020 respectively. At 20 Gy irradiated cells showed higher optical density than the control group. After irradiation, apoptosis was not observed but G2 arrest was observed in the NHOK cells. 1 day after 10 Gy irradiation, the expression of p53 remained unchanged, the p21 WAF1/Cip1 increased and the mdm2 decreased. The expression of bax, bcl-2, cyclin B1, and cyclin D remained unchanged. These results indicate that NHOK responds to irradiation by G2 arrest, which is possibly mediated by the expression of p21 WAF1/Cip1 , and that cell necrosis occurs by high dose irradiation.

  19. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  20. Phosphorylation variation during the cell cycle scales with structural propensities of proteins.

    Directory of Open Access Journals (Sweden)

    Stefka Tyanova

    Full Text Available Phosphorylation at specific residues can activate a protein, lead to its localization to particular compartments, be a trigger for protein degradation and fulfill many other biological functions. Protein phosphorylation is increasingly being studied at a large scale and in a quantitative manner that includes a temporal dimension. By contrast, structural properties of identified phosphorylation sites have so far been investigated in a static, non-quantitative way. Here we combine for the first time dynamic properties of the phosphoproteome with protein structural features. At six time points of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels, whereas regions with predominantly regular secondary structures retain more constant phosphorylation levels. The two groups show preferences for different amino acids in their kinase recognition motifs - proline and other disorder-associated residues are enriched in the former group and charged residues in the latter. Furthermore, these preferences scale with the degree of disorderedness, from regular to irregular and to disordered structures. Our results suggest that the structural organization of the region in which a phosphorylation site resides may serve as an additional control mechanism. They also imply that phosphorylation sites are associated with different time scales that serve different functional needs.

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

    Directory of Open Access Journals (Sweden)

    Amrita A Nagle

    Full Text Available 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

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

    Science.gov (United States)

    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

  3. The cell cycle regulator CCDC6 is a key target of RNA-binding protein EWS.

    Directory of Open Access Journals (Sweden)

    Sujitha Duggimpudi

    Full Text Available Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma.

  4. The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

    International Nuclear Information System (INIS)

    Zhang Weifang; Li Jing; Kanginakudru, Sriramana; Zhao Weiming; Yu Xiuping; Chen, Jason J.

    2010-01-01

    HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.

  5. Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.

    Science.gov (United States)

    Martínez-de la Cruz, Enrique; García-Ramírez, Elpidio; Vázquez-Ramos, Jorge M; Reyes de la Cruz, Homero; López-Bucio, José

    2015-03-15

    Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

    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.

  7. Expression of cell cycle proteins according to HPV status in oral squamous cell carcinoma affecting young patients: a pilot study.

    Science.gov (United States)

    Miranda Galvis, Marisol; Freitas Jardim, Juscelino; Kaminagakura, Estela; Santos-Silva, Alan Roger; Paiva Fonseca, Felipe; Paes Almeida, Oslei; Ajudarte Lopes, Marcio; Lópes Pinto, Clóvis; Kowalski, Luiz Paulo

    2018-04-01

    Tobacco and alcohol consumption are considered the main risk factors for oral squamous cell carcinoma (OSCC); however, the role of these factors in patients younger than 40 years is controversial, so it has been suggested that genomic instability and high-risk human papillomavirus (HR-HPV) infection may be contributing factors to oral carcinogenesis at a young age. Therefore, the aim of this study was to evaluate the immunoexpression of cell cycle proteins according HPV status in OSCC affecting young patients. A tissue microarray construction based on 34 OSCC samples from young patients (factor receptor, p53, and p16 antibodies. The clinicopathologic features and the immunoexpression of all tested proteins were similar in both groups. Patients with HPV-related OSSC tended to have better cancer-specific survival (CSS; 39% vs 60% 5-y CSS), and overall survival (OS; 29.2% vs 60% 5-year OS). However, this difference was not statistically significant. No significant difference exists in the expression of cell cycle proteins studied between HR-HPV DNA-positive and HR-HPV DNA-negative OSCC affecting young patients. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Immunohistochemical study of the expression of cell cycle regulating proteins at different stages of bladder cancer

    DEFF Research Database (Denmark)

    Primdahl, Hanne; von der Maase, Hans; Sørensen, Flemming Brandt

    2002-01-01

    ; group 1, 23 patients with recurrent stage Ta (non-invasive) tumors; group 2, 22 patients presenting at their first admission with T2-4 (muscle invasive) tumors; group 3, 24 patients who experienced disease progression from Ta or T1 (invasive in connective tissue) to a higher stage......PURPOSE: The cell cycle is known to be deregulated in cancer. We therefore analyzed the expression of the cell cycle related proteins p21, p27, p16, Rb, and L-myc by immunohistochemical staining of bladder tumors.METHODS: The tissue material consisted of bladder tumors from three groups of patients......(kip1) ( P=0.03), Rb ( P=0.00002), and L-myc ( P=0.00000007) in muscle invasive tumors compared to noninvasive tumors. Tumors presenting as muscle invasive at first diagnosis had significantly lower levels of p16/CDKN2A ( P=0.01) when compared to muscle invasive tumors that followed Ta or T1 precursor...

  9. Cell cycle, differentiation and tissue-independent expression of ribosomal protein L37.

    Science.gov (United States)

    Su, S; Bird, R C

    1995-09-15

    A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level of hG1.16 during different cell-cycle phases was determined by Northern-blot analysis of cells synchronized by double-thymidine block and serum deprivation/refeeding. hG1.16 was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level of hG1.16 did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post-mitotic. Examination of other tissues revealed that the relative expression level of hG1.16 was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down-modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter-species Northern-blot analysis revealed that hG1.16 was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis of hG1.16 revealed a high level of similarity to rat ribosomal protein L37, identifying hG1.16 as a new member of this multigene family. The deduced amino acid sequence of hG1.16 was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predicted M(r) of 11,065). hG1.16 protein has a single C2-C2 zinc-finger-like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence of hG1.16, unique bovine and rat cDNAs were also isolated by 5'-rapid-amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed

  10. Cell cycle regulatory proteins and miRNAs in premalignant lesions and breast cancer

    OpenAIRE

    Björner, Sofie

    2013-01-01

    Early diagnosis and reliable prognosis and treatment prediction of breast cancer will ultimately lead to a decreased mortality rate. This can be achieved by identification of prognostic and treatment predictive biomarkers, and by understanding the mechanisms behind early changes in the breast. The cell cycle is a closely controlled process, involving multiple components with regulation on several levels. Loss of adequate cell proliferation control and cell cycle regulation is one of the ma...

  11. DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA.

    Science.gov (United States)

    Matsson, L

    2001-12-01

    A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F'(ξ) =- (κ/2) ξ(1 - ξ)(2 - ξ)between thepre-replication complexes (pre-RCs) boundby the origins in DNA, ξ = ϕ/N being the relativedisplacement of pre-RCs, ϕ the number of pre-RCs, N the number of replicons to be replicated,and κ the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the thresholdϕ = N by a switch ofsign of F''(ξ), fromattraction (-) and assembly in the G(1) phase (0force at ϕ = 2N, from repulsion inS phase back to attraction in G(2), when all primed replicons havebeen duplicated once. F'(0) = 0corresponds to a resting cell in theabsence of driving force at ϕ= 0. The model thus ensures that the DNAcontent in G(2) cells is exactlytwice that of G(1) cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.

  12. Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

    International Nuclear Information System (INIS)

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-01-01

    A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

  13. Uncovering SUMOylation Dynamics during Cell-Cycle Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein

    DEFF Research Database (Denmark)

    Schimmel, Joost; Eifler, Karolin; Sigurdsson, Jón Otti

    2014-01-01

    Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell......-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation. SUMOylation of FoxM1 peaks during G2 and M...... relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression....

  14. Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates

    Directory of Open Access Journals (Sweden)

    P. Borelli

    2009-06-01

    Full Text Available Protein energy malnutrition (PEM is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group were submitted to PEM with a low-protein diet (4% or were fed a control diet (20% protein ad libitum. When the experimental group had lost about 20% of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

  15. UNcleProt (Universal Nuclear Protein database of barley): The first nuclear protein database that distinguishes proteins from different phases of the cell cycle

    Czech Academy of Sciences Publication Activity Database

    Blavet, Nicolas; Uřinovská, J.; Jeřábková, Hana; Chamrád, I.; Vrána, Jan; Lenobel, R.; Beinhauer, D.; Šebela, M.; Doležel, Jaroslav; Petrovská, Beáta

    2017-01-01

    Roč. 8, č. 1 (2017), s. 70-80 ISSN 1949-1034 R&D Projects: GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : cicer-arietinum l. * rice oryza-sativa * chromatin-associated protein s * proteomic analysis * mitotic chromosomes * dehydration * localization * chickpea * network * phosphoproteome * barley * cell cycle * database * flow-cytometry * localization * mass spectrometry * nuclear proteome * nucleus Subject RIV: CE - Biochemistry OBOR OECD: Cell biology Impact factor: 2.387, year: 2016

  16. Endo- and exocytic rate constants for spontaneous and protein kinase C-activated T cell receptor cycling

    DEFF Research Database (Denmark)

    Menné, Charlotte; Møller Sørensen, Tine; Siersma, Volkert

    2002-01-01

    To determine the rate constants of spontaneous and activated TCR cycling, we examined TCR endo- and exocytosis in the human T cell line Jurkat by three different methods. Using a simple kinetic model for TCR cycling and non-linear regression analyses, we found that the spontaneous endocytic rate...... constant of the TCR was low (approximately 0.012 min(-1)) whereas the spontaneous exocytic rate constant was similar to that of other cycling receptors (approximately 0.055 min(-1)). Following protein kinase C activation (PKC) the endocytic rate constant was increased tenfold (to approximately 0.128 min(-1......)) whereas the exocytic rate constant was unaffected. Thus, the TCR becomes a rapidly cycling receptor with kinetics similar to classical cycling receptors subsequent to PKC activation. This results in a reduction of the half-life of cell surface expressed TCR from approximately 58 to 6 min and allows rapid...

  17. Effect of zeranol on expression of apoptotic and cell cycle proteins in murine placentae

    International Nuclear Information System (INIS)

    Wang, Yanfei; Li, Lu; Wang, C.C.; Leung, Lai K.

    2013-01-01

    Highlights: • Zeranol administered at 1–100 mg/kg/day demonstrated an increasing trend of fetal resorption and early delivery in mice. • Placental expression of Cdk2 and 4, Cyclin D1 and Bcl-xL were reduced mostly in mice treated with ≥10 mg zeranol/kg/day. • Increased pErk-1 and 2 were also observed in the placentae of zeranol-treated mice. - Abstract: Mycotoxins are chemicals produced by fungus and many of them are toxic to humans. Zeranol is a mycotoxin used to promote growth in cattle in North America; yet such a practice draws concern about the residual compound in meat in European countries. In the present study, the toxicity of zeranol was tested in a mouse model for reproduction. Pregnant ICR mice were given p.o. daily doses of zeranol at 0, 1, 10, 100 mg/kg for 4 days (from E13.5 to E16.5). Increased rates of fetal resorption at late gestation (E17.5) and preterm birth (< E18.5) were observed in mice treated with zeranol. The apparent factors causing these perinatal conditions were subsequently investigated. Perturbation of cell death or proliferation-related proteins might deter the growth and maintenance of the placentae, and the subsequent fetal resorption and preterm birth. Placental tissue isolated from pregnant mice at E17.5 showed that the expressions of Cdk2 and 4, Cyclin D1 and Bcl-xL were reduced in zeranol-treatment groups. The downregulations might signify growth or maintenance failure in the placentae. Furthermore, reduction in the signaling proteins Erk-1/2 in the placentae could trigger the decrease in the cell cycle/apoptosis proteins. In addition, relaxin is associated with preterm labor. An increase in placental Relaxin-1 expression could also contribute to early delivery in this study. Result of the current study suggested that exposure to zeranol might introduce adverse effect in pregnancy

  18. HNRNPLL stabilizes mRNAs for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

    Science.gov (United States)

    Sakuma, Keiichiro; Sasaki, Eiichi; Kimura, Kenya; Komori, Koji; Shimizu, Yasuhiro; Yatabe, Yasushi; Aoki, Masahiro

    2018-06-05

    HNRNPLL (heterogeneous nuclear ribonucleoprotein L-like), an RNA-binding protein that regulates alternative splicing of pre-mRNAs, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here we show that HNRNPLL promotes cell cycle progression and hence proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed by three-fold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3, and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 hours of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3, and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3, and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNAs encoding regulators of DNA replication and promotes colorectal cancer cell proliferation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R.

    Science.gov (United States)

    Li, Ge; Park, Hyeon U; Liang, Dong; Zhao, Richard Y

    2010-07-07

    Cell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive. To examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV. These data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.

  20. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    Directory of Open Access Journals (Sweden)

    Liang Dong

    2010-07-01

    Full Text Available Abstract Background Cell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive. Results To examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU and ultraviolet light (UV also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV. Conclusions These data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.

  1. Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

    Science.gov (United States)

    Emanuele, Michael J; Ciccia, Alberto; Elia, Andrew E H; Elledge, Stephen J

    2011-06-14

    The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

  2. Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression*

    Science.gov (United States)

    Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

    2011-01-01

    Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. PMID:21543332

  3. Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.

    Science.gov (United States)

    Bruni, R; Roizman, B

    1998-11-01

    The herpes simplex virus 1 infected cell protein 22 (ICP22), the product of the alpha22 gene, is a nucleotidylylated and phosphorylated nuclear protein with properties of a transcriptional factor required for the expression of a subset of viral genes. Here, we report the following. (i) ICP22 interacts with a previously unknown cellular factor designated p78 in the yeast two-hybrid system. The p78 cDNA encodes a polypeptide with a distribution of leucines reminiscent of a leucine zipper. (ii) In uninfected and infected cells, antibody to p78 reacts with two major bands with an apparent Mr of 78,000 and two minor bands with apparent Mrs of 62, 000 and 55,000. (ii) p78 also interacts with ICP22 in vitro. (iii) In uninfected cells, p78 was dispersed largely in the nucleoplasm in HeLa cells and in the nucleoplasm and cytoplasm in HEp-2 cells. After infection, p78 formed large dense bodies which did not colocalize with the viral regulatory protein ICP0. (iv) Accumulation of p78 was cell cycle dependent, being highest very early in S phase. (v) The accumulation of ICP22 in synchronized cells was highest in early S phase, in contrast to the accumulation of another protein, ICP27, which was relatively independent of the cell cycle. (vi) In the course of the cell cycle, ICP22 was transiently modified in an aberrant fashion, and this modification coincided with expression of p78. The results suggest that ICP22 interacts with and may be stabilized by cell cycle-dependent proteins.

  4. Lamprey immune protein-1 (LIP-1) from Lampetra japonica induces cell cycle arrest and cell death in HeLa cells.

    Science.gov (United States)

    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 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.

  5. Delayed expression of cell cycle proteins contributes to astroglial scar formation and chronic inflammation after rat spinal cord contusion

    Directory of Open Access Journals (Sweden)

    Wu Junfang

    2012-07-01

    Full Text Available Abstract Background Traumatic spinal cord injury (SCI induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model. Methods Adult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury. Results Immunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins − including cyclin D1 and E, CDK4, E2F5 and PCNA − for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX − a key component of NADPH oxidase − up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16. Conclusions These data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar

  6. Cell-cycle protein expression in a population-based study of ovarian and endometrial cancers

    Directory of Open Access Journals (Sweden)

    Ashley S. Felix

    2015-02-01

    Full Text Available Aberrant expression of cyclin-dependent kinase (CDK inhibitors is implicated in the carcinogenesis of many cancers, including ovarian and endometrial cancers. We examined associations between CDK inhibitor expression, cancer risk factors, tumor characteristics, and survival outcomes among ovarian and endometrial cancer patients enrolled in a population-based case control study. Expression (negative vs. positive of three CDK inhibitors (p16, p21, p27 and ki67 was examined with immunohistochemical staining of tissue microarrays. Logistic regression was used to estimate adjusted odds ratios (ORs and 95% confidence intervals (CIs for associations between biomarkers, risk factors, and tumor characteristics. Survival outcomes were available for ovarian cancer patients and examined using Kaplan-Meier plots and Cox proportional hazards regression. Among ovarian cancer patients (n=175, positive p21 expression was associated with endometrioid tumors (OR=12.22, 95% CI=1.45-102.78 and higher overall survival (log-rank p=0.002. In Cox models adjusted for stage, grade, and histology, the association between p21 expression and overall survival was borderline significant (hazard ratio=0.65, 95% CI=0.42-1.05. Among endometrial cancer patients (n=289, positive p21 expression was inversely associated with age (OR ≥ 65 years of age=0.25, 95% CI=0.07-0.84 and current smoking status (OR: 0.33, 95% CI 0.15, 0.72 compared to negative expression. Our study showed heterogeneity in expression of cell-cycle proteins associated with risk factors and tumor characteristics of gynecologic cancers. Future studies to assess these markers of etiological classification and behavior may be warranted.

  7. Altered expression of the cell cycle regulatory protein cyclin D1 in the rat dentate gyrus after adrenalectomy-induced granular cell lass

    NARCIS (Netherlands)

    Postigo, JA; Van der Werf, YD; Korf, J; Krugers, HJ

    1998-01-01

    The loss of dentate gyrus (DG) granular cells after removal of the rat adrenal glands (ADX) is mediated by a process that is apoptotic in nature. The present study was initiated to compare changes in the immunocytochemical distribution of the cell-cycle regulatory protein cyclin D1, which has been

  8. Overexpression of Zwint predicts poor prognosis and promotes the proliferation of hepatocellular carcinoma by regulating cell-cycle-related proteins

    Directory of Open Access Journals (Sweden)

    Ying H

    2018-02-01

    Full Text Available Hanning Ying,1,2 Zhiyao Xu,3 Mingming Chen,1,2 Senjun Zhou,1,2 Xiao Liang,1,2 Xiujun Cai1,2 1Department of General Surgery, 2Key Laboratory of Endoscopic Technique Research of Zhejiang Province, 3Central Lab of Biomedical Research Center, School of Medicine, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China Introduction: Zwint, a centromere-complex component required for the mitotic spindle checkpoint, has been reported to be overexpressed in different human cancers, but it has not been studied in human hepatocellular carcinoma (HCC.Materials and methods: The role of Zwint in hepatocellular carcinoma cell proliferation capacities was evaluated by using cell counting kit-8 (CCK8, flow cytometry, clone formation and tumor formation assay in nude mice. Western blot analysis and qPCR assay were performed to assess Zwint interacting with cell-cycle-related proteins.Results: We report that ZWINT mRNA and protein expression were upregulated in HCC samples and cell lines. An independent set of 106 HCC-tissue pairs and corresponding noncancerous tissues was evaluated for Zwint expression using immunohistochemistry, and elevated Zwint expression in HCC tissues was significantly correlated with clinicopathological features, such as tumor size and number. Kaplan–Meier survival and Cox regression analysis revealed that high expression of Zwint was correlated with poor overall survival and a greater tendency for tumor recurrence. Ectopic expression of Zwint promoted HCC-cell proliferation, and Zwint expression affected the expression of several cell-cycle proteins, including PCNA, cyclin B1, Cdc25C and CDK1.Conclusion: Our findings suggest that upregulation of Zwint may contribute to the progression of HCC and may be a prognostic biomarker and potential therapeutic target for treating HCC. Keywords: Zwint, hepatocellular carcinoma, HCC, prognosis, cell proliferation, cell cycle

  9. 17-Allylamino-17-demethoxygeldanamycin induces downregulation of critical Hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells

    International Nuclear Information System (INIS)

    Karkoulis, Panagiotis K; Stravopodis, Dimitrios J; Margaritis, Lukas H; Voutsinas, Gerassimos E

    2010-01-01

    17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin antibiotic, specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in cellular signaling. In this study, we have investigated the effect of 17-AAG on the regulation of Hsp90-dependent signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cell lines. We have used MTT-based assays, FACS analysis, Western blotting, semi-quantitative RT-PCR, immunocytochemistry and scratch-wound assay in RT4, RT112 and T24 human urinary bladder cancer cell lines. We have demonstrated that, upon 17-AAG treatment, bladder cancer cells are arrested in the G1 phase of the cell cycle and eventually undergo apoptotic cell death in a dose-dependent manner. Furthermore, 17-AAG administration was shown to induce a pronounced downregulation of multiple Hsp90 protein clients and other downstream effectors, such as IGF-IR, Akt, IKK-α, IKK-β, FOXO1, ERK1/2 and c-Met, resulting in sequestration-mediated inactivation of NF-κB, reduced cell proliferation and decline of cell motility. In total, we have clearly evinced a dose-dependent and cell type-specific effect of 17-AAG on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of multiple Hsp90 clients and subsequent disruption of signaling integrity

  10. Arabidopsis EST1/SMG7-like protein is a novel regulator of meiotic cell cycle progression

    Czech Academy of Sciences Publication Activity Database

    Riehs, N.; Akimcheva, S.; Bulánková, P.; Idol, R.; Široký, Jiří; Shippen, D.; Schweizer, D.; Říha, K.

    2007-01-01

    Roč. 274, č. 1 (2007), s. 71 ISSN 1742-464X. [32nd FEBS Congress - Molecular Machines. 07.07.2007-12.07.2007, Vienna] R&D Projects: GA ČR(CZ) GA522/06/0380 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : meiosis * Arabidopsis * cell cycle Subject RIV: BO - Biophysics

  11. A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks.

    Directory of Open Access Journals (Sweden)

    Teeraphan Laomettachit

    Full Text Available To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a "standard component" modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with "standard components" can capture in quantitative detail many essential properties of cell cycle control in budding yeast.

  12. Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

    Science.gov (United States)

    Qiao, F; Moss, A; Kupfer, G M

    2001-06-29

    Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.

  13. Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

    Science.gov (United States)

    1999-07-01

    centrifugal elutriation, with a purity of -80% as shown by micro - ethyl acetate and separated on thin-layer chromatography plates (Whatman, scopic...Spain. (2) Servicio Bioquimica, Hosp. La Paz. Madrid. ICER protein is elevated in mHR6b-/- (murine Rad6B) fibroblasts Spain. and in human cells

  14. p53 functions as a cell cycle control protein in osteosarcomas.

    Science.gov (United States)

    Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

    1990-11-01

    Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

  15. p53 functions as a cell cycle control protein in osteosarcomas.

    Science.gov (United States)

    Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

    1990-01-01

    Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae. Images PMID:2233717

  16. A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

    Science.gov (United States)

    Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-12-28

    Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. 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.

    Science.gov (United States)

    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.

  18. 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

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

    2012-09-10

    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 cell 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.

  19. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    Science.gov (United States)

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  20. Analysis of protein incorporation of radioactive isotopes in the Chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Pipkin, J.L.; Anson, J.F.; Hinson, W.G.; Schol, H.; Burns, E.R.; Casciano, D.A.

    1986-03-01

    The patterns of (3H)-leucine and (32P)-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of (3H)-leucine- and (32P)-phosphate-labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.

  1. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    International Nuclear Information System (INIS)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-01-01

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  2. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  3. The centrosome protein NEDD1 as a potential pharmacological target to induce cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Etievant Chantal

    2009-02-01

    Full Text Available Abstract Background NEDD1 is a protein that binds to the gamma-tubulin ring complex, a multiprotein complex at the centrosome and at the mitotic spindle that mediates the nucleation of microtubules. Results We show that NEDD1 is expressed at comparable levels in a variety of tumor-derived cell lines and untransformed cells. We demonstrate that silencing of NEDD1 expression by treatment with siRNA has differential effects on cells, depending on their status of p53 expression: p53-positive cells arrest in G1, whereas p53-negative cells arrest in mitosis with predominantly aberrant monopolar spindles. However, both p53-positive and -negative cells arrest in mitosis if treated with low doses of siRNA against NEDD1 combined with low doses of the inhibitor BI2536 against the mitotic kinase Plk1. Simultaneous reduction of NEDD1 levels and inhibition of Plk1 act in a synergistic manner, by potentiating the anti-mitotic activity of each treatment. Conclusion We propose that NEDD1 may be a promising target for controlling cell proliferation, in particular if targeted in combination with Plk1 inhibitors.

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

    Science.gov (United States)

    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.

  5. Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells.

    Science.gov (United States)

    Boutant, Emmanuel; Fitterer, Chantal; Ritzenthaler, Christophe; Heinlein, Manfred

    2009-10-01

    Cell-to-cell movement of Tobacco mosaic virus (TMV) involves the interaction of virus-encoded 30-kDa movement protein (MP) with microtubules. In cells behind the infection front that accumulate high levels of MP, this activity is reflected by the formation of stabilized MP/microtubule complexes. The ability of MP to bind along and stabilize microtubules is conserved upon expression in mammalian cells. In mammalian cells, the protein also leads to inhibition of mitosis and cell division through a microtubule-independent process correlated with the loss of centrosomal gamma-tubulin and of centrosomal microtubule-nucleation activity. Since MP has the capacity to interact with plant factors involved in microtubule nucleation and dynamics, we used inducible expression in BY-2 cells to test whether MP expression inhibits mitosis and cell division also in plants. We demonstrate that MP:GFP associates with all plant microtubule arrays and, unlike in mammalian cells, does not interfere with mitosis. Thus, MP function and the interaction of MP with factors of the cytoskeleton do not entail an inhibition of mitosis in plants. We also report that the protein targets primary plasmodesmata in BY-2 cells immediately upon or during cytokinesis and that the accumulation of MP in plasmodesmata occurs in the presence of inhibitors of the cytoskeleton and the secretory pathway.

  6. 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)].

    Science.gov (United States)

    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.

  7. Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs

    International Nuclear Information System (INIS)

    Savas, Sevtap; Ozcelik, Hilmi

    2005-01-01

    Phosphorylation is a reversible post-translational modification that affects the intrinsic properties of proteins, such as structure and function. Non-synonymous single nucleotide polymorphisms (nsSNPs) result in the substitution of the encoded amino acids and thus are likely to alter the phosphorylation motifs in the proteins. In this study, we used the web-based NetPhos tool to predict candidate nsSNPs that either introduce or remove putative phosphorylation sites in proteins that act in DNA repair and cell cycle pathways. Our results demonstrated that a total of 15 nsSNPs (16.9%) were likely to alter the putative phosphorylation patterns of 14 proteins. Three of these SNPs (CDKN1A-S31R, OGG1-S326C, and XRCC3-T241M) have already found to be associated with altered cancer risk. We believe that this set of nsSNPs constitutes an excellent resource for further molecular and genetic analyses. The novel systematic approach used in this study will accelerate the understanding of how naturally occurring human SNPs may alter protein function through the modification of phosphorylation mechanisms and contribute to disease susceptibility

  8. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Karin [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden); Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Grawé, Jan [Department of Genetics and Pathology, Uppsala University, Uppsala 75185 (Sweden); McKinney-Freeman, Shannon L. [Department of Hematology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Daley, George Q. [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Welsh, Michael, E-mail: michael.welsh@mcb.uu.se [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden)

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via

  9. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    International Nuclear Information System (INIS)

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-01-01

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  10. Piperlongumine decreases cell proliferation and the expression of cell cycle-associated proteins by inhibiting Akt pathway in human lung cancer cells.

    Science.gov (United States)

    Seok, Jin Sil; Jeong, Chang Hee; Petriello, Michael C; Seo, Han Geuk; Yoo, Hyunjin; Hong, Kwonho; Han, Sung Gu

    2018-01-01

    Piperlongumine (PL) is an alkaloid of a pepper plant found in Southeast Asia. PL is known to induce selective toxicity towards a variety of cancer cell types. To explore the possible anti-lung cancer effects of PL, A549 cells were treated with PL (0-40 μM) for 24 h. Alterations in the expression of cell cycle-associated proteins (cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and retinoblastoma (Rb)) and intracellular signaling molecules (extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, p38 and nuclear factor-κB (NF-κB)) were examined in cells following treatment of PL using Western blot analysis. Results showed that proliferation of cells were significantly decreased by PL in a dose-dependent manner. Flow cytometry results demonstrated increased number of cells in G1 phase in PL (40 μM)-treated group. Reactive oxygen species was significantly increased in cells treated with PL at 20-40 μM. The expression of cyclin D1, CDK4, CDK6 and p-Rb were markedly decreased in cells treated with PL at 40 μM. Treatment of cells with PL suppressed phosphorylation of Akt but increased ERK1/2 phosphorylation. Treatment of PL significantly decreased nuclear translocation of NF-κB p65 in cells. These results suggest that PL possesses antiproliferative properties in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

    Science.gov (United States)

    Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

    2018-03-02

    Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

  12. Cell growth and division cycle

    International Nuclear Information System (INIS)

    Darzynkiewicz, Z.

    1986-01-01

    The concept of the cell cycle in its present form was introduced more than three decades ago. Studying incorporation of DNA precursors by autoradiography, these authors observed that DNA synthesis in individual cells was discontinuous and occupied a discrete portion of the cell life (S phase). Mitotic division was seen to occur after a certain period of time following DNA replication. A distinct time interval between mitosis and DNA replication was also apparent. Thus, the cell cycle was subdivided into four consecutive phases, G/sub 1/, S, G/sub 2/, and M. The G/sub 1/ and G/sub 2/ phases represented the ''gaps'' between mitosis and the start of DNA replication, and between the end of DNA replication and the onset of mitosis, respectively. The cell cycle was defined as the interval between the midpoint of mitosis and the midpoint of the subsequent mitosis of the daughter cell(s). The authors' present knowledge on the cell cycle benefited mostly from the development of four different techniques: autoradiography, time-lapse cinematography, cell synchronization and flow cytometry. Of these, autoradiography has been the most extensively used, especially during the past two decades. By providing a means to analyse incorporation of precursors of DNA, RNA or proteins by individual cells and, in combination with various techniques of cell synchronization, autoradiography yielded most of the data fundamental to the current understanding of the cell cycle-related phenomena. Kinetics of cell progression through the cell cycle could be analysed in great detail after development of such sophisticated autoradiographic approaches as measurements of the fraction of labeled mitoses (''FLM curves'') or multiple sequential cell labelling with /sup 3/H- and /sup 14/C-TdR

  13. Korean Byungkyul - Citrus platymamma Hort.et Tanaka flavonoids induces cell cycle arrest and apoptosis, regulating MMP protein expression in Hep3B hepatocellular carcinoma cells.

    Science.gov (United States)

    Hong, Gyeong Eun; Lee, Ho Jeong; Kim, Jin A; Yumnam, Silvia; Raha, Suchismita; Venkatarame Gowda Saralamma, Venu; Heo, Jeong Doo; Lee, Sang Joon; Kim, Eun Hee; Won, Chun Kil; Kim, Gon Sup

    2017-02-01

    Citrus platymamma Hort.et Tanaka is an indigenous fruit of Jeju island in Korea. In this study the bioactivity of C. platymamma flavonoids were evaluated on human hepatoma Hep3B cell lines. Eleven flavonoids were identified from the peels of C. platymamma Hort.et Tanaka through high-performance liquid chromatography-Tandem mass spectrometry and the anticancer effect of these C. platymamma flavonoids on human hepatoma Hep3B were studied. Chromatin condensation was observed in Hep3B cells treated with C. platymamma flavonoids. DNA fragmentation was confirmed through agarose gel electrophoresis and TUNEL assay. An increase in the total apoptotic cells and G2/M cell cycle arrest with decreased protein expression of CDC25C, CDK1, cyclin B1 and p21 were observed in Hep3B cells treated with flavonoids of C. platymamma. Further, protein expression of Bcl-XL, Bax, caspase-3 and -9 were also modulated by C. platymamma flavonoids treatment indicating that cell death is through intrinsic apoptotic pathway. Moreover, C. platymamma flavonoids also regulated the phosphorylation of MAPKs, PI3K, and Akt in Hep3B cells. Relevant to inhibiting metastasis, C. platymamma treatment reduced wound closure of Hep3B cells and the protein expression of matrix metalloproteinase-2 and -9 were reduced in C. platymamma treated cells. The results show that C. platymamma flavonoids induce cell cycle arrest and apoptosis following activation of MAPKs and suppression of PI3K/Akt pathway which eventually inhibits cell migration in Hep3B cells. The finding provides evidence on biochemical activities of C. platymamma Hort.et Tanaka, which would be an essential agent for hepatocellular carcinoma (HCC) treatment.

  14. Transfer-messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces

    DEFF Research Database (Denmark)

    Barends, Sharief; Zehl, Martin; Bialek, Sylwia

    2010-01-01

    coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A...... functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress....

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

    Science.gov (United States)

    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

  16. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    International Nuclear Information System (INIS)

    Zhong, Wenbin; Zhou, You; Li, Jiwei; Mysore, Raghavendra; Luo, Wei; Li, Shiqian; Chang, Mau-Sun; Olkkonen, Vesa M.; Yan, Daoguang

    2014-01-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle

  17. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Wenbin [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Zhou, You [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Li, Jiwei [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Mysore, Raghavendra [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Luo, Wei; Li, Shiqian [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Chang, Mau-Sun [Institute of Biochemical Sciences, National Taiwan University, No. 1, Taipei, Taiwan (China); Olkkonen, Vesa M. [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Yan, Daoguang, E-mail: tydg@jnu.edu.cn [Department of Biotechnology, Jinan University, Guangzhou 510632 (China)

    2014-04-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle.

  18. Expression of hypoxia-inducible factor-1α and cell cycle proteins in invasive breast cancer are estrogen receptor related

    International Nuclear Information System (INIS)

    Bos, Reinhard; Diest, Paul J van; Groep, Petra van der; Shvarts, Avi; Greijer, Astrid E; Wall, Elsken van der

    2004-01-01

    The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. Previous studies showed that concentrations of its subunit HIF-1α, as a surrogate for HIF-1 activity, are increased during breast carcinogenesis and can independently predict prognosis in breast cancer. During carcinogenesis, the cell cycle is progressively deregulated, and proliferation rate is a strong prognostic factor in breast cancer. In this study we undertook a detailed evaluation of the relationships between HIF-1α and cell cycle-associated proteins. In a representative estrogen receptor (ER) group of 150 breast cancers, the expression of HIF-1α, vascular endothelial growth factor, the ER, HER-2/neu, Ki-67, cyclin A, cyclin D 1 , p21, p53, and Bcl-2 was investigated by immunohistochemistry. High concentrations (5% or more) of HIF-1α were associated with increased proliferation as shown by positive correlations with Ki-67 (P < 0.001) and the late S–G2-phase protein cyclin A (P < 0.001), but not with the G1-phase protein cyclin D 1 . High HIF-1α concentrations were also strongly associated with p53 positivity (P < 0.001) and loss of Bcl-2 expression (P = 0.013). No association was found between p21 and HIF-1α (P = 0.105) in the whole group of patients. However, the subgroup of ER-positive cancers was characterized by a strong positive association between HIF-1α and p21 (P = 0.023), and HIF-1α lacked any relation with proliferation. HIF-1α overexpression is associated with increased proliferation, which might explain the adverse prognostic impact of increased concentrations of HIF-1α in invasive breast cancer. In ER-positive tumors, HIF-1α is associated with p21 but not against proliferation. This shows the importance of further functional analysis to unravel the role of HIF-1 in late cell cycle progression, and the link between HIF-1, p21, and ER

  19. Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization

    International Nuclear Information System (INIS)

    Waldhuber, Megan G.; Bateson, Michael; Tan, Judith; Greenway, Alison L.; McPhee, Dale A.

    2003-01-01

    The human immunodeficiency virus type 1 (HIV-1) Vpr protein is known to arrest the cell cycle in G 2 /M and induce apoptosis following arrest. The functions of Vpr relative to its location in the cell remain unresolved. We now demonstrate that the location and function of Vpr are dependent on the makeup of fusion proteins and that the functions of G 2 /M arrest and apoptosis are separable. Using green fluorescence protein mutants (EGFP or EYFP), we found that fusion at either the N- or C-terminus compromised the ability of Vpr to arrest cell cycling, relative to that of His-Vpr or wild-type protein. Additionally, utilizing the ability to specifically identify cells expressing the fusion proteins, we confirm that Vpr can induce apoptosis, but appears to be independent of cell-cycle arrest in G 2 /M. Both N- and C-terminal Vpr/EYFP fusion proteins induced apoptosis but caused minimal G 2 /M arrest. These studies with Vpr fusion proteins indicate that the functions of Vpr leading to G 2 /M arrest and apoptosis are separable and that fusion of Vpr to EGFP or EYFP affected the localization of the protein. Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr

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

    Directory of Open Access Journals (Sweden)

    Xiaotong Li

    2018-03-01

    Full Text Available 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.

  1. Hepatitis C virus core protein expression leads to biphasic regulation of the p21 cdk inhibitor and modulation of hepatocyte cell cycle

    International Nuclear Information System (INIS)

    Nguyen, Hau; Mudryj, Maria; Guadalupe, Moraima; Dandekar, Satya

    2003-01-01

    Hepatitis C virus (HCV) Core protein is implicated in viral pathogenesis by the modulation of hepatocyte gene expression and function. To determine the effect of Core protein on the cell-cycle control of hepatocytes, a HepG2 cell line containing a Flag-tagged Core under the control of an inducible promoter was generated. Initial Core protein expression included the presence of unprocessed (191 aa) and processed (173 aa) forms of the Core proteins with the processed form becoming dominant later. Expression of the 191 aa form of Core protein corresponded to an increase in the expression of the p21, a decrease in cdk2-dependent kinase activity, and a decrease in the percentage of cells in S-phase along with an accumulation of cells in the G 0 /G 1 phase of the cell cycle. As the processed form accumulated, the p21 levels started to decline, suggesting that Core protein regulates p21 expression in a biphasic manner. These findings implicate Core protein in potentially modulating hepatocyte cell cycle differentially in the early stages of infection through biphasic regulation of p21 cdk kinase inhibitor

  2. Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

    1980-01-01

    A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

  3. A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

    Science.gov (United States)

    Sellers, W R; Rodgers, J W; Kaelin, W G

    1995-01-01

    An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

  4. Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

    Science.gov (United States)

    Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

    2012-04-15

    Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more

  5. Roles of Rad51 protein in homologous recombination in mammalian cells: relation with repair, replication and cell cycle

    International Nuclear Information System (INIS)

    Lambert, S.

    2001-01-01

    Homologous recombination (HR) is a fundamental process, allowing a faithful repair. In mammalian, MmRAD51, which is the homologue of Saccharomyces cerevisiae ScRAD51 key protein for HR, is an essential gene. This work is based on the characterisation of viable hyper and hypo-recombinant cell lines specifically affected in the Rad51 pathway. By expressing wild type and dominant negative forms of MmRad51, we demonstrated that Rad51 pathway participates to the repair by HR to induced DNA damages. However, inhibition of the Rad 51 pathway does not affect cell viability, spontaneously or after irradiation, whereas, radiation induced HR is inhibited. In the presence of DNA damages during late S and G2/M phase, inhibition of Rad51 pathway induced chromosomal aberrations, leading to a transient arrest in mitosis. This arrest is associated with an increased of cell death. However, a fraction of cells can escape from this transient arrest by forming tetraploid cells, associated with an absence of chromalid separation. Thus, in response to impaired Rad51 pathway, mitotic checkpoints seems to play an essential role. In line with this, we showed that the essential function of Rad51 is p53-dependent, which is in agreement with the role of p53 in tetraploidy inhibition. Our results suggest that the Rad51 protein could participate to the control of mitotic checkpoints and thus to the maintenance of genetic stability. This function could involve other Rad51 partners such as the tumour suppressors BRCA1, BRCA2 and p53. (author) [fr

  6. Origins of the protein synthesis cycle

    Science.gov (United States)

    Fox, S. W.

    1981-01-01

    Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

  7. p53 functions as a cell cycle control protein in osteosarcomas.

    OpenAIRE

    Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

    1990-01-01

    Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfect...

  8. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    DEFF Research Database (Denmark)

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA repli...

  9. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    International Nuclear Information System (INIS)

    Jeong, Jin Boo; Jeong, Hyung Jin

    2010-01-01

    Research highlights: → 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. → 2M4VP inhibited hyper-phosphorylation of Rb protein. → 2M4VP induced cell cycle arrest from G1 to S. → 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. → 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  10. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  11. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    International Nuclear Information System (INIS)

    Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C.

    2006-01-01

    Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G 1 /S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the

  12. F-box protein FBXO31 is a dedicated checkpoint protein to facilitate cell cycle arrest through activation of regulators in radiation induced DNA damage

    International Nuclear Information System (INIS)

    Santra, Manas Kumar

    2017-01-01

    In response to radiation-induced DNA damage, eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Previous study showed that induction of G1 arrest in response to radiation induced DNA damage is minimally a two-step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability. We elucidated the molecular mechanism of slow and fast response of radiation induced DDR. We showed that FBXO31, a member of F-box family proteins, plays important role in DDR induced by ionizing radiation. We show that FBXO31 is responsible for promoting MDM2 degradation following radiation. FBXO31 interacts with and directs the degradation of MDM2 in ATM dependent phosphorylation of MDM2. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage

  13. Effects of low dose radiation on tumor apoptosis, cell cycle progression and changes of apoptosis-related protein bcl-2 in tumor-bearing mice

    International Nuclear Information System (INIS)

    Yu Hongsheng; Fei Conghe; Shen Fangzhen; Liang Jun

    2003-01-01

    Objective: To study the effect of low dose radiation (LDR) on tumor apoptosis, cell cycle progression and changes of apoptosis-related protein bcl-2 in tumor-bearing mice. Methods: Kunming stain male mice were implanted with S180 sarcoma cells in the left inguen subcutaneously as an in situ experimental animal model. Seven days after implantation, the mice were given 75 mGy whole-body γ-irradiation. At 24 and 48 h after irradiation, all mice were sacrificed to measure the tumor volume, and tumor cell apoptosis, cell cycle progression were analyzed by flow cytometry. The expression of apoptosis-related protein bcl-2 and the apoptotic rate of tumor cells were observed by immunohistochemistry and electron microscopy. Results: Tumor growth was significantly slowed down after LDR (P 1 phase and the expression of bcl-2 protein decreased at 24 h. Apoptotic rate of tumor cells increased significantly at 48 h after LDR. Conclusion: LDR could cause a G 1 -phase arrest and increase the apoptosis of tumor cells through the low level of apoptosis-related protein bcl-2 in the tumor-bearing mice. The organized immune function and anti-tumor ability are markedly increased after LDR. The study provides practical evidence of clinical application to cancer treatment

  14. Sodium arsenite alters cell cycle and MTHFR, MT1/2, and c-Myc protein levels in MCF-7 cells

    International Nuclear Information System (INIS)

    Ruiz-Ramos, Ruben; Lopez-Carrillo, Lizbeth; Albores, Arnulfo; Hernandez-Ramirez, Raul U.; Cebrian, Mariano E.

    2009-01-01

    There is limited available information on the effects of arsenic on enzymes participating in the folate cycle. Therefore, our aim was to evaluate the effects of sodium arsenite on the protein levels of methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR) and its further relationship with the expression MT1/2 and c-myc in MCF-7 cells. Arsenite treatment (0-10 μM) for 4 h decreased MTHFR levels in a concentration-dependent fashion without significant effects on DHFR. The effects on MTHFR were observed at arsenite concentrations not significantly affecting cell viability. We also observed an increase in S-phase recruitment at all concentrations probed. Lower concentrations (< 5 μM) induced cell proliferation, showing a high proportion of BrdU-stained cells, indicating a higher DNA synthesis rate. However, higher concentrations (≥ 5 μM) or longer treatment periods induced apoptosis. Arsenite also induced dose-dependent increases in MT1/2 and c-Myc protein levels. The levels of MTHFR were inversely correlated to MT1/2 and c-Myc overexpression and increased S-phase recruitment. Our findings indicate that breast epithelial cells are responsive to arsenite and suggest that exposure may pose a risk for breast cancer. The reductions in MTHFR protein levels contribute to understand the mechanisms underlying the induction of genes influencing growth regulation, such as c-myc and MT1/2. However, further research is needed to ascertain if the effects here reported following short-time and high-dose exposure are relevant for human populations chronically exposed to low arsenic concentrations.

  15. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

    International Nuclear Information System (INIS)

    Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

    1990-01-01

    The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

  16. Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle.

    Science.gov (United States)

    Bozhkova, V P; Budayova, M; Kvasnicka, P; Cigankova, N; Chorvat, D

    1994-12-01

    Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.

  17. Borealin/Dasra B is a cell cycle-regulated chromosomal passenger protein and its nuclear accumulation is linked to poor prognosis for human gastric cancer

    International Nuclear Information System (INIS)

    Chang, J.-L.; Chen, T.-H.; Wang, C.-F.; Chiang, Y.-H.; Huang, Y.-L.; Wong, F.-H.; Chou, C.-K.; Chen, C.-M.

    2006-01-01

    Chromosomal passenger proteins including Aurora B, Survivin, and Borealin/Dasra B, also called CDCA8/FLJ10468, are known to play crucial roles during mitosis and cell division. Inappropriate chromosomal segregation and cell division may cause auneuploidy leading to cancer. However, it is still unclear how the expression of chromosomal passenger proteins may be linked to cancer. In this study, we demonstrated that Borealin is a cell cycle-regulated gene and is upregulated at G2-M phases of the cell cycle. We showed that Borealin interacts with Survivin but not with Aurora B. The interaction domain of Survivin in Borealin was mapped to the N-terminal 92 amino-acid residues of Borealin. To examine the linkage between expression of Borealin and cancer, we performed immunohistochemistry analysis using anti-Borealin specific antibody on the paraffin-embedded gastric cancer tissues. Our results showed that Borealin expression is significantly correlated with Survivin (P = 0.003) and Ki67 (P = 0.007) in gastric cancer. Interestingly, an increased nuclear Borealin level reveals borderline association with a poor survival rate (P = 0.047). Taken together, our results demonstrated that Borealin is a cell cycle-regulated chromosomal passenger protein and its aberrant expression is linked to a poor prognosis for gastric cancer

  18. Peroxisome proliferator-activated receptors (PPAR) agonists affect cell viability, apoptosis and expression of cell cycle related proteins in cell lines of glial brain tumors

    Czech Academy of Sciences Publication Activity Database

    Straková, N.; Ehrmann, J.; Bartoš, Jan; Malíková, J.; Doležel, Jaroslav; Kolář, Z.

    2005-01-01

    Roč. 52, - (2005), s. 126-136 ISSN 0028-2685 Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z5038910 Keywords : PPAR * glioplasma * cell cycle Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.731, year: 2005

  19. The Multiple Faces of Prostaglandin E2 G-Protein Coupled Receptor Signaling during the Dendritic Cell Life Cycle

    NARCIS (Netherlands)

    de Keijzer, Sandra; Meddens, Marjolein B.M.; Torensma, Ruurd; Cambi, A.

    2013-01-01

    Many processes regulating immune responses are initiated by G-protein coupled receptors (GPCRs) and report biochemical changes in the microenvironment. Dendritic cells (DCs) are the most potent antigen-presenting cells and crucial for the regulation of innate and adaptive immune responses. The lipid

  20. Analysis of cell cycle regulated and regulating proteins following exposure of lung derived cells to sub-lethal doses of a-rays

    Science.gov (United States)

    Trani, D.; Claudio, P. P.; Cassone, M.; Lucchetti, C.; D'Agostino, L.; Caputi, M.; Giordano, A.

    Introduction Since the last century mankind had to face an increased exposure to man made and natural sources of radiation Radiation represents a therapeutic instrument for radiosensitive cancers as well as a cytotoxic agent for normal human tissues The effects of prolonged exposure to low doses of high energy radiation are still not well-known at the molecular and clinical level Understanding their molecular effects will aid in developing more tailored therapeutic strategies as well as implementing radio-protective measures essential prerequisite for the long-time permanence of men in space Objective of the study The general aim of this study was to evaluate the susceptibility and the response of lung epithelial cells to DNA damage induced by ionizing radiations We decided to study a panel of epithelial bronchial cell lines because of their fast-growth rate and their prominent exposure to both environmental and medical radiations The specific objective of our study was to qualitatively and semi-quantitatively assess the involvement and behaviour of selected genes in DNA damage DNA-repair mechanisms and apoptosis which follow radiation exposure with the aim to determine the involvement of the most promising targets for the early detection of radiation-mediated lung damage before chronic disease develops Methods Four epithelial cell lines one normal and three neoplastic were selected in order to detect and compare survival cell cycle and protein expression differences related to their different genetic asset

  1. Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21WAF1/CIP1

    International Nuclear Information System (INIS)

    Kim, Mi Jin; Yoo, Young A.; Kim, Hyung Jung; Kang, Seongman; Kim, Yong Geon; Kim, Jun Suk; Yoo, Young Do

    2005-01-01

    Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27 Kip1 in the absence of p53. This study found that MRPL41 mediates the p21 WAF1/CIP1 -mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21 WAF1/CIP1 and p27 Kip1 levels under the growth inhibitory conditions

  2. Cell cycle-dependent changes in localization of a 210 k Da microtubule-interacting protein in Leishmania

    Czech Academy of Sciences Publication Activity Database

    Libusová, Lenka; Dráberová, Eduarda; Juliano, C.; Viklický, Vladimír; Fiori, P.; Cappuccinelli, P.; Dráber, Pavel

    2002-01-01

    Roč. 96, č. 4 (2002), s. 226-227 [Sigma-Aldrich konference mladých chemiků, biochemiků a molekulárních biologů. 22.05.2002-25.05.2002, Velké Meziříčí] R&D Projects: GA ČR GA304/00/0553; GA AV ČR IAA5052004 Keywords : Leishmania * cell cycle * microtubule Subject RIV: EB - Genetics ; Molecular Biology

  3. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

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

    International Nuclear Information System (INIS)

    Lemiere, Sylvie; Azar, Rania; Belloc, Francis; Guersel, Demir; Pyronnet, Stephane; Bikfalvi, Andreas; Auguste, Patrick

    2008-01-01

    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 the 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

  5. MadR1, a Mycobacterium tuberculosis cell cycle stress response protein that is a member of a widely conserved protein class of prokaryotic, eukaryotic and archeal origin.

    Science.gov (United States)

    Crew, Rebecca; Ramirez, Melissa V; England, Kathleen; Slayden, Richard A

    2015-05-01

    Stress-induced molecular programs designed to stall division progression are nearly ubiquitous in bacteria, with one well-known example being the participation of the SulA septum inhibiting protein in the SOS DNA damage repair response. Mycobacteria similarly demonstrate stress-altered growth kinetics, however no such regulators have been found in these organisms. We therefore set out to identify SulA-like regulatory proteins in Mycobacterium tuberculosis. A bioinformatics modeling-based approach led to the identification of rv2216 as encoding for a protein with weak similarity to SulA, further analysis distinguished this protein as belonging to a group of uncharacterized growth promoting proteins. We have named the mycobacterial protein encoded by rv2216 morphology altering division regulator protein 1, MadR1. Overexpression of madR1 modulated cell length while maintaining growth kinetics similar to wild-type, and increased the proportion of bent or V-form cells in the population. The presence of MadR1-GFP at regions of cellular elongation (poles) and morphological differentiation (V-form) suggests MadR1 involvement in phenotypic heterogeneity and longitudinal cellular growth. Global transcriptional analysis indicated that MadR1 functionality is linked to lipid editing programs required for growth and persistence. This is the first report to differentiate the larger class of these conserved proteins from SulA proteins and characterizes MadR1 effects on the mycobacterial cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells.

    OpenAIRE

    Andreassen, P R; Margolis, R L

    1992-01-01

    BHK cells blocked at any of several points in the cell cycle override their drug-induced arrest and proceed in the cycle when exposed concurrently to the protein kinase inhibitor 2-aminopurine (2-AP). For cells arrested at various points in interphase, 2-AP-induced cell cycle progression is made evident by arrival of the drug-treated cell population in mitosis. Cells that have escaped from mimosine G1 arrest, from hydroxyurea or aphidicolin S-phase arrest, or from VM-26-induced G2 arrest subs...

  7. Cell Cycle Deregulation in Ewing's Sarcoma Pathogenesis

    Science.gov (United States)

    Kowalewski, Ashley A.; Randall, R. Lor; Lessnick, Stephen L.

    2011-01-01

    Ewing's sarcoma is a highly aggressive pediatric tumor of bone that usually contains the characteristic chromosomal translocation t(11;22)(q24;q12). This translocation encodes the oncogenic fusion protein EWS/FLI, which acts as an aberrant transcription factor to deregulate target genes necessary for oncogenesis. One key feature of oncogenic transformation is dysregulation of cell cycle control. It is therefore likely that EWS/FLI and other cooperating mutations in Ewing's sarcoma modulate the cell cycle to facilitate tumorigenesis. This paper will summarize current published data associated with deregulation of the cell cycle in Ewing's sarcoma and highlight important questions that remain to be answered. PMID:21052502

  8. Cell Cycle Deregulation in Ewing's Sarcoma Pathogenesis

    Directory of Open Access Journals (Sweden)

    Ashley A. Kowalewski

    2011-01-01

    Full Text Available Ewing's sarcoma is a highly aggressive pediatric tumor of bone that usually contains the characteristic chromosomal translocation t(11;22(q24;q12. This translocation encodes the oncogenic fusion protein EWS/FLI, which acts as an aberrant transcription factor to deregulate target genes necessary for oncogenesis. One key feature of oncogenic transformation is dysregulation of cell cycle control. It is therefore likely that EWS/FLI and other cooperating mutations in Ewing's sarcoma modulate the cell cycle to facilitate tumorigenesis. This paper will summarize current published data associated with deregulation of the cell cycle in Ewing's sarcoma and highlight important questions that remain to be answered.

  9. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene

    International Nuclear Information System (INIS)

    Knight, G.B.; Gudas, J.M.; Pardee, A.B.

    1987-01-01

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene

  10. Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride.

    Science.gov (United States)

    Barbieri, F; Sparatore, F; Cagnoli, M; Bruzzo, C; Novelli, F; Alama, A

    2001-03-14

    Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

  11. Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

    Science.gov (United States)

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2013-01-01

    Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816

  12. Induction of cell cycle arrest and apoptosis with downregulation of Hsp90 client proteins and histone modification by 4β-hydroxywithanolide E isolated from Physalis peruviana.

    Science.gov (United States)

    Park, Eun-Jung; Sang-Ngern, Mayuramas; Chang, Leng Chee; Pezzuto, John M

    2016-06-01

    Physalis peruviana (Solanaceae) is used for culinary and medicinal purposes. We currently report withanolides, isolated from P. peruviana, inhibit the growth of colon cancer monolayer and spheroid cultures. A detailed mechanistic evaluation was performed with 4β-hydroxywithanolide E (4HWE). Treatment of HT-29 cells with low concentrations of 4HWE inhibited growth while enhancing levels of p21(Waf1/Cip1) and reducing levels of several cell cycle-related proteins. Apoptosis was induced at higher concentrations. In addition, 4HWE treatment downregulated the levels of Hsp90 client proteins. Nuclear sirtuin 1 (SIRT1) was increased and histone H3 acetylated at lysine 9 was decreased. An additional consequence of SIRT1 elevation in the nucleus may be inhibition of c-Jun activity. The expression of 21 genes was altered, including downregulation of PTGS2, and this correlated with reduced protein levels of cyclooxygenase-2 (COX-2). Overall, efficacious induction of G0/G1 cell cycle arrest at low concentrations, and induction of apoptosis at higher concentrations are interesting 4HWE-mediated phenomena that are accompanied by a complex array of molecular events. Considering the worldwide prevalence of colon cancer, and the unique mode of action mediated by 4HWE, it is reasonable to investigate additional mechanistic details and the potential utility of this compound. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Regulation of the cell cycle by irradiation

    International Nuclear Information System (INIS)

    Akashi, Makoto

    1995-01-01

    The molecular mechanism of cell proliferation is extremely complex; deregulation results in neoplastic transformation. In eukaryotes, proliferation of cells is finely regulated through the cell cycle. Studies have shown that the cell cycle is regulated by s series of enzymes known as cyclin-dependent kinases (CDKs). The activities of CDKs are controlled by their association with regulatory subunits, cyclins; the expression of cyclins and the activation of the different cyclin-CDK complexes are required for the cell to cycle. Thus, the cell cycle is regulated by activating and inhibiting phosphorylation of the CDK subunits and this program has internal check points at different stages of the cell cycle. When cells are exposed to external insults such as DNA damaging agents, negative regulation of the cell cycle occurs; arrest in either G1 or G2 stage is induced to prevent the cells from prematurely entering into the next stage before DNA is repaired. Recently, a potent inhibitor of CDKs, which inhibits the phosphorylation of retinoblastoma susceptibility (Rb) gene product by cyclin A-CDK2, cyclin E-CDK2, cyclin D1-CDK4, and cyclin D2-CDK4 complexes has been identified. This protein named WAF1, Sdi1, Cip1, or p21 (a protein of Mr 21,000) contains a p53-binding site in its promoter and studies have reported that the expression of WAF1 was directly regulated by p53; cells with loss of p53 activity due to mutational alteration were unable to induce WAF1. This chapter will be focused on the mechanisms of the cell cycle including inhibitors of CDKs, and the induction of WAF1 by irradiation through a pathway independent of p53 will be also described. (author)

  14. The Multiple Faces of Prostaglandin E2 G-Protein Coupled Receptor Signaling during the Dendritic Cell Life Cycle

    Directory of Open Access Journals (Sweden)

    Alessandra Cambi

    2013-03-01

    Full Text Available Many processes regulating immune responses are initiated by G-protein coupled receptors (GPCRs and report biochemical changes in the microenvironment. Dendritic cells (DCs are the most potent antigen-presenting cells and crucial for the regulation of innate and adaptive immune responses. The lipid mediator Prostaglandin E2 (PGE2 via four GPCR subtypes (EP1-4 critically regulates DC generation, maturation and migration. The role of PGE2 signaling in DC biology was unraveled by the characterization of EP receptor subtype expression in DC progenitor cells and DCs, the identification of the signaling pathways initiated by these GPCR subtypes and the classification of DC responses to PGE2 at different stages of differentiation. Here, we review the advances in PGE2 signaling in DCs and describe the efforts still to be made to understand the spatio-temporal fine-tuning of PGE2 responses by DCs.

  15. Cell Cycle Control by PTEN.

    Science.gov (United States)

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

    2017-07-21

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

  16. Cell cycle arrest induced by radiation

    International Nuclear Information System (INIS)

    Okaichi, Yasuo; Matsumoto, Hideki; Ohnishi, Takeo

    1994-01-01

    It is known that various chemical reactions, such as cell cycle arrest, DNA repair and cell killing, can occur within the cells when exposed to ionizing radiation and ultraviolet radiation. Thus protein dynamics involved in such chemical reactions has received considerable attention. In this article, cell cycle regulation is first discussed in terms of the G2/M-phase and the G1/S-phase. Then, radiation-induced cell cycle arrest is reviewed. Cell cycle regulation mechanism involved in the G2 arrest, which is well known to occur when exposed to radiation, has recently been investigated using yeasts. In addition, recent study has yielded a noticeable finding that the G1 arrest can occur with intracellular accumulation of p53 product following ionization radiation. p53 is also shown to play an extremely important role in both DNA repair and cell killing due to DNA damage. Studies on the role of genes in protein groups induced by radiation will hold promise for the elucidation of cell cycle mechanism. (N.K.) 57 refs

  17. The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1

    International Nuclear Information System (INIS)

    Gupta, Sanjay; Takhar, Param Parkash S; Degenkolbe, Roland; Heng Koh, Choon; Zimmermann, Holger; Maolin Yang, Christopher; Guan Sim, Khe; I-Hong Hsu, Stephen; Bernard, Hans-Ulrich

    2003-01-01

    The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ('low-risk types'), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ('high-risk types'), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared

  18. Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

    Science.gov (United States)

    Petojevic, Tatjana; Pesavento, James J.; Costa, Alessandro; Liang, Jingdan; Wang, Zhijun; Berger, James M.; Botchan, Michael R.

    2015-01-01

    DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel. PMID:25561522

  19. Interlink between cholesterol & cell cycle in prostate carcinoma

    Directory of Open Access Journals (Sweden)

    Govind Singh

    2017-01-01

    Interpretation & conclusions: The present findings along with increased expression of cell cycle protein cyclin E in the cell nucleus of the tumour tissue suggested the possibility of an intriguing role of cholesterol in the mechanism of cell cycle process of prostate cell proliferation.

  20. The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability

    Directory of Open Access Journals (Sweden)

    Teresa L.F. Ho

    2016-04-01

    Full Text Available Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability.

  1. Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK and FOXO1.

    Directory of Open Access Journals (Sweden)

    Tamás Fodor

    Full Text Available Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK jointly with methotrexate (MTX, a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.

  2. A hybrid mammalian cell cycle model

    Directory of Open Access Journals (Sweden)

    Vincent Noël

    2013-08-01

    Full Text Available Hybrid modeling provides an effective solution to cope with multiple time scales dynamics in systems biology. Among the applications of this method, one of the most important is the cell cycle regulation. The machinery of the cell cycle, leading to cell division and proliferation, combines slow growth, spatio-temporal re-organisation of the cell, and rapid changes of regulatory proteins concentrations induced by post-translational modifications. The advancement through the cell cycle comprises a well defined sequence of stages, separated by checkpoint transitions. The combination of continuous and discrete changes justifies hybrid modelling approaches to cell cycle dynamics. We present a piecewise-smooth version of a mammalian cell cycle model, obtained by hybridization from a smooth biochemical model. The approximate hybridization scheme, leading to simplified reaction rates and binary event location functions, is based on learning from a training set of trajectories of the smooth model. We discuss several learning strategies for the parameters of the hybrid model.

  3. Molecular biological mechanism II. Molecular mechanisms of cell cycle regulation

    International Nuclear Information System (INIS)

    Jung, T.

    2000-01-01

    The cell cycle in eukaryotes is regulated by central cell cycle controlling protein kinase complexes. These protein kinase complexes consist of a catalytic subunit from the cyclin-dependent protein kinase family (CDK), and a regulatory subunit from the cyclin family. Cyclins are characterised by their periodic cell cycle related synthesis and destruction. Each cell cycle phase is characterised by a specific set of CDKs and cyclins. The activity of CDK/cyclin complexes is mainly regulated on four levels. It is controlled by specific phosphorylation steps, the synthesis and destruction of cyclins, the binding of specific inhibitor proteins, and by active control of their intracellular localisation. At several critical points within the cell cycle, named checkpoints, the integrity of the cellular genome is monitored. If damage to the genome or an unfinished prior cell cycle phase is detected, the cell cycle progression is stopped. These cell cycle blocks are of great importance to secure survival of cells. Their primary importance is to prevent the manifestation and heritable passage of a mutated genome to daughter cells. Damage sensing, DNA repair, cell cycle control and apoptosis are closely linked cellular defence mechanisms to secure genome integrity. Disregulation in one of these defence mechanisms are potentially correlated with an increased cancer risk and therefore in at least some cases with an increased radiation sensitivity. (orig.) [de

  4. An autoradiographic study of the synthesis of nucleic acids and protein during the cell cycle of synchronously dividing antheridial filaments in Chara vulgaris L

    Energy Technology Data Exchange (ETDEWEB)

    Olszewska, M J; Godlewski, M [Lodz Univ. (Poland)

    1972-01-01

    The synthesis of DNA, RNA, and protein in successive mitotic cycles of the synchronously dividing antheridial filaments of Chara vulgaris was studied autoradiographically. In all the generations examined, which enter the next mitosis, i.e., in the 4-, 8-, 16-, and 32-cell generations, the synthesis of DNA begins as early as telephase and continues into the early stages of interphase. The telephase cells of the 32-cell filaments do not incorporate //sup 3/H/thymidine, because the cells which arise from them do not divide but are transformed into spermatozoa. The DNA synthesis is accompanied by intense synthesis of RNA. The intensity of radioactivity calculated for 100 ..mu../sup 2/ of the area of the nucleus and cytoplasm is similar in all the generations, whereas the radioactivity induced by the incorporation of /8-/sup 14/C/adenine and//sup 3/H/phenylalanine calculated for one cell decreases proportionally to the reduction of the volume of the cytoplasm and nucleus in successive generations. (auth)

  5. Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p53 and p21/WAF1 proteins in skin of mice

    International Nuclear Information System (INIS)

    Saxena, Neha; Ansari, Kausar M.; Kumar, Rahul; Dhawan, Alok; Dwivedi, Premendra D.; Das, Mukul

    2009-01-01

    Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40-160 μg/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 μg/100 μl acetone) for 24-72 h caused (a) DNA damage in skin cells showing significant increase (34-63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8-10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p 21/WAF1 (3.6-3.9 fold), pro apoptotic protein Bax (1.3-2.6) and tumor suppressor wild type p 53 (2.8-3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p 53 mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications

  6. A nuclear glutathione cycle within the cell cycle.

    Science.gov (United States)

    Diaz Vivancos, Pedro; Wolff, Tonja; Markovic, Jelena; Pallardó, Federico V; Foyer, Christine H

    2010-10-15

    The complex antioxidant network of plant and animal cells has the thiol tripeptide GSH at its centre to buffer ROS (reactive oxygen species) and facilitate cellular redox signalling which controls growth, development and defence. GSH is found in nearly every compartment of the cell, including the nucleus. Transport between the different intracellular compartments is pivotal to the regulation of cell proliferation. GSH co-localizes with nuclear DNA at the early stages of proliferation in plant and animal cells. Moreover, GSH recruitment and sequestration in the nucleus during the G1- and S-phases of the cell cycle has a profound impact on cellular redox homoeostasis and on gene expression. For example, the abundance of transcripts encoding stress and defence proteins is decreased when GSH is sequestered in the nucleus. The functions of GSHn (nuclear GSH) are considered in the present review in the context of whole-cell redox homoeostasis and signalling, as well as potential mechanisms for GSH transport into the nucleus. We also discuss the possible role of GSHn as a regulator of nuclear proteins such as histones and PARP [poly(ADP-ribose) polymerase] that control genetic and epigenetic events. In this way, a high level of GSH in the nucleus may not only have an immediate effect on gene expression patterns, but also contribute to how cells retain a memory of the cellular redox environment that is transferred through generations.

  7. Rhizoma Dioscoreae extract protects against alveolar bone loss by regulating the cell cycle: A predictive study based on the protein‑protein interaction network.

    Science.gov (United States)

    Zhang, Zhi-Guo; Song, Chang-Heng; Zhang, Fang-Zhen; Chen, Yan-Jing; Xiang, Li-Hua; Xiao, Gary Guishan; Ju, Da-Hong

    2016-06-01

    Rhizoma Dioscoreae extract (RDE) exhibits a protective effect on alveolar bone loss in ovariectomized (OVX) rats. The aim of this study was to predict the pathways or targets that are regulated by RDE, by re‑assessing our previously reported data and conducting a protein‑protein interaction (PPI) network analysis. In total, 383 differentially expressed genes (≥3‑fold) between alveolar bone samples from the RDE and OVX group rats were identified, and a PPI network was constructed based on these genes. Furthermore, four molecular clusters (A‑D) in the PPI network with the smallest P‑values were detected by molecular complex detection (MCODE) algorithm. Using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) tools, two molecular clusters (A and B) were enriched for biological process in Gene Ontology (GO). Only cluster A was associated with biological pathways in the IPA database. GO and pathway analysis results showed that cluster A, associated with cell cycle regulation, was the most important molecular cluster in the PPI network. In addition, cyclin‑dependent kinase 1 (CDK1) may be a key molecule achieving the cell‑cycle‑regulatory function of cluster A. From the PPI network analysis, it was predicted that delayed cell cycle progression in excessive alveolar bone remodeling via downregulation of CDK1 may be another mechanism underling the anti‑osteopenic effect of RDE on alveolar bone.

  8. Do lipids shape the eukaryotic cell cycle?

    Science.gov (United States)

    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.

  9. The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

    Science.gov (United States)

    1996-08-01

    that my statement of work (SOW) for the current project omitted many of the tasks that had to be carried out in order to get the lab up and running...that we knew that we could stabilize wild-type p53 in ML-1 cells along with the possibility of being able to get an excellent elutriation profile with...nuclear protein extract was immunoprecipitated with PAb421 cross-linked to ProteinA -Sepharose beads and analysed by SDS-PAGE Western blot analysis with

  10. Dynamic ubiquitin signaling in cell cycle regulation.

    Science.gov (United States)

    Gilberto, Samuel; Peter, Matthias

    2017-08-07

    The cell division cycle is driven by a collection of enzymes that coordinate DNA duplication and separation, ensuring that genomic information is faithfully and perpetually maintained. The activity of the effector proteins that perform and coordinate these biological processes oscillates by regulated expression and/or posttranslational modifications. Ubiquitylation is a cardinal cellular modification and is long known for driving cell cycle transitions. In this review, we emphasize emerging concepts of how ubiquitylation brings the necessary dynamicity and plasticity that underlie the processes of DNA replication and mitosis. New studies, often focusing on the regulation of chromosomal proteins like DNA polymerases or kinetochore kinases, are demonstrating that ubiquitylation is a versatile modification that can be used to fine-tune these cell cycle events, frequently through processes that do not involve proteasomal degradation. Understanding how the increasing variety of identified ubiquitin signals are transduced will allow us to develop a deeper mechanistic perception of how the multiple factors come together to faithfully propagate genomic information. Here, we discuss these and additional conceptual challenges that are currently under study toward understanding how ubiquitin governs cell cycle regulation. © 2017 Gilberto and Peter.

  11. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA)

    International Nuclear Information System (INIS)

    Nicol, Alcina F; Pirmez, Claude; Pires, Andréa Rodrigues Cordovil; Souza, Simone R de; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; Silva, José R Lapa e

    2008-01-01

    The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm 2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm 2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development

  12. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA).

    Science.gov (United States)

    Nicol, Alcina F; Pires, Andréa Rodrigues Cordovil; de Souza, Simone R; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; Lapa e Silva, José R; Pirmez, Claude

    2008-10-07

    The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development.

  13. Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

    Science.gov (United States)

    Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

    2016-07-01

    Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  14. Lobaplatin arrests cell cycle progression in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Chen Chang-Jie

    2010-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored. Methods Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR and protein (Western blot levels. The phosphorylation status of cyclin-dependent kinases (CDKs and retinoblastoma (Rb protein was also examined using Western blot analysis. Results Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1, pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27. Conclusion Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

  15. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Carrasco, L.; Bravo, R.

    1986-01-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with [ 3 H]glucosamine were detected in vaccinia-infected HeLa cells

  16. Sex and estrous cycle-dependent rapid protein kinase signaling actions of estrogen in distal colonic cells.

    LENUS (Irish Health Repository)

    O'Mahony, Fiona

    2008-10-01

    Previous studies from our laboratory demonstrated that 17beta-estradiol (E2) rapidly inhibits Cl(-) secretion in rat and human distal colonic epithelium. The inhibition has been shown to occur via targeting of a basolateral K(+) channel identified as the KCNQ1 (KvLQT1) channel. E2 indirectly modulates the channel activity via a cascade of second messengers which are rapidly phosphorylated in response to E2. The anti-secretory mechanism may be the manner by which E2 induces fluid retention in the intestine during periods of high circulating plasma E2. Here we review the sex-dependent and estrous cycle regulation of this novel rapid response to E2. The inhibition of KCNQ1 channel activity and Cl(-) secretion will be of interest in the future in the investigation of the retentive effects of estrogen in female tissue and also in the study of secretory disorders and drugable targets of the intestine.

  17. A quantitative characterization of the yeast heterotrimeric G protein cycle

    Science.gov (United States)

    Yi, Tau-Mu; Kitano, Hiroaki; Simon, Melvin I.

    2003-01-01

    The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. PMID:12960402

  18. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

    Directory of Open Access Journals (Sweden)

    Elena Favaro

    2010-04-01

    Full Text Available Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1 and a microRNA, hsa-miR-210 (miR-210 which is associated with a poor prognosis.In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

  19. Segmentation and classification of cell cycle phases in fluorescence imaging.

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

    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 (IEevasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens. PMID:25144360

  1. A Method to Design Synthetic Cell-Cycle Networks

    International Nuclear Information System (INIS)

    Ke-Ke, Miao

    2009-01-01

    The interactions among proteins, DNA and RNA in an organism form elaborate cell-cycle networks which govern cell growth and proliferation. Understanding the common structure of cell-cycle networks will be of great benefit to science research. Here, inspired by the importance of the cell-cycle regulatory network of yeast which has been studied intensively, we focus on small networks with 11 nodes, equivalent to that of the cell-cycle regulatory network used by Li et al. [Proc. Natl. Acad. Sci. USA 101(2004)4781] Using a Boolean model, we study the correlation between structure and function, and a possible common structure. It is found that cascade-like networks with a great number of interactions between nodes are stable. Based on these findings, we are able to construct synthetic networks that have the same functions as the cell-cycle regulatory network. (condensed matter: structure, mechanical and thermal properties)

  2. Tumor cell surface proteins

    International Nuclear Information System (INIS)

    Kennel, S.J.; Braslawsky, G.R.; Flynn, K.; Foote, L.J.; Friedman, E.; Hotchkiss, J.A.; Huang, A.H.L.; Lankford, P.K.

    1982-01-01

    Cell surface proteins mediate interaction between cells and their environment. Unique tumor cell surface proteins are being identified and quantified in several tumor systems to address the following questions: (i) how do tumor-specific proteins arise during cell transformation; (ii) can these proteins be used as markers of tumor cell distribution in vivo; (iii) can cytotoxic drugs be targeted specifically to tumor cells using antibody; and (iv) can solid state radioimmunoassay of these proteins provide a means to quantify transformation frequencies. A tumor surface protein of 180,000 M/sub r/ (TSP-180) has been identified on cells of several lung carcinomas of BALB/c mice. TSP-180 was not detected on normal lung tissue, embryonic tissue, or other epithelial or sarcoma tumors, but it was found on lung carcinomas of other strains of mice. Considerable amino acid sequence homology exists among TSP-180's from several cell sources, indicating that TSP-180 synthesis is directed by normal cellular genes although it is not expressed in normal cells. The regulation of synthesis of TSP-180 and its relationship to normal cell surface proteins are being studied. Monoclonal antibodies (MoAb) to TSP-180 have been developed. The antibodies have been used in immunoaffinity chromatography to isolate TSP-180 from tumor cell sources. This purified tumor antigen was used to immunize rats. Antibody produced by these animals reacted at different sites (epitopes) on the TSP-180 molecule than did the original MoAb. These sera and MoAb from these animals are being used to identify normal cell components related to the TSP-180 molecule

  3. A low protein diet during pregnancy provokes a lasting shift of hepatic expression of genes related to cell cycle throughout ontogenesis in a porcine model

    Directory of Open Access Journals (Sweden)

    Oster Michael

    2012-03-01

    Full Text Available Abstract Background In rodent models and in humans the impact of gestational diets on the offspring's phenotype was shown experimentally and epidemiologically. Adverse environmental conditions during fetal development provoke an intrauterine adaptive response termed 'fetal programming', which may lead to both persistently biased responsiveness to extrinsic factors and permanent consequences for the organismal phenotype. This leads to the hypothesis that the offspring's transcriptome exhibits short-term and long-term changes, depending on the maternal diet. In order to contribute to a comprehensive inventory of genes and functional networks that are targets of nutritional programming initiated during fetal life, we applied whole-genome microarrays for expression profiling in a longitudinal experimental design covering prenatal, perinatal, juvenile, and adult ontogenetic stages in a porcine model. Pregnant sows were fed either a gestational low protein diet (LP, 6% CP or an adequate protein diet (AP, 12% CP. All offspring was nursed by foster sows receiving standard diets. After weaning, all offspring was fed standard diets ad libitum. Results Analyses of the hepatic gene expression of the offspring at prenatal (94 dies post conceptionem, dpc and postnatal stages (1, 28, 188 dies post natum, dpn included comparisons between dietary groups within stages as well as comparisons between ontogenetic stages within diets to separate diet-specific transcriptional changes and maturation processes. We observed differential expression of genes related to lipid metabolism (e.g. Fatty acid metabolism, Biosynthesis of steroids, Synthesis and degradation of ketone bodies, FA elongation in mitochondria, Bile acid synthesis and cell cycle regulation (e.g. Mitotic roles of PLK, G1/S checkpoint regulation, G2/M DNA damage checkpoint regulation. Notably, at stage 1 dpn no regulation of a distinct pathway was found in LP offspring. Conclusions The transcriptomic

  4. Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

    Science.gov (United States)

    2012-01-01

    Background Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Methods Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. Results We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. Conclusions We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin. PMID:22280307

  5. Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

    International Nuclear Information System (INIS)

    Lee, Seung Joon; Langhans, Sigrid A

    2012-01-01

    Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin

  6. Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

    Directory of Open Access Journals (Sweden)

    Lee Seung Joon

    2012-01-01

    Full Text Available Abstract Background Curcumin (diferuloylmethane, the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC, is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Methods Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. Results We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. Conclusions We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to

  7. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    Science.gov (United States)

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  8. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

    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. These 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.

  9. Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of gammaH2AX- and NBS1-positive repair foci

    Czech Academy of Sciences Publication Activity Database

    Suchánková, Jana; Kozubek, Stanislav; Legartová, Soňa; Sehnalová, Petra; Kuntzinger, T.; Bártová, Eva

    2015-01-01

    Roč. 107, č. 12 (2015), s. 440-454 ISSN 0248-4900 R&D Projects: GA ČR(CZ) GBP302/12/G157; GA ČR(CZ) GA13-07822S Institutional support: RVO:68081707 Keywords : Cell cycle * DNA repair * Interphase Subject RIV: BO - Biophysics Impact factor: 2.552, year: 2015

  10. Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    Full Text Available The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

  11. Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

    Directory of Open Access Journals (Sweden)

    Laura L Quinn

    2014-08-01

    Full Text Available 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 (IEcycle phase-specific effects that provide mechanistic

  12. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.

    2010-01-01

    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  13. RBP-J-interacting and tubulin-associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7 pathway

    International Nuclear Information System (INIS)

    Wang, Haihe; Yang, Zhanchun; Liu, Chunbo; Huang, Shishun; Wang, Hongzhi; Chen, Yingli; Chen, Guofu

    2014-01-01

    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. 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

  14. An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

    Science.gov (United States)

    Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

    2018-01-01

    Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving

  15. Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

    Directory of Open Access Journals (Sweden)

    Claudia Scotti

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

  16. VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle

    Czech Academy of Sciences Publication Activity Database

    Horníková, L.; Fraiberk, M.; Man, Petr; Janovec, V.; Forstová, J.

    2017-01-01

    Roč. 284, č. 2 (2017), s. 301-323 E-ISSN 1742-4658 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1509 Grant - others:Ministerstvo pro místní rozvoj(CZ) CZ.2.16./3.1.00/24023 Institutional support: RVO:61388971 Keywords : cell cycle arrest * chaperone Hsp90 * microtubules Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology

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

    Science.gov (United States)

    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.

  18. NONO couples the circadian clock to the cell cycle.

    Science.gov (United States)

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

    2013-01-29

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

  19. Cell cycle control by components of cell anchorage

    OpenAIRE

    Gad, Annica

    2005-01-01

    Extracellular factors, such as growth factors and cell anchorage to the extracellular matrix, control when and where cells may proliferate. This control is abolished when a normal cell transforms into a tumour cell. The control of cell proliferation by cell anchorage was elusive and less well studied than the control by growth factors. Therefore, we aimed to clarify at what points in the cell cycle and through which molecular mechanisms cell anchorage controls cell cycle pro...

  20. Cell cycle control by the thyroid hormone in neuroblastoma cells

    International Nuclear Information System (INIS)

    Garcia-Silva, Susana; Perez-Juste, German; Aranda, Ana

    2002-01-01

    The thyroid hormone (T3) blocks proliferation and induces differentiation of neuroblastoma N2a-β cells that overexpress the β1 isoform of the T3 receptor. An element in the region responsible for premature termination of transcription mediates a rapid repression of c-myc gene expression by T3. The hormone also causes a decrease of cyclin D1 gene transcription, and is able to antagonize the activation of the cyclin D1 promoter by Ras. In addition, a strong and sustained increase of the levels of the cyclin kinase inhibitor (CKI) p27 Kip1 are found in T3-treated cells. The increased levels of p27 Kip1 lead to a marked inhibition of the kinase activity of the cyclin-CDK2 complexes. As a consequence of these changes, retinoblastoma proteins are hypophosphorylated in T3-treated N2a-β cells, and progression through the restriction point in the cell cycle is blocked

  1. Chromatin association of UHRF1 during the cell cycle

    KAUST Repository

    Al-Gashgari, Bothayna

    2017-05-01

    Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a nuclear protein that associates with chromatin. Regardless of the various functions of UHRF1 in the cell, one of its more important functions is its role in the maintenance of DNA methylation patterns by the recruitment of DNMT1. Studies on UHRF1 based on this function have revealed the importance of UHRF1 during the cell cycle. Moreover, based on different studies various factors were described to be involved in the regulation of UHRF1 with different functionalities that can control its binding affinity to different targets on chromatin. These factors are regulated differently in a cell cycle specific manner. In light of this, we propose that UHRF1 has different binding behaviors during the cell cycle in regard to its association with chromatin. In this project, we first analyzed the binding behavior of endogenous UHRF1 from different unsynchronized cell systems in pull-down assays with peptides and oligonucleotides. Moreover, to analyze UHRF1 binding behavior during the cell cycle, we used two different approaches. First we sorted Jurkat and HT1080 cells based on their cell cycle stage using FACS analysis. Additionally, we synchronized HeLa cells to different stages of the cell cycle by chemical treatments, and used extracts from cellsorting and cell synchronization experiments for pull-down assays. We observed that UHRF1 in different cell systems has different preferences in regard to its binding to H3 unmodified and H3K9me3. Moreover, we detected that UHRF1, in general, displays different patterns between different stages of cell cycle; however, we cannot draw a final model for UHRF1 binding pattern during cell cycle.

  2. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  3. Evolution of cell cycle control: same molecular machines, different regulation

    DEFF Research Database (Denmark)

    de Lichtenberg, Ulrik; Jensen, Thomas Skøt; Brunak, Søren

    2007-01-01

    Decades of research has together with the availability of whole genomes made it clear that many of the core components involved in the cell cycle are conserved across eukaryotes, both functionally and structurally. These proteins are organized in complexes and modules that are activated or deacti......Decades of research has together with the availability of whole genomes made it clear that many of the core components involved in the cell cycle are conserved across eukaryotes, both functionally and structurally. These proteins are organized in complexes and modules that are activated...... for assembling the same molecular machines just in time for action....

  4. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

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

  5. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes.

    Science.gov (United States)

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    Spermatogenesis, a study of germ cell development, is a long, orderly, and well-defined process occurring in seminiferous tubules of the testis. It is a temporal event whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa over a period of several weeks. Spermatogenesis is characterized by three specific functional phases: proliferation, meiosis, and differentiation, and it involves spermatogonia, spermatocytes, and spermatids. Germ cells at steps of development form various cellular associations or stages, with 6, 12, and 14 specific stages being identified in human, mouse, and rat, respectively. The stages evolve over time in a given area of the seminiferous tubule forming a cycle of the seminiferous epithelium that has a well-defined duration for a given species. In this part, we discuss the proliferation and meiotic phase whereby spermatogonia undergo several mitotic divisions to form spermatocytes that undergo two meiotic divisions to form haploid spermatids. In the rat, spermatogonia can be subdivided into several classes: stem cells (A(s)), proliferating cells (A(pr), A(al)), and differentiating cells (A(1)-A(4), In, B). They are dependent on a specific microenvironment (niche) contributed by Sertoli, myoid, and Leydig cells for proper development. Spermatogonia possess several surface markers whereby they can be identified from each other. During meiosis, spermatocytes undergo chromosomal pairing, synapsis, and genetic exchange as well as transforming into haploid cells following meiosis. The meiotic cells form specific structural entities such as the synaptonemal complex and sex body. Many genes involved in spermatogonial renewal and the meiotic process have been identified and shown to be essential for this event. Copyright 2009 Wiley-Liss, Inc.

  6. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

    Science.gov (United States)

    Vielfort, Katarina; Weyler, Linda; Söderholm, Niklas; Engelbrecht, Mattias; Löfmark, Sonja; Aro, Helena

    2013-01-01

    We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells. PMID:23675492

  7. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

    Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

  8. Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

    Science.gov (United States)

    Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

    2003-11-15

    Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

  9. A Dominant-Negative PPARγ Mutant Promotes Cell Cycle Progression and Cell Growth in Vascular Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Joey Z. Liu

    2009-01-01

    Full Text Available PPARγ ligands have been shown to have antiproliferative effects on many cell types. We herein report that a synthetic dominant-negative (DN PPARγ mutant functions like a growth factor to promote cell cycle progression and cell proliferation in human coronary artery smooth muscle cells (CASMCs. In quiescent CASMCs, adenovirus-expressed DN-PPARγ promoted G1→S cell cycle progression, enhanced BrdU incorporation, and increased cell proliferation. DN-PPARγ expression also markedly enhanced positive regulators of the cell cycle, increasing Rb and CDC2 phosphorylation and the expression of cyclin A, B1, D1, and MCM7. Conversely, overexpression of wild-type (WT or constitutively-active (CA PPARγ inhibited cell cycle progression and the activity and expression of positive regulators of the cell cycle. DN-PPARγ expression, however, did not up-regulate positive cell cycle regulators in PPARγ-deficient cells, strongly suggesting that DN-PPARγ effects on cell cycle result from blocking the function of endogenous wild-type PPARγ. DN-PPARγ expression enhanced phosphorylation of ERK MAPKs. Furthermore, the ERK specific-inhibitor PD98059 blocked DN-PPARγ-induced phosphorylation of Rb and expression of cyclin A and MCM7. Our data thus suggest that DN-PPARγ promotes cell cycle progression and cell growth in CASMCs by modulating fundamental cell cycle regulatory proteins and MAPK mitogenic signaling pathways in vascular smooth muscle cells (VSMCs.

  10. Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

    Science.gov (United States)

    Ly, Tony; Endo, Aki; Lamond, Angus I

    2015-01-02

    Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

  11. Cell cycle control by a minimal Cdk network.

    Directory of Open Access Journals (Sweden)

    Claude Gérard

    2015-02-01

    Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

  12. Transcriptional Waves in the Yeast Cell Cycle

    OpenAIRE

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

  13. Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae.

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    Full Text Available Dinoflagellates are the major causative agents of harmful algal blooms in the coastal zone, which has resulted in adverse effects on the marine ecosystem and public health, and has become a global concern. Knowledge of cell cycle regulation in proliferating cells is essential for understanding bloom dynamics, and so this study compared the protein profiles of Prorocentrum donghaiense at different cell cycle phases and identified differentially expressed proteins using 2-D fluorescence difference gel electrophoresis combined with MALDI-TOF-TOF mass spectrometry. The results showed that the synchronized cells of P. donghaiense completed a cell cycle within 24 hours and cell division was phased with the diurnal cycle. Comparison of the protein profiles at four cell cycle phases (G1, S, early and late G2/M showed that 53 protein spots altered significantly in abundance. Among them, 41 were identified to be involved in a variety of biological processes, e.g. cell cycle and division, RNA metabolism, protein and amino acid metabolism, energy and carbon metabolism, oxidation-reduction processes, and ABC transport. The periodic expression of these proteins was critical to maintain the proper order and function of the cell cycle. This study, to our knowledge, for the first time revealed the major biological processes occurring at different cell cycle phases which provided new insights into the mechanisms regulating the cell cycle and growth of dinoflagellates.

  14. Expression of the Long Intergenic Non-Protein Coding RNA 665 (LINC00665) Gene and the Cell Cycle in Hepatocellular Carcinoma Using The Cancer Genome Atlas, the Gene Expression Omnibus, and Quantitative Real-Time Polymerase Chain Reaction.

    Science.gov (United States)

    Wen, Dong-Yue; Lin, Peng; Pang, Yu-Yan; Chen, Gang; He, Yun; Dang, Yi-Wu; Yang, Hong

    2018-05-05

    BACKGROUND Long non-coding RNAs (lncRNAs) have a role in physiological and pathological processes, including cancer. The aim of this study was to investigate the expression of the long intergenic non-protein coding RNA 665 (LINC00665) gene and the cell cycle in hepatocellular carcinoma (HCC) using database analysis including The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and quantitative real-time polymerase chain reaction (qPCR). MATERIAL AND METHODS Expression levels of LINC00665 were compared between human tissue samples of HCC and adjacent normal liver, clinicopathological correlations were made using TCGA and the GEO, and qPCR was performed to validate the findings. Other public databases were searched for other genes associated with LINC00665 expression, including The Atlas of Noncoding RNAs in Cancer (TANRIC), the Multi Experiment Matrix (MEM), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) networks. RESULTS Overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. Overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR=1.47795%; CI: 1.046-2.086). Bioinformatics analysis identified 469 related genes and further analysis supported a hypothesis that LINC00665 regulates pathways in the cell cycle to facilitate the development and progression of HCC through ten identified core genes: CDK1, BUB1B, BUB1, PLK1, CCNB2, CCNB1, CDC20, ESPL1, MAD2L1, and CCNA2. CONCLUSIONS Overexpression of the lncRNA, LINC00665 may be involved in the regulation of cell cycle pathways in HCC through ten identified hub genes.

  15. Regulation of cell cycle progression by cell-cell and cell-matrix forces

    NARCIS (Netherlands)

    Uroz, Marina; Wistorf, Sabrina; Serra-Picamal, Xavier; Conte, Vito; Sales-Pardo, Marta; Roca-Cusachs, Pere; Guimerà, Roger; Trepat, Xavier

    2018-01-01

    It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces 1-12 . However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression

  16. Dihydromyricetin induces cell cycle arrest and apoptosis in melanoma SK-MEL-28 cells.

    Science.gov (United States)

    Zeng, Guofang; Liu, Jie; Chen, Hege; Liu, Bin; Zhang, Qingyu; Li, Mingyi; Zhu, Runzhi

    2014-06-01

    Dihydromyricetin (DHM) exhibits multiple pharmacological activities; however, the role of DHM in anti-melanoma activities and the underlying molecular mechanisms are unclear. The aim of the present study was to evaluate the effects of DHM on cell proliferation, cell cycle distribution and apoptosis in the human melanoma SK-MEL-28 cell line, and to explore the related mechanisms. The effect of DHM on cell proliferation was investigated by MTT assay, and cell cycle distribution was determined by flow cytometry. TUNEL assay was used to evaluate DHM-mediated apoptosis, and western blotting was applied to examine expression levels of p53, p21, Cdc25A, Cdc2, P-Cdc2, Bax, IKK-α, NF-κB p65, p38 and P-p38 proteins. The results revealed that DHM suppressed cell proliferation of SK-MEL-28 cells in a concentration- and time-dependent manner, and caused cell cycle arrest at the G1/S phase. DHM increased the production of p53 and p21 proteins and downregulated the production of Cdc25A, Cdc2 and P-Cdc2 proteins, which induced cell cycle arrest. Additionally, DHM significantly induced the apoptosis of SK-MEL-28 cells, and enhanced the expression levels of Bax proteins and decreased the protein levels of IKK-α, NF-κB (p65) and P-p38. The results suggest that DHM may be a novel and effective candidate agent to inhibit the growth of melanoma.

  17. Molecular machinery of signal transduction and cell cycle regulation in Plasmodium

    OpenAIRE

    Koyama, Fernanda C.; Chakrabarti, Debopam; Garcia, Célia R.S.

    2009-01-01

    The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is co...

  18. Toona Sinensis Extracts Induced Cell Cycle Arrest and Apoptosis in the Human Lung Large Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Cheng-Yuan Wang

    2010-02-01

    Full Text Available Toona sinensis extracts have been shown to exhibit anti-cancer effects in human ovarian cancer cell lines, human promyelocytic leukemia cells and human lung adenocarcinoma. Its safety has also been confirmed in animal studies. However, its anti-cancer properties in human lung large cell carcinoma have not been studied. Here, we used a powder obtained by freeze-drying the super-natant of centrifuged crude extract from Toona sinensis leaves (TSL-1 to treat the human lung carcinoma cell line H661. Cell viability was evaluated by the 3-(4-,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. Flow cytometry analysis revealed that TSL-1 blocked H661 cell cycle progression. Western blot analysis showed decreased expression of cell cycle proteins that promote cell cycle progression, including cyclin-dependent kinase 4 and cyclin D1, and increased the expression of proteins that inhibit cell cycle progression, including p27. Furthermore, flow cytometry analysis showed that TSL-1 induced H661 cell apoptosis. Western blot analysis showed that TSL-1 reduced the expression of the anti-apoptotic protein B-cell lymphoma 2, and degraded the DNA repair protein, poly(ADP-ribose polymerase. TSL-1 shows potential as a novel therapeutic agent or for use as an adjuvant for treating human lung large cell carcinoma.

  19. Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control

    DEFF Research Database (Denmark)

    Ozaki, Shogo; Schalch-Moser, Annina; Zumthor, Ludwig

    2014-01-01

    that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal......A to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit...

  20. Inhibition of the oncogenic fusion protein EWS-FLI1 causes G2-M cell cycle arrest and enhanced vincristine sensitivity in Ewing's sarcoma.

    Science.gov (United States)

    Zöllner, Stefan K; Selvanathan, Saravana P; Graham, Garrett T; Commins, Ryan M T; Hong, Sung Hyeok; Moseley, Eric; Parks, Sydney; Haladyna, Jessica N; Erkizan, Hayriye V; Dirksen, Uta; Hogarty, Michael D; Üren, Aykut; Toretsky, Jeffrey A

    2017-10-03

    Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergistic therapeutic effect. YK-4-279 rapidly induced G 2 -M arrest, increased the abundance of cyclin B1, and decreased EWS-FLI1-mediated generation of microtubule-associated proteins, which rendered cells more susceptible to microtubule depolymerization by vincristine. YK-4-279 reduced the expression of the EWS-FLI1 target gene encoding the ubiquitin ligase UBE2C, which, in part, contributed to the increase in cyclin B1. YK-4-279 also increased the abundance of proapoptotic isoforms of MCL1 and BCL2, presumably through inhibition of alternative splicing by EWS-FLI1, thus promoting cell death in response to vincristine. Thus, a combination of vincristine and YK-4-279 might be therapeutically effective in ES patients. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  1. Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

    Directory of Open Access Journals (Sweden)

    Peng Xu

    2017-03-01

    Full Text Available Human parvovirus B19 (B19V infection of primary human erythroid progenitor cells (EPCs arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2 within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

  2. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 5: intercellular junctions and contacts between germs cells and Sertoli cells and their regulatory interactions, testicular cholesterol, and genes/proteins associated with more than one germ cell generation.

    Science.gov (United States)

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    In the testis, cell adhesion and junctional molecules permit specific interactions and intracellular communication between germ and Sertoli cells and apposed Sertoli cells. Among the many adhesion family of proteins, NCAM, nectin and nectin-like, catenins, and cadherens will be discussed, along with gap junctions between germ and Sertoli cells and the many members of the connexin family. The blood-testis barrier separates the haploid spermatids from blood borne elements. In the barrier, the intercellular junctions consist of many proteins such as occludin, tricellulin, and claudins. Changes in the expression of cell adhesion molecules are also an essential part of the mechanism that allows germ cells to move from the basal compartment of the seminiferous tubule to the adluminal compartment thus crossing the blood-testis barrier and well-defined proteins have been shown to assist in this process. Several structural components show interactions between germ cells to Sertoli cells such as the ectoplasmic specialization which are more closely related to Sertoli cells and tubulobulbar complexes that are processes of elongating spermatids embedded into Sertoli cells. Germ cells also modify several Sertoli functions and this also appears to be the case for residual bodies. Cholesterol plays a significant role during spermatogenesis and is essential for germ cell development. Lastly, we list genes/proteins that are expressed not only in any one specific generation of germ cells but across more than one generation. Copyright 2009 Wiley-Liss, Inc.

  3. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Kwak

    2016-01-01

    Full Text Available Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC. In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin. Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC.

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

    Science.gov (United States)

    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.

  5. Development of cell-cycle checkpoint therapy for solid tumors.

    Science.gov (United States)

    Tamura, Kenji

    2015-12-01

    Cellular proliferation is tightly controlled by several cell-cycle checkpoint proteins. In cancer, the genes encoding these proteins are often disrupted and cause unrestrained cancer growth. The proteins are over-expressed in many malignancies; thus, they are potential targets for anti-cancer therapies. These proteins include cyclin-dependent kinase, checkpoint kinase, WEE1 kinase, aurora kinase and polo-like kinase. Cyclin-dependent kinase inhibitors are the most advanced cell-cycle checkpoint therapeutics available. For instance, palbociclib (PD0332991) is a first-in-class, oral, highly selective inhibitor of CDK4/6 and, in combination with letrozole (Phase II; PALOMA-1) or with fulvestrant (Phase III; PALOMA-3), it has significantly prolonged progression-free survival, in patients with metastatic estrogen receptor-positive, HER2-negative breast cancer, in comparison with that observed in patients using letrozole, or fulvestrant alone, respectively. In this review, we provide an overview of the current compounds available for cell-cycle checkpoint protein-directed therapy for solid tumors. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Cell Cycle Regulation of Stem Cells by MicroRNAs.

    Science.gov (United States)

    Mens, Michelle M J; Ghanbari, Mohsen

    2018-06-01

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

  7. Fuel cell hybrid taxi life cycle analysis

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Patricia, E-mail: patricia.baptista@ist.utl.pt [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Ribau, Joao; Bravo, Joao; Silva, Carla [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Adcock, Paul; Kells, Ashley [Intelligent Energy, Charnwood Building, HolywellPark, Ashby Road, Loughborough, LE11 3GR (United Kingdom)

    2011-09-15

    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO{sub 2} emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO{sub 2} emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: > A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. > The hydrogen powered vehicles have the lowest energy consumption and CO{sub 2} emissions results. > A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  8. Fuel cell hybrid taxi life cycle analysis

    International Nuclear Information System (INIS)

    Baptista, Patricia; Ribau, Joao; Bravo, Joao; Silva, Carla; Adcock, Paul; Kells, Ashley

    2011-01-01

    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO 2 emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO 2 emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: → A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. → The hydrogen powered vehicles have the lowest energy consumption and CO 2 emissions results. → A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  9. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Science.gov (United States)

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-07-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  10. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Anna Oliva

    2005-07-01

    Full Text Available Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast. The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  11. Cell cycle kinetics and radiation therapy

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1975-01-01

    Radiation therapy as currently practiced involves the subtle largely empirical art of balancing the recurrence of cancer due to undertreatment against severe damage to local tissues due to overtreatment. Therapeutic results too often fall short of desired success rates; yet, the therapist is continually tantalized to the likelihood that a slight shift of therapeutic ratio favoring normal tissue over cancer would have a profoundly beneficial effect. The application of cell cycle kinetics to radiation therapy is one hope for improving the therapeutic ratio; but, as I will try to show, kinetic approaches are complex, poorly understood, and presently too elusive to elicit confidence or to be used clinically. Their promise lies in their diversity and in the magnitude of our ignorance about how they work and how they should be used. Potentially useful kinetic approaches to therapy can be grouped into three classes. The first class takes advantage of intracyclic differential sensitivity, an effect involving the metabolism and biology of the cell cycle; its strategies are based on synchronization of cells over intervals of hours to days. The second class involves the distinction between cycling and noncycling cells; its strategies are based on the resistance of noncycling cells to cycle-linked radiation sensitizers and chemotherapeutic agents. The third class uses cell repopulation between fractions; its strategies are based on the relative growth rates of tumor and relevant normal tissue before and after perturbation

  12. Cell Cycle Inhibition To Treat Sleeping Sickness

    Directory of Open Access Journals (Sweden)

    Conrad L. Epting

    2017-09-01

    Full Text Available 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.

  13. Cell-cycle phase specificity of chloroethylnitrosoureas

    International Nuclear Information System (INIS)

    Linfoot, P.A.

    1986-01-01

    Although the cancer chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is considered a non-cell cycle phase specific drug, it has been shown to produce differential cell killing in G 1 , S, and G 2 /M phase cells, with S phase cells appearing relatively resistant. Studies of cell cycle phase specific cell killing produced by nitrosoureas with different chemical reactivities, clearly indicated that the ability of compounds to cross-link DNA was important in determining their phase specificity. Cells that lacked guanine O 6 -alkytransferase activity showed similar patterns of BCNU phase specificity regardless of their intrinsic sensitivity to BCNU. DNA inter-strand cross-linking, as measured by alkaline elution, was similar in cells exposed to BCNU in G 1 or S phase. 3 H [1-chloroethyl-1nitrosourea] binding to DNA was the same in G 1 , S and G 2 /M phase cells indicating that phase-specific differences in drug uptake and intracellular drug dose were not responsible for phase specific cell kill. These studies suggest that cross-link lesions, other than DNA inter-strand cross-links, and/or effects on DNA repair, other than guanine O 6 -alkyltransferase, are additional important determinants of BCNU phase specific cell killing

  14. Certain amplified genomic-DNA fragments (AGFs) may be involved in cell cycle progression and chloroquine is found to induce the production of cell-cycle-associated AGFs (CAGFs) in Plasmodium falciparum

    OpenAIRE

    Li, Gao-De

    2015-01-01

    It is well known that cyclins are a family of proteins that control cell-cycle progression by activating cyclin-dependent kinase. Based on our experimental results, we propose here a novel hypothesis that certain amplified genomic-DNA fragments (AGFs) may also be required for the cell cycle progression of eukaryotic cells and thus can be named as cell-cycle-associated AGFs (CAGFs). Like fluctuation in cyclin levels during cell cycle progression, these CAGFs are amplified and degraded at diffe...

  15. Control points within the cell cycle

    International Nuclear Information System (INIS)

    Van't Hof, J.

    1984-01-01

    Evidence of the temporal order of chromosomal DNA replication argues favorably for the view that the cell cycle is controlled by genes acting in sequence whose time of expression is determined by mitosis and the amount of nuclear DNA (2C vs 4C) in the cell. Gl and G2 appear to be carbohydrate dependent in that cells starved of either carbohydrate of phosphate fail to make these transitions. Cells deprived of nitrate, however, fail only at Gl to S transition indicating that the controls that operate in G1 differ from those that operate in G2. 46 references, 5 figures

  16. Cell cycle and apoptosis genes in atherosclerosis

    NARCIS (Netherlands)

    Boesten, Lianne Simone Mirjam

    2006-01-01

    The work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder occurring in the large and medium-sized arteries of the body. Although in the beginning 90s promising

  17. Roles of Apicomplexan protein kinases at each life cycle stage.

    Science.gov (United States)

    Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya

    2012-06-01

    Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yoshinori Kagawa

    Full Text Available 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.

  2. Cell cycle regulation of hematopoietic stem or progenitor cells.

    Science.gov (United States)

    Hao, Sha; Chen, Chen; Cheng, Tao

    2016-05-01

    The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

  3. Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

    Science.gov (United States)

    Ma, Shi-Hong; Chen, George G; Ye, Caiguo; Leung, Billy C S; Ho, Rocky L K; Lai, Paul B S

    2011-01-01

    tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Modeling of SONOS Memory Cell Erase Cycle

    Science.gov (United States)

    Phillips, Thomas A.; MacLeod, Todd C.; Ho, Fat H.

    2011-01-01

    Utilization of Silicon-Oxide-Nitride-Oxide-Silicon (SONOS) nonvolatile semiconductor memories as a flash memory has many advantages. These electrically erasable programmable read-only memories (EEPROMs) utilize low programming voltages, have a high erase/write cycle lifetime, are radiation hardened, and are compatible with high-density scaled CMOS for low power, portable electronics. In this paper, the SONOS memory cell erase cycle was investigated using a nonquasi-static (NQS) MOSFET model. Comparisons were made between the model predictions and experimental data.

  5. Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

    International Nuclear Information System (INIS)

    Gupta, Anu; Yang, L.-X.; Chen, L.-C.

    2002-01-01

    Purpose: Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes, Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4A overexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizing radiation. Methods and Materials: The normal mammary epithelial cell line MCF10A was stably transfected with full-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selected and treated with either 8 Gy of ionizing radiation or 7 J/M 2 of UV radiation. The profile of cell cycle progression and the accumulation of several cell cycle proteins were analyzed. Results: We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint in response to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizing radiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in the mock-transfected MCF10A cells, but not in the Cul-4A transfectants. Conclusion: Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly through disruption of cell cycle control

  6. Curcumin Induces Autophagy, Apoptosis, and Cell Cycle Arrest in Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yaping Zhu

    2017-01-01

    Full Text Available Objective. Curcumin is an active extract from turmeric. The aim of this study was to identify the underlying mechanism of curcumin on PCa cells and the role of autophagy in this process. Methods. The inhibitory effect of curcumin on the growth of PANC1 and BxPC3 cell lines was detected by CCK-8 assay. Cell cycle distribution and apoptosis were tested by flow cytometry. Autophagosomes were tested by cell immunofluorescence assay. The protein expression was detected by Western blot. The correlation between LC3II/Bax and cell viability was analyzed. Results. Curcumin inhibited the cell proliferation in a dose- and time-dependent manner. Curcumin could induce cell cycle arrest at G2/M phase and apoptosis of PCa cells. The autophagosomes were detected in the dosing groups. Protein expression of Bax and LC3II was upregulated, while Bcl2 was downregulated in the high dosing groups of curcumin. There was a significant negative correlation between LC3II/Bax and cell viability. Conclusions. Autophagy could be triggered by curcumin in the treatment of PCa. Apoptosis and cell cycle arrest also participated in this process. These findings imply that curcumin is a multitargeted agent for PCa cells. In addition, autophagic cell death may predominate in the high concentration groups of curcumin.

  7. Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence.

    Science.gov (United States)

    Brown, Robert W B; Sharma, Aabha I; Engman, David M

    2017-04-01

    Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include - targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites - Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia - have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.

  8. SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Cao, Rubo; Ding, Qian; Li, Pindong; Xue, Jun; Zou, Zhenwei; Huang, Jing; Peng, Gang

    2013-01-01

    Radioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1). A NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression. Compared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression. SHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance

  9. Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

    International Nuclear Information System (INIS)

    Sun, Ting; Zhang, Zizhu; Li, Bin; Chen, Guilin; Xie, Xueshun; Wei, Yongxin; Wu, Jie; Zhou, Youxin; Du, Ziwei

    2013-01-01

    Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma

  10. Cell cycle-dependent changes in localization of a 210-kDa microtubule-interacting protein in .I.Leishmania./I..

    Czech Academy of Sciences Publication Activity Database

    Libusová, Lenka; Dráberová, Eduarda; Juliano, C.; Viklický, Vladimír; Fiori, P. L.; Cappuccinelli, P.; Dráber, Petr

    2001-01-01

    Roč. 266, č. 2 (2001), s. 270-278 ISSN 0014-4827 R&D Projects: GA ČR GA304/00/0553; GA AV ČR IAA5052004 Keywords : microtubule-associated proteins * Leishmania-antibody Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.096, year: 2001

  11. Analysis of cell-cycle regulation following exposure of lung-derived cells to γ-rays

    Science.gov (United States)

    Trani, D.; Lucchetti, C.; Cassone, M.; D'Agostino, L.; Caputi, M.; Giordano, A.

    Acute exposure of mammalian cells to ionizing radiation results in a delay of cell-cycle progression and/or augmentation of apoptosis. Following ionizing radiation-induced DNA damage, cell-cycle arrest in the G1- or G2-phase of the cell-cycle prevents or delays DNA replication or mitosis, providing time for the DNA repair machinery to exert its function. Deregulation or failing of cell-cycle checkpoints and/or DNA repair mechanisms may lead normal cells bearing chromosome mutations to acquire neoplastic autonomy, which in turn can trigger the onset of cancer. Existing studies have focused on the impact of p53 status on the radiation response of lung cancer (LC) cell lines in terms of both cell-cycle regulation and apoptosis, while no comparative studies have been performed on the radiation response of lung derived normal and cancerous epithelial cells. To investigate the radiation response in normal and cancerous phenotypes, along with the role and impact of p53 status, and possible correlations with pRb/p105 or other proteins involved in carcinogenesis and cell-cycle regulation, we selected two lung-derived epithelial cell lines, one normal (NL20, p53 wild-type) and one non-small cell lung cancer (NSCLC), H358 (known to be p53-deficient). We compared the levels of γ-induced cell proliferation ability, cell-cycle arrest, apoptotic index, and expression levels of cell-cycle regulating and regulated proteins. The different cell sensitivity, apoptotic response and protein expression profiles resulting from our study for NL20 and H358 cells suggest that still unknown mechanisms involving p53, pRb/p105 and their target molecules might play a pivotal role in determining cell sensitivity and resistance upon exposure to ionizing radiation.

  12. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  13. A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

    Science.gov (United States)

    Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

    2012-01-01

    In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

  14. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

    KAUST Repository

    Hudik, Elodie

    2014-07-18

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

  15. Effect of dihydroartemisinin on the cell cycle progress of irradiated human cervical cancer cell line and its mechanism

    International Nuclear Information System (INIS)

    Chen Xialin; Ji Rong; Cao Jianping; Zhu Wei; Fan Sanjun; Wang Jianfang; Cao Jianping

    2010-01-01

    Objective: To observe the changes of cell cycle on cancer cells after dihydroartemisinin and X-ray irradiation. Methods: Human HeLa cells of cervical cancer with p53 mutation was used and human SiHa cells of cervical cancer with wild p53 was used as control. Flow cytometry was used to detect the effect of dihydroartemisinin (20 and 100 μmol/L) and irradiation (6 Gy)on cell cycle. Western blot was used to measure the levels of cell cycle protein. Results: G 2 arrest was observed in irradiated HeLa cells, which the proportion of cells in G 2 phase was increased from 14.45% to 73.58% after 6 Gy X-ray irradiation, but it was abrogated by dihydroartemisinin from 73. 58% to 48.31% in HeLa cells, and it had no change on the SiHa cells. The elevated Wee1 protein and the lowered Cyclin B1 protein were observed with the G 2 arrest severity. The expression of radiation-induced Wee1 protein was suppressed and the Cyclin B1 protein was increased after dihydroartemisinin treatment, which was in accordance with the abrogation of radiation-induced G 2 delay. Conclusions: The main effect of irradiation on cell cycle of p53 mutated HeLa cells is G 2 arrest. Dihydroartemisinin could abrogate it, which is associated with the changes of Wee1 protein and Cyclin B1 protein. In Siha cells, the main effect of irradiation on cell cycle is G 1 arrest, and dihydroartemisinin has no effect on it. (authors)

  16. Endothelial nitric oxide synthase deficiency influences normal cell cycle progression and apoptosis in trabecular meshwork cells

    Directory of Open Access Journals (Sweden)

    Qiong Liao

    2016-06-01

    Full Text Available AIM: To clarify how the endothelial nitric oxide synthase (eNOS, NOS3 make effect on outflow facility through the trabecular meshwork (TM. METHODS: Inhibition of NOS3 gene expression in human TM cells were conducted by three siRNAs. Then the mRNA and protein levels of NOS3 in siRNA-treated and negative control (NC cells were determined, still were the collagen, type IV, alpha 1 (COL4A1 and fibronectin 1 by real-time PCR and Western blot analysis. In addition, NOS3 concentrations in culture supernatant fluids of TM cells were measured. Cell cycle and cell apoptosis analysis were performed using flow cytometry. RESULTS: The mRNA level of NOS3 was decreased by three different siRNA interference, similar results were obtained not only of the relative levels of NOS3 protein, but also the expression levels of COL4A1 and fibronectin 1. The number of cells in S phase was decreased, while contrary result was obtained in G2 phase. The number of apoptotic cells in siRNA-treated groups were significant increased compared to the NC samples. CONCLUSION: Abnormal NOS3 expression can make effects on the proteins levels of extracellular matrix component (e.g. fibronectin 1 and COL4A1. Reduced NOS3 restrains the TM cell cycle progression at the G2/M-phase transition and induced cell apoptosis.

  17. Redox regulation of cell proliferation: Bioinformatics and redox proteomics approaches to identify redox-sensitive cell cycle regulators.

    Science.gov (United States)

    Foyer, Christine H; Wilson, Michael H; Wright, Megan H

    2018-03-29

    Plant stem cells are the foundation of plant growth and development. The balance of quiescence and division is highly regulated, while ensuring that proliferating cells are protected from the adverse effects of environment fluctuations that may damage the genome. Redox regulation is important in both the activation of proliferation and arrest of the cell cycle upon perception of environmental stress. Within this context, reactive oxygen species serve as 'pro-life' signals with positive roles in the regulation of the cell cycle and survival. However, very little is known about the metabolic mechanisms and redox-sensitive proteins that influence cell cycle progression. We have identified cysteine residues on known cell cycle regulators in Arabidopsis that are potentially accessible, and could play a role in redox regulation, based on secondary structure and solvent accessibility likelihoods for each protein. We propose that redox regulation may function alongside other known posttranslational modifications to control the functions of core cell cycle regulators such as the retinoblastoma protein. Since our current understanding of how redox regulation is involved in cell cycle control is hindered by a lack of knowledge regarding both which residues are important and how modification of those residues alters protein function, we discuss how critical redox modifications can be mapped at the molecular level. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Thomas Zerjatke

    2017-05-01

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

  20. Impact of cycling cells and cell cycle regulation on Hydra regeneration.

    Science.gov (United States)

    Buzgariu, Wanda; Wenger, Yvan; Tcaciuc, Nina; Catunda-Lemos, Ana-Paula; Galliot, Brigitte

    2018-01-15

    Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Temporal controls of the asymmetric cell division cycle in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Shenghua Li

    2009-08-01

    Full Text Available The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella.

  2. Temporal controls of the asymmetric cell division cycle in Caulobacter crescentus.

    Science.gov (United States)

    Li, Shenghua; Brazhnik, Paul; Sobral, Bruno; Tyson, John J

    2009-08-01

    The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella).

  3. Maid (GCIP) is involved in cell cycle control of hepatocytes

    DEFF Research Database (Denmark)

    Sonnenberg-Riethmacher, Eva; Wüstefeld, Torsten; Miehe, Michaela

    2007-01-01

    . Therefore, we studied the role of Maid during cell cycle progression after partial hepatectomy (PH). Lack of Maid expression after PH was associated with a delay in G1/S-phase progression as evidenced by delayed cyclinA expression and DNA replication in Maid-deficient mice. However, at later time points......The function of Maid (GCIP), a cyclinD-binding helix-loop-helix protein, was analyzed by targeted disruption in mice. We show that Maid function is not required for normal embryonic development. However, older Maid-deficient mice-in contrast to wild-type controls--develop hepatocellular carcinomas...

  4. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  5. Alteration of cell cycle progression by Sindbis virus infection

    International Nuclear Information System (INIS)

    Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa; Shirasawa, Hiroshi

    2015-01-01

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G 1 phase preferred to proliferate during S/G 2 phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G 1 phase than in cells infected during S/G 2 phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases

  6. Dynamics of the cell-cycle network under genome-rewiring perturbations

    International Nuclear Information System (INIS)

    Katzir, Yair; Elhanati, Yuval; Braun, Erez; Averbukh, Inna

    2013-01-01

    The cell-cycle progression is regulated by a specific network enabling its ordered dynamics. Recent experiments supported by computational models have shown that a core of genes ensures this robust cycle dynamics. However, much less is known about the direct interaction of the cell-cycle regulators with genes outside of the cell-cycle network, in particular those of the metabolic system. Following our recent experimental work, we present here a model focusing on the dynamics of the cell-cycle core network under rewiring perturbations. Rewiring is achieved by placing an essential metabolic gene exclusively under the regulation of a cell-cycle's promoter, forcing the cell-cycle network to function under a multitasking challenging condition; operating in parallel the cell-cycle progression and a metabolic essential gene. Our model relies on simple rate equations that capture the dynamics of the relevant protein–DNA and protein–protein interactions, while making a clear distinction between these two different types of processes. In particular, we treat the cell-cycle transcription factors as limited ‘resources’ and focus on the redistribution of resources in the network during its dynamics. This elucidates the sensitivity of its various nodes to rewiring interactions. The basic model produces the correct cycle dynamics for a wide range of parameters. The simplicity of the model enables us to study the interface between the cell-cycle regulation and other cellular processes. Rewiring a promoter of the network to regulate a foreign gene, forces a multitasking regulatory load. The higher the load on the promoter, the longer is the cell-cycle period. Moreover, in agreement with our experimental results, the model shows that different nodes of the network exhibit variable susceptibilities to the rewiring perturbations. Our model suggests that the topology of the cell-cycle core network ensures its plasticity and flexible interface with other cellular processes

  7. Allyl isothiocyanate affects the cell cycle of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Signe Elisabeth Åsberg

    2015-05-01

    Full Text Available Isothiocyanates (ITCs are degradation products of glucosinolates present in members of the Brassicaceae family acting as herbivore repellents and antimicrobial compounds. Recent results indicate that allyl ITC (AITC has a role in defense responses such as glutathione depletion, ROS generation and stomatal closure. In this study we show that exposure to non-lethal concentrations of AITC causes a shift in the cell cycle distribution of Arabidopsis thaliana leading to accumulation of cells in S-phases and a reduced number of cells in non-replicating phases. Furthermore, transcriptional analysis revealed an AITC-induced up-regulation of the gene encoding cyclin-dependent kinase A while several genes encoding mitotic proteins were down-regulated, suggesting an inhibition of mitotic processes. Interestingly, visualization of DNA synthesis indicated that exposure to AITC reduced the rate of DNA replication. Taken together, these results indicate that non-lethal concentrations of AITC induce cells of A. thaliana to enter the cell cycle and accumulate in S-phases, presumably as a part of a defensive response. Thus, this study suggests that AITC has several roles in plant defense and add evidence to the growing data supporting a multifunctional role of glucosinolates and their degradation products in plants.

  8. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  9. Reprogramming cells with synthetic proteins.

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  10. Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses.

    Directory of Open Access Journals (Sweden)

    Lei Wang

    Full Text Available Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days. Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease.

  11. P27 in cell cycle control and cancer

    DEFF Research Database (Denmark)

    Møller, Michael Boe

    2000-01-01

    In order to survive, cells need tight control of cell cycle progression. The control mechanisms are often lost in human cancer cells. The cell cycle is driven forward by cyclin-dependent kinases (CDKs). The CDK inhibitors (CKIs) are important regulators of the CDKs. As the name implies, CKIs were...

  12. Cell division cycle 20 overexpression predicts poor prognosis for patients with lung adenocarcinoma.

    Science.gov (United States)

    Shi, Run; Sun, Qi; Sun, Jing; Wang, Xin; Xia, Wenjie; Dong, Gaochao; Wang, Anpeng; Jiang, Feng; Xu, Lin

    2017-03-01

    The cell division cycle 20, a key component of spindle assembly checkpoint, is an essential activator of the anaphase-promoting complex. Aberrant expression of cell division cycle 20 has been detected in various human cancers. However, its clinical significance has never been deeply investigated in non-small-cell lung cancer. By analyzing The Cancer Genome Atlas database and using some certain online databases, we validated overexpression of cell division cycle 20 in both messenger RNA and protein levels, explored its clinical significance, and evaluated the prognostic role of cell division cycle 20 in non-small-cell lung cancer. Cell division cycle 20 expression was significantly correlated with sex (p = 0.003), histological classification (p overexpression of cell division cycle 20 was significantly associated with bigger primary tumor size (p = 0.0023), higher MKI67 level (r = 0.7618, p Overexpression of cell division cycle 20 is associated with poor prognosis in lung adenocarcinoma patients, and its overexpression can also be used to identify high-risk groups. In conclusion, cell division cycle 20 might serve as a potential biomarker for lung adenocarcinoma patients.

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

    Science.gov (United States)

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

    2012-08-01

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

  14. ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

    DEFF Research Database (Denmark)

    Høj, Berit Rahbek; la Cour, Peter Jonas Marstrand; Mollerup, Jens

    2009-01-01

    downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate...... that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.......ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2...

  15. Proteomic Analysis of the Cell Cycle of Procylic Form Trypanosoma brucei.

    Science.gov (United States)

    Crozier, Thomas W M; Tinti, Michele; Wheeler, Richard J; Ly, Tony; Ferguson, Michael A J; Lamond, Angus I

    2018-06-01

    We describe a single-step centrifugal elutriation method to produce synchronous Gap1 (G1)-phase procyclic trypanosomes at a scale amenable for proteomic analysis of the cell cycle. Using ten-plex tandem mass tag (TMT) labeling and mass spectrometry (MS)-based proteomics technology, the expression levels of 5325 proteins were quantified across the cell cycle in this parasite. Of these, 384 proteins were classified as cell-cycle regulated and subdivided into nine clusters with distinct temporal regulation. These groups included many known cell cycle regulators in trypanosomes, which validates the approach. In addition, we identify 40 novel cell cycle regulated proteins that are essential for trypanosome survival and thus represent potential future drug targets for the prevention of trypanosomiasis. Through cross-comparison to the TrypTag endogenous tagging microscopy database, we were able to validate the cell-cycle regulated patterns of expression for many of the proteins of unknown function detected in our proteomic analysis. A convenient interface to access and interrogate these data is also presented, providing a useful resource for the scientific community. Data are available via ProteomeXchange with identifier PXD008741 (https://www.ebi.ac.uk/pride/archive/). © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

    Directory of Open Access Journals (Sweden)

    Yoshinori Ohno

    Full Text Available Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

  17. The cell cycle inhibitor p27Kip¹ controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist.

    Science.gov (United States)

    Menchón, Cristina; Edel, Michael J; Izpisua Belmonte, Juan Carlos

    2011-05-01

    The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27(Kip)¹ cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27(Kip)¹ in hESC lead to a G₁phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27(Kip)¹ caused an elongated/scatter cell-like phenotype involving up-regulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27(Kip)¹ protein occupies the Twist1 gene promoter and manipulation of p27(Kip)¹ by gain and loss of function is associated with Twist gene expression changes. These results define p27(Kip)¹ expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27(Kip)¹ in controlling an epithelial to mesenchymal transition (EMT) in hESC.

  18. Architecture and inherent robustness of a bacterial cell-cycle control system.

    Science.gov (United States)

    Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

    2008-08-12

    A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

  19. Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma.

    Science.gov (United States)

    Peponi, Evangelia; Drakos, Elias; Reyes, Guadalupe; Leventaki, Vasiliki; Rassidakis, George Z; Medeiros, L Jeffrey

    2006-12-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

  20. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    Science.gov (United States)

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are

  1. Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chiaro, Christopher, E-mail: cchiaro@tcmedc.org [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States); Lazarova, Darina L., E-mail: dlazarova@tcmedc.org [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States); Bordonaro, Michael, E-mail: mbordonaro@tcmedc.org [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116, does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that

  2. Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

    International Nuclear Information System (INIS)

    Chiaro, Christopher; Lazarova, Darina L.; Bordonaro, Michael

    2012-01-01

    Highlights: ► We investigate mechanisms responsible for butyrate resistance in colon cancer cells. ► Tcf3 modulates butyrate’s effects on Wnt activity and cell growth in resistant cells. ► Tcf3 modulation of butyrate’s effects differ by cell context. ► Cell cycle factors are overexpressed in the resistant cells. ► Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116, does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G 1 to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.

  3. Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

    Directory of Open Access Journals (Sweden)

    Shunsuke Nojiri

    2014-03-01

    Full Text Available Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP, p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD with appropriate software (ModFit LT; BD. The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore. The mRNA levels of AFP relative to Alb(−: Alb(−, Alb(+, and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−, and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(− and p = 0.004 for Prionex, and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(− and Prionex, and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+. More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+ than in Alb(− (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−, Alb

  4. KOH concentration effect on cycle life of nickel-hydrogen cells. III - Cycle life test

    Science.gov (United States)

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

    1988-01-01

    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.

  5. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

    Science.gov (United States)

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  6. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 4: intercellular bridges, mitochondria, nuclear envelope, apoptosis, ubiquitination, membrane/voltage-gated channels, methylation/acetylation, and transcription factors.

    Science.gov (United States)

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic. Copyright 2009 Wiley-Liss, Inc.

  7. Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells

    International Nuclear Information System (INIS)

    Su, Miaoxian; Chung, Hau Yin; Li, Yaolan

    2011-01-01

    Highlights: → Deoxyelephantopin (ESD) inhibited cell proliferation in the human nasopharyngeal cancer CNE cells. → ESD induced cell cycle arrest in S and G2/M phases via modulation of cell cycle regulatory proteins. → ESD triggered apoptosis by dysfunction of mitochondria and induction of both intrinsic and extrinsic apoptotic signaling pathways. → ESD also triggered Akt, ERK, and JNK signaling pathways. -- Abstract: Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential (ΔΨm), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

  8. Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Miaoxian [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Chung, Hau Yin, E-mail: anthonychung@cuhk.edu.hk [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Li, Yaolan [Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou (China); Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Guangzhou (China)

    2011-07-29

    Highlights: {yields} Deoxyelephantopin (ESD) inhibited cell proliferation in the human nasopharyngeal cancer CNE cells. {yields} ESD induced cell cycle arrest in S and G2/M phases via modulation of cell cycle regulatory proteins. {yields} ESD triggered apoptosis by dysfunction of mitochondria and induction of both intrinsic and extrinsic apoptotic signaling pathways. {yields} ESD also triggered Akt, ERK, and JNK signaling pathways. -- Abstract: Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential ({Delta}{Psi}m), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

  9. Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle

    DEFF Research Database (Denmark)

    Grabon, Aby; Orłowski, Adam; Tripathi, Ashutosh

    2017-01-01

    . However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical...... analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability......Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling...

  10. Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

    Science.gov (United States)

    Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

    2014-09-29

    Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

  11. Reprogramming cells with synthetic proteins

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Yang

    2015-06-01

    Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  12. Reprogramming cells with synthetic proteins

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

  13. Cell cycle checkpoints: reversible when possible, irreversible when needed

    NARCIS (Netherlands)

    Krenning, L.

    2015-01-01

    Cell cycle checkpoints are reversible in nature, and can prevent progression into the next cell cycle phase if needed. In the case of DNA damage, cells can prevent progression from G1 into S phase, and from G2 into mitosis in the presence of DNA double strand breaks. Following DNA repair, these

  14. DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2013-09-01

    Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

  15. Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation

    Directory of Open Access Journals (Sweden)

    Brandon M. Invergo

    2017-11-01

    Full Text Available Summary: The transmission of malaria parasites to mosquitoes relies on the rapid induction of sexual reproduction upon their ingestion into a blood meal. Haploid female and male gametocytes become activated and emerge from their host cells, and the males enter the cell cycle to produce eight microgametes. The synchronized nature of gametogenesis allowed us to investigate phosphorylation signaling during its first minute in Plasmodium berghei via a high-resolution time course of the phosphoproteome. This revealed an unexpectedly broad response, with proteins related to distinct cell cycle events undergoing simultaneous phosphoregulation. We implicate several protein kinases in the process, and we validate our analyses on the plant-like calcium-dependent protein kinase 4 (CDPK4 and a homolog of serine/arginine-rich protein kinases (SRPK1. Mutants in these kinases displayed distinct phosphoproteomic disruptions, consistent with differences in their phenotypes. The results reveal the central role of protein phosphorylation in the atypical cell cycle regulation of a divergent eukaryote. : Invergo et al. measure a phosphoproteomic time course during a life cycle transition of a malarial parasite. They observed broad phosphoregulation on a sub-minute scale, including simultaneous regulation of replication- and mitosis-related proteins. Their analyses reveal conserved phosphorylation patterns, and they highlight functional roles of specific protein kinases during this process. Keywords: gametogenesis, proteomics, signal transduction, ARK2, CRK5

  16. Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

    Science.gov (United States)

    Song, Shuichuan; Du, Leiwen; Jiang, Hao; Zhu, Xinhai; Li, Jinhui; Xu, Ji

    2016-10-18

    BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.

  17. Cell cycle controls: potential targets for chemical carcinogens?

    OpenAIRE

    Afshari, C A; Barrett, J C

    1993-01-01

    The progression of the cell cycle is controlled by the action of both positive and negative growth regulators. The key players in this activity include a family of cyclins and cyclin-dependent kinases, which are themselves regulated by other kinases and phosphatases. Maintenance of balanced cell cycle controls may be directly linked to genomic stability. Loss of the check-points involved in cell cycle control may result in unrepaired DNA damage during DNA synthesis or mitosis leading to genet...

  18. Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration

    NARCIS (Netherlands)

    Wagner, Ines; Wang, Heng; Weissert, Philipp M.; Straube, Werner L.; Shevchenko, Anna; Gentzel, Marc; Brito, Goncalo; Tazaki, Akira; Oliveira, Catarina; Sugiura, Takuji; Shevchenko, Andrej; Simon, Andras; Drechsel, David N.; Tanaka, Elly M.

    2017-01-01

    Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell

  19. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    International Nuclear Information System (INIS)

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1996-01-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation mediated by UV radiation. In these experiments, we investigated the effects of exposure to 254 nm radiation on cell cycle progression in the rabbit lens epithelial cell line N/N1003A. The RNA was harvested at various times following exposure to UV (254 nm) radiation and analyzed by dot-blot and northern blot hybridizations. These results revealed that during the first 6 h following exposure of the cells to UV, there was, associated with decreasing dose, a decrease in accumulation of transcripts specific for histones H3 and H4 and an increase in the mRNA encoding protein kinase C and β- and γ-actin. Using flow cytometry, we detected an accumulation of cells in G1/S phase of the cell cycle 1 h following exposure to 254 nm radiation. The observed changes in gene expression, especially the decreased accumulation of histone transcripts reported here, may play a role in UV-induced inhibition of cell cycle progression. (Author)

  20. [Effects of HSP90 inhibitor 17-AAG on cell cycle and apoptosis of human gastric cancer cell lines SGC-7901].

    Science.gov (United States)

    Chen, Meini; Xu, Jinghong; Zhao, Jumei

    2013-02-01

    To study the effect of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on cell proliferation and apoptosis of human cancer SGC-7901 cells and explore the mechanisms. The inhibitory effect of 17-AAG on the proliferation and morphology of SGC-7901 cells was assessed with MTT assay and DNA-PI staining, respectively. Flow cytometry was employed to analyze the changes in cell cycle and apoptosis of the cells following 17-AAG exposure. The cellular expression of Fas protein was detected by immunohistochemistry. 17-AAG significantly suppressed the proliferation of SGC-7901 cells in a time- and dose-dependent manner. After treatment with 17-AAG for 48 h, SGC-7901 cells showed cell cycle arrested at G(2)/M stage, and the cell apoptosis rate increased with the 17-AAG concentration. The expression of Fas protein in the cytoplasm of SGC-7901 cells increased gradually with the increase of 17-AAG concentration. 17-AAG can induce apoptosis, alters the cell cycle distribution and up-regulates the expression of Fas protein in SGC-7901 cells to suppress the cell proliferation.

  1. Cell Cycle Phase Abnormalities Do Not Account for Disordered Proliferation in Barrett's Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Pierre Lao-Sirieix

    2004-11-01

    Full Text Available Barrett's esophagus (BE epithelium is the precursor lesion for esophageal adenocarcinoma. Cell cycle proteins have been advocated as biomarkers to predict the malignant potential in BE. However, whether disruption of the cell cycle plays a causal role in Barrett's carcinogenesis is not clear. Specimens from the Barrett's dysplasia—carcinoma sequence were immunostained for cell cycle phase markers (cyclin D1 for G1; cyclin A for S, G2, and M; cytoplasmic cyclin B1 for G2; and phosphorylated histone 3 for M phase and expressed as a proportion of proliferating cells. Flow cytometric analysis of the cell cycle phase of prospective biopsies was also performed. The proliferation status of nondysplastic BE was similar to gastric antrum and D2, but the proliferative compartment extended to the luminal surface. In dysplastic samples, the number of proliferating cells correlated with the degree of dysplasia (P < .001. The overall levels of cyclins A and B1 correlated with the degree of dysplasia (P < .001. However, the cell cycle phase distribution measured with both immunostaining and flow cytometry was conserved during all stages of BE, dysplasia, and cancer. Hence, the increased proliferation seen in Barrett's carcinogenesis is due to abnormal cell cycle entry or exit, rather than a primary abnormality within the cell cycle.

  2. Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

    Science.gov (United States)

    Koyama, Fernanda C; Chakrabarti, Debopam; Garcia, Célia R S

    2009-05-01

    The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.

  3. Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

    Directory of Open Access Journals (Sweden)

    Giddings Ian

    2011-06-01

    Full Text Available Abstract Background Benzo[a]pyrene (BaP is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP 1A1 and CYP1B1, which are involved in BaP metabolism. Results Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE, the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. Conclusions This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell

  4. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

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    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

  5. A data integration approach for cell cycle analysis oriented to model simulation in systems biology

    Directory of Open Access Journals (Sweden)

    Mosca Ettore

    2007-08-01

    Full Text Available Abstract Background The cell cycle is one of the biological processes most frequently investigated in systems biology studies and it involves the knowledge of a large number of genes and networks of protein interactions. A deep knowledge of the molecular aspect of this biological process can contribute to making cancer research more accurate and innovative. In this context the mathematical modelling of the cell cycle has a relevant role to quantify the behaviour of each component of the systems. The mathematical modelling of a biological process such as the cell cycle allows a systemic description that helps to highlight some features such as emergent properties which could be hidden when the analysis is performed only from a reductionism point of view. Moreover, in modelling complex systems, a complete annotation of all the components is equally important to understand the interaction mechanism inside the network: for this reason data integration of the model components has high relevance in systems biology studies. Description In this work, we present a resource, the Cell Cycle Database, intended to support systems biology analysis on the Cell Cycle process, based on two organisms, yeast and mammalian. The database integrates information about genes and proteins involved in the cell cycle process, stores complete models of the interaction networks and allows the mathematical simulation over time of the quantitative behaviour of each component. To accomplish this task, we developed, a web interface for browsing information related to cell cycle genes, proteins and mathematical models. In this framework, we have implemented a pipeline which allows users to deal with the mathematical part of the models, in order to solve, using different variables, the ordinary differential equation systems that describe the biological process. Conclusion This integrated system is freely available in order to support systems biology research on the cell cycle and

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2018-06-01

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

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

    Science.gov (United States)

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

    2017-11-06

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

  9. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

    Science.gov (United States)

    Reinhardt, H Christian; Yaffe, Michael B

    2013-09-01

    Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

  10. Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells.

    Science.gov (United States)

    Chang, Hsin-Yi; Shih, Meng-Her; Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

    2013-01-01

    There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3-5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G(2)/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G(2)/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.

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

    Science.gov (United States)

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

    2015-03-10

    Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle-dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle-dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A(Cdc55)) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.

  12. Playing with the cell cycle to build the spinal cord.

    Science.gov (United States)

    Molina, Angie; Pituello, Fabienne

    2017-12-01

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

  13. Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

    2018-03-26

    Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

  14. HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

    Science.gov (United States)

    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

  15. Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei.

    Science.gov (United States)

    McDermott, Suzanne M; Guo, Xuemin; Carnes, Jason; Stuart, Kenneth

    2015-10-09

    Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3'-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. A Spatial Control for Correct Timing of Gene Expression during the Escherichia coli Cell Cycle

    Directory of Open Access Journals (Sweden)

    Yuan Yao

    2016-12-01

    Full Text Available Temporal transcriptions of genes are achieved by different mechanisms such as dynamic interaction of activator and repressor proteins with promoters, and accumulation and/or degradation of key regulators as a function of cell cycle. We find that the TorR protein localizes to the old poles of the Escherichia coli cells, forming a functional focus. The TorR focus co-localizes with the nucleoid in a cell-cycle-dependent manner, and consequently regulates transcription of a number of genes. Formation of one TorR focus at the old poles of cells requires interaction with the MreB and DnaK proteins, and ATP, suggesting that TorR delivery requires cytoskeleton organization and ATP. Further, absence of the protein–protein interactions and ATP leads to loss in function of TorR as a transcription factor. We propose a mechanism for timing of cell-cycle-dependent gene transcription, where a transcription factor interacts with its target genes during a specific period of the cell cycle by limiting its own spatial distribution.

  17. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes.

    Science.gov (United States)

    Santos, Alberto; Wernersson, Rasmus; Jensen, Lars Juhl

    2015-01-01

    The eukaryotic cell division cycle is a highly regulated process that consists of a complex series of events and involves thousands of proteins. Researchers have studied the regulation of the cell cycle in several organisms, employing a wide range of high-throughput technologies, such as microarray-based mRNA expression profiling and quantitative proteomics. Due to its complexity, the cell cycle can also fail or otherwise change in many different ways if important genes are knocked out, which has been studied in several microscopy-based knockdown screens. The data from these many large-scale efforts are not easily accessed, analyzed and combined due to their inherent heterogeneity. To address this, we have created Cyclebase--available at http://www.cyclebase.org--an online database that allows users to easily visualize and download results from genome-wide cell-cycle-related experiments. In Cyclebase version 3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNA and protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web interface, designed around an overview figure that summarizes all the cell-cycle-related data for a gene. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Irradiation And Papillomavirus E2 Proteins On Hela Cells

    International Nuclear Information System (INIS)

    Abderrafi, B.

    2005-01-01

    Exposure to relatively high doses ionizing radiation activates cellular responses that impair cell survival. These responses, for which the p53 protein plays a central role, form the basis for cancer radiotherapy. However, the efficacy of radiation treatments on cell killing is often reduced as a consequence of the frequent inactivation of the p53 protein in cancer cells. Loss of p53 protein is associated with later stages of most human tumors and resistance to anticancer agents. Carcinomas are frequent malignant tumors in humans. The majority of cervical carcinomas are etiologically linked to the presence of HPV virus (Human Papillomavirus). In carcinoma tumor cells, as well as in their derived-cell lines such as HeLa cells, the p53 protein is generally not detected due to its degradation by the product of the HPV-associated oncogenic E6 gene. Another characteristic of HPV-positive cervical cancer cells is the loss of the regulatory viral E2 gene expression as a consequence of viral DNA integration into the cellular genome. Reintroduction of E2 expression in HeLa cells reactivates p53, due to a negative effect on the expression of E6 protein, with a concomitant arrest of cell proliferation at the phase G1 of the cell cycle and delay in cell division via the repression of E2F-target genes. To elucidate whether reactivation of p53 would improve the cell killing effect of ionizing radiation in cancer cells, we studied the combined effects of radiation and E2 expression on the cell cycle distribution in HeLa cells

  19. Variety in intracellular diffusion during the cell cycle

    DEFF Research Database (Denmark)

    Selhuber-Unkel, C.; Yde, P.; Berg-Sørensen, Kirstine

    2009-01-01

    During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast...... Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent...... a that is also linked to the viscoelastic moduli of the cytoplasm. The exponent a was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences...

  20. Effects of arsenite on cell cycle progression in a human bladder cancer cell line

    International Nuclear Information System (INIS)

    Hernandez-Zavala, A.; Cordova, E.; Razo, L.M. del; Cebrian, M.E.; Garrido, E.

    2005-01-01

    Bladder cancer is one of the most important diseases associated with arsenic (As) exposure in view of its high prevalence and mortality rate. Experimental studies have shown that As exposure induces cell proliferation in the bladder of sodium arsenite (iAsIII) subchronically treated mice. However, there is little available information on its effects on the cell cycle of bladder cells. Thus, our purpose was to evaluate the effects of iAsIII on cell cycle progression and the response of p53 and p21 on the human-derived epithelial bladder cell line HT1197. iAsIII treatment (1-10 μM) for 24 h induced a dose-dependent increase in the proportion of cells in S-phase, which reached 65% at the highest dose. A progressive reduction in cell proliferation was also observed. BrdU was incorporated to cellular DNA in an interrupted form, suggesting an incomplete DNA synthesis. The time-course of iAsIII effects (10 μM) showed an increase in p53 protein content and a transient increase in p21 protein levels accompanying the changes in S-phase. These effects were correlated with iAs concentrations inside the cells, which were not able to metabolize inorganic arsenic. Our findings suggest that p21 was not able to block CDK2-cyclin E complex activity and was therefore unable to arrest cells in G1 allowing their progression into the S-phase. Further studies are needed to ascertain the mechanisms underlying the effects of iAsIII on the G1 to S phase transition in bladder cells

  1. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  2. The p75NTR tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

    International Nuclear Information System (INIS)

    Khwaja, Fatima; Tabassum, Arshia; Allen, Jeff; Djakiew, Daniel

    2006-01-01

    The p75 neurotrophin receptor (p75 NTR ) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75 NTR retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (ΔDD) dominant-negative antagonist of p75 NTR showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75 NTR -dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75 NTR expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75 NTR rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75 NTR was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75 NTR -dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75 NTR expressing prostate cancer cells

  3. The p75{sup NTR} tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Khwaja, Fatima [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057-1436 (United States); Tabassum, Arshia [Toronto Western Hospital, Toronto, ON, M5T258 (Canada); Allen, Jeff [National Center for Complementary and Alternative Medicine, N.I.H., Bethesda, MD 20892 (United States); Djakiew, Daniel [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057-1436 (United States) and Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057-1436 (United States)

    2006-03-24

    The p75 neurotrophin receptor (p75{sup NTR}) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75{sup NTR} retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted ({delta}DD) dominant-negative antagonist of p75{sup NTR} showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75{sup NTR}-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75{sup NTR} expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75{sup NTR} rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75{sup NTR} was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75{sup NTR}-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75{sup NTR} expressing prostate cancer cells.

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

    Science.gov (United States)

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

    2015-06-01

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

  5. Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

    Science.gov (United States)

    Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

    2012-01-01

    Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

  6. Boletus edulis biologically active biopolymers induce cell cycle arrest in human colon adenocarcinoma cells.

    Science.gov (United States)

    Lemieszek, Marta Kinga; Cardoso, Claudia; Ferreira Milheiro Nunes, Fernando Hermínio; Ramos Novo Amorim de Barros, Ana Isabel; Marques, Guilhermina; Pożarowski, Piotr; Rzeski, Wojciech

    2013-04-25

    The use of biologically active compounds isolated from edible mushrooms against cancer raises global interest. Anticancer properties are mainly attributed to biopolymers including mainly polysaccharides, polysaccharopeptides, polysaccharide proteins, glycoproteins and proteins. In spite of the fact that Boletus edulis is one of the widely occurring and most consumed edible mushrooms, antitumor biopolymers isolated from it have not been exactly defined and studied so far. The present study is an attempt to extend this knowledge on molecular mechanisms of their anticancer action. The mushroom biopolymers (polysaccharides and glycoproteins) were extracted with hot water and purified by anion-exchange chromatography. The antiproliferative activity in human colon adenocarcinoma cells (LS180) was screened by means of MTT and BrdU assays. At the same time fractions' cytotoxicity was examined on the human colon epithelial cells (CCD 841 CoTr) by means of the LDH assay. Flow cytometry and Western blotting were applied to cell cycle analysis and protein expression involved in anticancer activity of the selected biopolymer fraction. In vitro studies have shown that fractions isolated from Boletus edulis were not toxic against normal colon epithelial cells and in the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells. The best results were obtained in the case of the fraction designated as BE3. The tested compound inhibited cancer cell proliferation which was accompanied by cell cycle arrest in the G0/G1-phase. Growth inhibition was associated with modulation of the p16/cyclin D1/CDK4-6/pRb pathway, an aberration of which is a critical step in the development of many human cancers including colon cancer. Our results indicate that a biopolymer BE3 from Boletus edulis possesses anticancer potential and may provide a new therapeutic/preventive option in colon cancer chemoprevention.

  7. Connecting the nucleolus to the cell cycle and human disease.

    Science.gov (United States)

    Tsai, Robert Y L; Pederson, Thoru

    2014-08-01

    Long known as the center of ribosome synthesis, the nucleolus is connected to cell cycle regulation in more subtle ways. One is a surveillance system that reacts promptly when rRNA synthesis or processing is impaired, halting cell cycle progression. Conversely, the nucleolus also acts as a first-responder to growth-related stress signals. Here we review emerging concepts on how these "infraribosomal" links between the nucleolus and cell cycle progression operate in both forward and reverse gears. We offer perspectives on how new cancer therapeutic designs that target this infraribosomal mode of cell growth control may shape future clinical progress. © FASEB.

  8. Repressive histone methylation regulates cardiac myocyte cell cycle exit.

    Science.gov (United States)

    El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

    2018-05-22

    Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.

  9. Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

    International Nuclear Information System (INIS)

    Bhadriraju, Kiran; Hansen, Linda K.

    2004-01-01

    Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation

  10. The Cell Cycle: An Activity Using Paper Plates to Represent Time Spent in Phases of the Cell Cycle

    Science.gov (United States)

    Scherer, Yvette D.

    2014-01-01

    In this activity, students are given the opportunity to combine skills in math and geometry for a biology lesson in the cell cycle. Students utilize the data they collect and analyze from an online onion-root-tip activity to create a paper-plate time clock representing a 24-hour cell cycle. By dividing the paper plate into appropriate phases of…

  11. Responses of genes involved in cell cycle control to diverse DNA damaging chemicals in human lung adenocarcinoma A549 cells

    Directory of Open Access Journals (Sweden)

    Gooderham Nigel J

    2005-08-01

    Full Text Available Abstract Background Many anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The molecular mechanisms of DNA damage-induced cell cycle alteration are not well understood. We have studied the effects of etoposide (an anticancer agent, cryptolepine (CLP, a cytotoxic alkaloid, benzo [a]pyrene (BaP, a carcinogenic polycyclic aromatic hydrocarbon and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP, a cooked-meat derived carcinogen on the expression of cell cycle regulatory genes to understand the molecular mechanisms of the cell cycle disturbance. Results A549 cells were treated with DMSO or chemicals for up to 72 h and periodically sampled for cell cycle analysis, mRNA and protein expression. DMSO treated cells showed a dominant G1 peak in cell cycle at all times examined. Etoposide and CLP both induced G2/M phase arrest yet the former altered the expression of genes functioning at multiple phases, whilst the latter was more effective in inhibiting the expression of genes in G2-M transition. Both etoposide and CLP induced an accumulation of p53 protein and upregulation of p53 transcriptional target genes. Neither BaP nor PhIP had substantial phase-specific cell cycle effect, however, they induced distinctive changes in gene expression. BaP upregulated the expression of CYP1B1 at 6–24 h and downregulated many cell cycle regulatory genes at 48–72 h. By contrast, PhIP increased the expression of many cell cycle regulatory genes. Changes in the expression of key mRNAs were confirmed at protein level. Conclusion Our experiments show that DNA damaging agents with different mechanisms of action induced distinctive changes in the expression pattern of a panel of cell cycle regulatory genes. We suggest that examining the genomic response to chemical exposure provides an exceptional opportunity to understand the molecular mechanism involved in cellular

  12. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  13. Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

    Directory of Open Access Journals (Sweden)

    Rahmberg Andrew R

    2011-05-01

    Full Text Available Abstract Background Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Results Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Conclusions Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

  14. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Jennifer L Bandura

    Full Text Available The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C, suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  15. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    Science.gov (United States)

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

  16. Distinct mechanisms act in concert to mediate cell cycle arrest.

    Science.gov (United States)

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

    2009-01-20

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

  17. Resveratrol induces cell cycle arrest and apoptosis in human eosinophils from asthmatic individuals.

    Science.gov (United States)

    Hu, Xin; Wang, Jing; Xia, Yu; Simayi, Mihereguli; Ikramullah, Syed; He, Yuanbing; Cui, Shihong; Li, Shuang; Wushouer, Qimanguli

    2016-12-01

    Eosinophils exert a number of inflammatory effects through the degranulation and release of intracellular mediators, and are considered to be key effector cells in allergic disorders, including asthma. In order to investigate the regulatory effects of the natural polyphenol, resveratrol, on eosinophils derived from asthmatic individuals, the cell counting Kit‑8 assay and flow cytometry analysis were used to determine cell proliferation and cell cycle progression in these cells, respectively. Cellular apoptosis was detected using annexin V-fluorescein isothiocyanate/propidium iodide double‑staining. The protein expression levels of p53, p21, cyclin‑dependent kinase 2 (CDK2), cyclin A, cyclin E, Bim, B‑cell lymphoma (Bcl)‑2 and Bcl‑2‑associated X protein (Bax) were measured by western blot analysis following resveratrol treatment. The results indicated that resveratrol effectively suppressed the proliferation of eosinophils from asthmatic patients in a concentration‑ and time‑dependent manner. In addition, resveratrol was observed to arrest cell cycle progression in G1/S phase by increasing the protein expression levels of p53 and p21, and concurrently reducing the protein expression levels of CDK2, cyclin A and cyclin E. Furthermore, resveratrol treatment significantly induced apoptosis in eosinophils, likely through the upregulation of Bim and Bax protein expression levels and the downregulation of Bcl‑2 protein expression. These findings suggested that resveratrol may be a potential agent for the treatment of asthma by decreasing the number of eosinophils.

  18. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido

    2007-09-15

    When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

  19. Brucella abortus Cell Cycle and Infection Are Coordinated.

    Science.gov (United States)

    De Bolle, Xavier; Crosson, Sean; Matroule, Jean-Yves; Letesson, Jean-Jacques

    2015-12-01

    Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

    Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

  1. Synchronization and Arrest of the Budding Yeast Cell Cycle Using Chemical and Genetic Methods.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture. Of note, cell growth and division are decoupled during arrest and block-release experiments. Cells will continue transcription, translation, and accumulation of protein while arrested. If allowed to reenter the cell cycle, cells will do so as a population of mixed, larger-than-normal cells. Despite this important caveat, many aspects of budding yeast physiology are accessible using these simple chemical and genetic tools. Described here are methods for the block and release of cells in G 1 phase and at the M/G 1 transition using α-factor mating pheromone and the temperature-sensitive cdc15-2 allele, respectively, in addition to methods for arresting the cell cycle in early S phase and at G 2 /M by using hydroxyurea and nocodazole, respectively. © 2017 Cold Spring Harbor Laboratory Press.

  2. Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death.

    Science.gov (United States)

    Ur Rahman, Muhammad Saif; Zhang, Ling; Wu, Lingyan; Xie, Yuqiong; Li, Chunchun; Cao, Jiang

    2017-01-01

    Severe side effects are major problems with chemotherapy of gastric cancer (GC). These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB) was used with other DNA linker agents mitomycin C (MMC), cisplatin (DDP), or cyclophosphamide (CTX) to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC 50 ) by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G 0 /G 1 and G 1 /S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC cells to the alkylating agents via arresting the cell cycle and enhancing cell death. This is of significant therapeutic importance in the reduction of side effects associated with these drugs.

  3. The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

    Directory of Open Access Journals (Sweden)

    Fuerst John A

    2009-01-01

    Full Text Available Abstract Background Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy. Results Using phase contrast and fluorescence light microscopy, and transmission electron microscopy, the cell division cycle of G. obscuriglobus was determined. During the budding process, the bud was formed and developed in size from one point of the mother cell perimeter until separation. The matured daughter cell acted as a new mother cell and started its own budding cycle while the mother cell can itself initiate budding repeatedly. Fluorescence microscopy of DAPI-stained cells of G. obscuriglobus suggested that translocation of the nucleoid and formation of the bud did not occur at the same time. Confocal laser scanning light microscopy applied to cells stained for membranes as

  4. Whi7 is an unstable cell-cycle repressor of the Start transcriptional program.

    Science.gov (United States)

    Gomar-Alba, Mercè; Méndez, Ester; Quilis, Inma; Bañó, M Carmen; Igual, J Carlos

    2017-08-24

    Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCF Grr1 ubiquitin-ligase, whose stability is cell cycle regulated by CDK1 phosphorylation. Importantly, Whi7 associates to G1/S gene promoters in late G1 acting as a repressor of SBF-dependent transcription. Our results demonstrate that Whi7 is a genuine paralog of Whi5. In fact, both proteins collaborate in Start repression bringing to light that yeast cells, as occurs in mammalian cells, rely on the combined action of multiple transcriptional repressors to block Start transition.The commitment of cells to a new cycle of division involves inactivation of the Start transcriptional repressor Whi5. Here the authors show that the sequence related protein Whi7 associates to G1/S gene promoters in late G1 and collaborates with Whi5 in Start repression.

  5. Timing the Generation of Distinct Retinal Cells by Homeobox Proteins

    Science.gov (United States)

    Decembrini, Sarah; Andreazzoli, Massimiliano; Vignali, Robert; Barsacchi, Giuseppina; Cremisi, Federico

    2006-01-01

    The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of “sensors” in which green fluorescent protein translation is under control of the 3′ untranslated region (UTR), we found that the 3′ UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities. PMID:16903786

  6. Timing the generation of distinct retinal cells by homeobox proteins.

    Directory of Open Access Journals (Sweden)

    Sarah Decembrini

    2006-09-01

    Full Text Available The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR, we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

  7. Uncovering Viral Protein-Protein Interactions and their Role in Arenavirus Life Cycle

    Directory of Open Access Journals (Sweden)

    Nora López

    2012-09-01

    Full Text Available The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article.

  8. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Bauer, K.D.

    1989-01-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

  9. Effect of specific silencing of EMMPRIN on the growth and cell cycle distribution of MCF-7 breast cancer cells.

    Science.gov (United States)

    Yang, X Q; Yang, J; Wang, R; Zhang, S; Tan, Q W; Lv, Q; Meng, W T; Mo, X M; Li, H J

    2015-12-02

    The extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin family and shows increased expression in tumor cells. We examined the effect of RNAi-mediated EMMPRIN gene silencing induced by lentiviral on the growth and cycle distribution of MCF-7 breast cancer cells. Lentiviral expressing EMMPRIN-short hairpin RNA were packaged to infect MCF-7 cells. The inhibition efficiency of EMMPRIN was validated by real-time fluorescent quantitation polymerase chain reaction and western blotting. The effect of EMMPRIN on cell proliferation ability was detected using the MTT assay and clone formation experiments. Changes in cell cycle were detected by flow cytometry. EMMPRIN-short hairpin RNA-packaged lentiviral significantly down-regulated EMMPRIN mRNA and protein expression, significantly inhibited cell proliferation and in vitro tumorigenicity, and induced cell cycle abnormalities. Cells in the G0/G1 and G2/M phases were increased, while cells in the S phase were decreased after infection of MCF-7 cells for 3 days. The EMMPRIN gene facilitates breast cancer cell malignant proliferation by regulating cell cycle distribution and may be a molecular target for breast cancer gene therapy.

  10. Imaging protein-protein interactions in living cells

    NARCIS (Netherlands)

    Hink, M.A.; Bisseling, T.; Visser, A.J.W.G.

    2002-01-01

    The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide

  11. RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Houcai; Yu, Jing; Zhang, Lixia; Xiong, Yuanyuan; Chen, Shuying; Xing, Haiyan; Tian, Zheng; Tang, Kejing; Wei, Hui; Rao, Qing; Wang, Min; Wang, Jianxiang, E-mail: wangjx@ihcams.ac.cn

    2014-04-18

    Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.

  12. Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle.

    Science.gov (United States)

    Wheelhouse, Nick; Sait, Michelle; Wilson, Kim; Aitchison, Kevin; McLean, Kevin; Smith, David G E; Longbottom, David

    2012-12-07

    It has been suggested that polymorphic membrane proteins (Pmps) belonging to the Type V autotransporter protein family play an important role in the pathogenesis of Chlamydia abortus (C. abortus; formerly Chlamydophila abortus) infection. In a previous study we demonstrated the expression of all the pmps at the transcriptional level. The purpose of this study was to measure the number of Pmp positive inclusions throughout the C. abortus developmental cycle to investigate heterogeneity in expression patterns. McCoy cells were infected with C. abortus and analysed for Pmp expression over a 72 h period by fluorescent immunocytochemistry. Pmp18D could be detected at all analysed time points, and could only be accurately quantified from 36 hpi while Pmp10G positive inclusions could be visualised from 36hpi. Expression of Pmps 13G, 16G and 17G could only be visualised later in the cycle and within less than half of visualised inclusions. These results indicate that while expression of specific Pmps is constitutive (Pmp18D), the pattern of expression of other Pmps is more variable. This suggests that different members of the Pmp family may play different roles within the developmental cycle of the organism, with some (Pmps10G and 18D) having roles throughout the cycle, while the heterogeneity of expression of others may aid in antigenic variation. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Discrete gene replication events drive coupling between the cell cycle and circadian clocks.

    Science.gov (United States)

    Paijmans, Joris; Bosman, Mark; Ten Wolde, Pieter Rein; Lubensky, David K

    2016-04-12

    Many organisms possess both a cell cycle to control DNA replication and a circadian clock to anticipate changes between day and night. In some cases, these two rhythmic systems are known to be coupled by specific, cross-regulatory interactions. Here, we use mathematical modeling to show that, additionally, the cell cycle generically influences circadian clocks in a nonspecific fashion: The regular, discrete jumps in gene-copy number arising from DNA replication during the cell cycle cause a periodic driving of the circadian clock, which can dramatically alter its behavior and impair its function. A clock built on negative transcriptional feedback either phase-locks to the cell cycle, so that the clock period tracks the cell division time, or exhibits erratic behavior. We argue that the cyanobacterium Synechococcus elongatus has evolved two features that protect its clock from such disturbances, both of which are needed to fully insulate it from the cell cycle and give it its observed robustness: a phosphorylation-based protein modification oscillator, together with its accompanying push-pull read-out circuit that responds primarily to the ratios of different phosphoform concentrations, makes the clock less susceptible to perturbations in protein synthesis; the presence of multiple, asynchronously replicating copies of the same chromosome diminishes the effect of replicating any single copy of a gene.

  14. Combination of ascorbate/epigallocatechin-3-gallate/gemcitabine synergistically induces cell cycle deregulation and apoptosis in mesothelioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Martinotti, Simona [Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale “Amedeo Avogadro”, viale T. Michel 11, 15121 Alessandria (Italy); Ranzato, Elia, E-mail: ranzato@unipmn.it [Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale “Amedeo Avogadro”, viale T. Michel 11, 15121 Alessandria (Italy); Parodi, Monica [IRCCS A.O.U. S. Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova (Italy); DI.ME.S., Università degli Studi di Genova, Via L. Alberti 2, 16132 Genova (Italy); Vitale, Massimo [IRCCS A.O.U. S. Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova (Italy); Burlando, Bruno [Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale “Amedeo Avogadro”, viale T. Michel 11, 15121 Alessandria (Italy)

    2014-01-01

    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, 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.

  15. Situational Awareness: Regulation of the Myb Transcription Factor in Differentiation, the Cell Cycle and Oncogenesis

    Energy Technology Data Exchange (ETDEWEB)

    George, Olivia L.; Ness, Scott A., E-mail: sness@salud.unm.edu [Department of Internal Medicine, Section of Molecular Medicine, University of New Mexico Health Sciences Center, MSC07 4025-CRF 121, 1 University of New Mexico, Albuquerque, NM 87131 (United States)

    2014-10-02

    This review summarizes the mechanisms that control the activity of the c-Myb transcription factor in normal cells and tumors, and discusses how c-Myb plays a role in the regulation of the cell cycle. Oncogenic versions of c-Myb contribute to the development of leukemias and solid tumors such as adenoid cystic carcinoma, breast cancer and colon cancer. The activity and specificity of the c-Myb protein seems to be controlled through changes in protein-protein interactions, so understanding how it is regulated could lead to the development of novel therapeutic strategies.

  16. Absence of p53 in Clara cells favours multinucleation and loss of cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Clarke Alan R

    2002-11-01

    Full Text Available Abstract Background The p53 oncosuppressor protein is a critical mediator of the response to injury in mammalian cells and is mutationally inactivated in the majority of lung malignancies. In this analysis, the effects of p53-deficiency were investigated in short-term primary cultures of murine bronchiolar Clara cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates. Results p53 null cultures displayed abnormal morphology; specifically, a high incidence of multinucleation, which increased with time in culture. Multinucleated cells were proficient in S phase DNA synthesis, as determined by BrdU incorporation. However, multinucleation did not reflect altered rates of S phase synthesis, which were similar between wild type and p53-/- cultures. Nucleation defects in p53-/- Clara cells associated with increased centrosome number, as determined by confocal microscopy of pericentrin-stained cultures, and may highlight a novel role of p53 in preserving genomic integrity in lung epithelial cells. Effects of p53-deficiency were also studied following exposure to DNA damage. A p53-dependent reduction in the BrdU index was observed in Clara cells following ionizing radiation. The reduction in BrdU index in wild type cells displayed serum-dependency, and occurred only in the absence of serum. Taken together, these findings demonstrate that in murine primary Clara cell culture, cell cycle arrest is a p53-mediated response to DNA damage, and that extracellular factors, such as serum, influence this response. Conclusion These findings highlight functions of wild type p53 protein in bipolar spindle formation, centrosome regulation, and growth control in bronchiolar Clara cells.

  17. Analysis of the Budding Yeast Cell Cycle by Flow Cytometry.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    DNA synthesis is one of the landmark events in the cell cycle: G 1 cells have one copy of the genome, S phase cells are actively engaged in DNA synthesis, and G 2 cells have twice as much nuclear DNA as G 1 cells. Cellular DNA content can be measured by staining with a fluorescent dye followed by a flow-cytometric readout. This method provides a quantitative measurement of cell cycle position on a cell-by-cell basis at high speed. Using flow cytometry, tens of thousands of single-cell measurements can be generated in a few seconds. This protocol details staining of cells of the budding yeast Saccharomyces cerevisiae for flow cytometry using Sytox Green dye in a method that can be scaled widely-from one sample to many thousands and operating on inputs ranging from 1 million to more than 100 million cells. Flow cytometry is preferred over light microscopy or Coulter analyses for the analysis of the cell cycle as DNA content and cell cycle position are being directly measured. © 2017 Cold Spring Harbor Laboratory Press.

  18. Studies on regulation of the cell cycle in fission yeast.

    Directory of Open Access Journals (Sweden)

    Miroslava Požgajová

    2015-05-01

    Full Text Available All living organisms including plants and animals are composed of millions of cells. These cells perform different functions for the organism although they possess the same chromosomes and carry the same genetic information. Thus, to be able to understand multicellular organism we need to understand the life cycle of individual cells from which the organism comprises. The cell cycle is the life cycle of a single cell in the plant or animal body. It involves series of events in which components of the cell doubles and afterwards equally segregate into daughter cells. Such process ensures growth of the organism, and specialized reductional cell division which leads to production of gamets, assures sexual reproduction. Cell cycle is divided in the G1, S, G2 and M phase. Two gap-phases (G1 and G2 separate S phase (or synthesis and M phase which stays either for mitosis or meiosis. Essential for normal life progression and reproduction is correct chromosome segregation during mitosis and meiosis. Defects in the division program lead to aneuploidy, which in turn leads to birth defects, miscarriages or cancer. Even thou, researchers invented much about the regulation of the cell cycle, there is still long way to understand the complexity of the regulatory machineries that ensure proper segregation of chromosomes. In this paper we would like to describe techniques and materials we use for our studies on chromosome segregation in the model organism Schizosaccharomyces pombe.

  19. Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

    International Nuclear Information System (INIS)

    Playle, L.C.

    2001-03-01

    The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

  20. 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    Directory of Open Access Journals (Sweden)

    Sakai Keiko

    2008-04-01

    Full Text Available Abstract Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.

  1. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes

    DEFF Research Database (Denmark)

    Santos Delgado, Alberto; Wernersson, Rasmus; Jensen, Lars Juhl

    2015-01-01

    3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNAand protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web interface...

  2. Isoalantolactone inhibits UM-SCC-10A cell growth via cell cycle arrest and apoptosis induction.

    Directory of Open Access Journals (Sweden)

    Minjun Wu

    Full Text Available Isoalantolactone is a sesquiterpene lactone compound isolated from the roots of Inula helenium L. Previous studies have demonstrated that isoalantolactone possesses antifungal, anti-bacterial, anti-helminthic and anti-proliferative properties in a variety of cells, but there are no studies concerning its effects on head and neck squamous cell carcinoma (HNSCC. In the present study, an MTT assay demonstrated that isoalantolactone has anti-proliferative activity against the HNSCC cell line (UM-SCC-10A. Immunostaining identified that this compound induced UM-SCC-10A cell apoptosis but not necrosis. To explain the molecular mechanisms underlying its effects, flow cytometry and western blot analysis showed that the apoptosis was associated with cell cycle arrest during the G1 phase, up-regulation of p53 and p21, and down-regulation of cyclin D. Furthermore, our results revealed that induction of apoptosis through a mitochondrial pathway led to up-regulation of pro-apoptotic protein expression (Bax, down-regulation of anti-apoptotic protein expression (Bcl-2, mitochondrial release of cytochrome c (Cyto c, reduction of mitochondrial membrane potential (MMP and activation of caspase-3 (Casp-3. Involvement of the caspase apoptosis pathway was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. Together, our findings suggest that isoalantolactone induced caspase-dependent apoptosis via a mitochondrial pathway and was associated with cell cycle arrest in the G1 phase in UM-SCC-10A cells. Therefore, isoalantolactone may become a potential drug for treating HNSCC.

  3. Drosophila cell cycle under arrest: uncapped telomeres plead guilty.

    Science.gov (United States)

    Cenci, Giovanni

    2009-04-01

    Telomeres are specialized structures that protect chromosome ends from degradation and fusion events. In most organisms, telomeres consist of short, repetitive G-rich sequences added to chromosome ends by a reverse transcriptase with an internal RNA template, called telomerase. Specific DNA-binding protein complexes associate with telomeric sequences preventing chromosome ends from being recognized as DNA double strand breaks (DSBs). Telomeres that lose their cap activate the DNA damage response (DDR) likewise DSBs and, if inappropriately repaired, generate telomeric fusions, which eventually lead to genome instability. In Drosophila there is not telomerase, and telomere length is maintained by transposition of three specialized retroelements. However, fly telomeres are protected by multi protein complexes like their yeast and vertebrate counterparts; these complexes bind chromosome ends in a sequence-independent fashion and are required to prevent checkpoint activation and end-to-end fusion. Uncapped Drosophila telomeres elicit a DDR just as dysfunctional human telomeres. Most interestingly, uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. BubR1 accumulations at chromosome ends trigger the SAC that inhibits the metaphase-to-anaphase transition. These findings, reviewed here, highlight an intriguing and unsuspected connection between telomeres and cell cycle regulation, providing a clue to understand human telomere function.

  4. Cellular plasticity enables adaptation to unforeseen cell-cycle rewiring challenges.

    Directory of Open Access Journals (Sweden)

    Yair Katzir

    Full Text Available The fundamental dynamics of the cell cycle, underlying cell growth and reproduction, were previously found to be robust under a wide range of environmental and internal perturbations. This property was commonly attributed to its network structure, which enables the coordinated interactions among hundreds of proteins. Despite significant advances in deciphering the components and autonomous interactions of this network, understanding the interfaces of the cell cycle with other major cellular processes is still lacking. To gain insight into these interfaces, we used the process of genome-rewiring in yeast by placing an essential metabolic gene HIS3 from the histidine biosynthesis pathway, under the exclusive regulation of different cell-cycle promoters. In a medium lacking histidine and under partial inhibition of the HIS3p, the rewired cells encountered an unforeseen multitasking challenge; the cell-cycle regulatory genes were required to regulate the essential histidine-pathway gene in concert with the other metabolic demands, while simultaneously driving the cell cycle through its proper temporal phases. We show here that chemostat cell populations with rewired cell-cycle promoters adapted within a short time to accommodate the inhibition of HIS3p and stabilized a new phenotypic state. Furthermore, a significant fraction of the population was able to adapt and grow into mature colonies on plates under such inhibiting conditions. The adapted state was shown to be stably inherited across generations. These adaptation dynamics were accompanied by a non-specific and irreproducible genome-wide transcriptional response. Adaptation of the cell-cycle attests to its multitasking capabilities and flexible interface with cellular metabolic processes and requirements. Similar adaptation features were found in our previous work when rewiring HIS3 to the GAL system and switching cells from galactose to glucose. Thus, at the basis of cellular plasticity is

  5. Cellular plasticity enables adaptation to unforeseen cell-cycle rewiring challenges.

    Science.gov (United States)

    Katzir, Yair; Stolovicki, Elad; Stern, Shay; Braun, Erez

    2012-01-01

    The fundamental dynamics of the cell cycle, underlying cell growth and reproduction, were previously found to be robust under a wide range of environmental and internal perturbations. This property was commonly attributed to its network structure, which enables the coordinated interactions among hundreds of proteins. Despite significant advances in deciphering the components and autonomous interactions of this network, understanding the interfaces of the cell cycle with other major cellular processes is still lacking. To gain insight into these interfaces, we used the process of genome-rewiring in yeast by placing an essential metabolic gene HIS3 from the histidine biosynthesis pathway, under the exclusive regulation of different cell-cycle promoters. In a medium lacking histidine and under partial inhibition of the HIS3p, the rewired cells encountered an unforeseen multitasking challenge; the cell-cycle regulatory genes were required to regulate the essential histidine-pathway gene in concert with the other metabolic demands, while simultaneously driving the cell cycle through its proper temporal phases. We show here that chemostat cell populations with rewired cell-cycle promoters adapted within a short time to accommodate the inhibition of HIS3p and stabilized a new phenotypic state. Furthermore, a significant fraction of the population was able to adapt and grow into mature colonies on plates under such inhibiting conditions. The adapted state was shown to be stably inherited across generations. These adaptation dynamics were accompanied by a non-specific and irreproducible genome-wide transcriptional response. Adaptation of the cell-cycle attests to its multitasking capabilities and flexible interface with cellular metabolic processes and requirements. Similar adaptation features were found in our previous work when rewiring HIS3 to the GAL system and switching cells from galactose to glucose. Thus, at the basis of cellular plasticity is the emergence of a yet

  6. Proliferation marker pKi-67 affects the cell cycle in a self-regulated manner.

    Science.gov (United States)

    Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Duchrow, Michael

    2002-01-01

    The proliferation marker pKi-67 is commonly used in research and pathology to detect proliferating cells. In a previous work, we found the protein to be associated with regulators of the cell cycle, controlling S-phase progression, as well as entry into and exit from mitosis. Here we investigate whether pKi-67 has a regulative effect on the cell cycle itself. For that purpose we cloned four fragments of pKi-67, together representing nearly the whole protein, and an N-terminal pKi-67 antisense oligonucleotide into a tetracycline inducible gene expression system. The sense fragments were C-terminally modified by addition of either a nuclear localization sequence (NLS) or a STOP codon to address the impact of their intracellular distribution. FACS based cell cycle analysis revealed that expression of nearly all pKi-67 domains and the antisense oligonucleotide led to a decreased amount of cells in S-phase and an increased number of cells in G(2)/M- and G(1)-phase. Subsequent analysis of the endogenous pKi-67 mRNA and protein levels revealed that the constructs with the most significant impact on the cell cycle were able to silence pKi-67 transcription as well. We conclude from the data that pKi-67 influences progression of S-phase and mitosis in a self-regulated manner and, therefore, effects the cell cycle checkpoints within both phases. Furthermore, we found pKi-67 mediates an anti-apoptotic effect on the cell and we verified that this marker, although it is a potential ribosomal catalyst, is not expressed in differentiated tissues with a high transcriptional activity. Copyright 2002 Wiley-Liss, Inc.

  7. Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

    Science.gov (United States)

    Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

    2018-03-05

    Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

  8. Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data

    DEFF Research Database (Denmark)

    Celis, J E; Gesser, B; Rasmussen, H H

    1990-01-01

    , mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat...

  9. Performances of Saft Lithium-Ion Cells in LEO Cycling

    Directory of Open Access Journals (Sweden)

    Prevot D.

    2017-01-01

    The article will thus present the whole LEO cycling results available for the two cells, and will provide afterwards the correlation status of Saft Li-ion Model (SLIM with all the experimental data acquired.

  10. Cellular Clocks : Coupled Circadian Dispatch and Cell Division Cycles

    NARCIS (Netherlands)

    Merrow, Martha; Roenneberg, Till

    2004-01-01

    Gating of cell division by the circadian clock is well known, yet its mechanism is little understood. Genetically tractable model systems have led to new hypotheses and questions concerning the coupling of these two cellular cycles.

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

    Science.gov (United States)

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

    2017-08-01

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

  12. Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle.

    Directory of Open Access Journals (Sweden)

    Selene Glück

    Full Text Available In infected cells rotavirus (RV replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2 involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.

  13. Role of Kupffer Cells in Thioacetamide-Induced Cell Cycle Dysfunction

    Directory of Open Access Journals (Sweden)

    Mirandeli Bautista

    2011-09-01

    Full Text Available It is well known that gadolinium chloride (GD attenuates drug-induced hepatotoxicity by selectively inactivating Kupffer cells. In the present study the effect of GD in reference to cell cycle and postnecrotic liver regeneration induced by thioacetamide (TA in rats was studied. Two months male rats, intraveously pretreated with a single dose of GD (0.1 mmol/Kg, were intraperitoneally injected with TA (6.6 mmol/Kg. Samples of blood and liver were obtained from rats at 0, 12, 24, 48, 72 and 96 h following TA intoxication. Parameters related to liver damage were determined in blood. In order to evaluate the mechanisms involved in the post-necrotic regenerative state, the levels of cyclin D and cyclin E as well as protein p27 and Proliferating Cell Nuclear Antigen (PCNA were determined in liver extracts because of their roles in the control of cell cycle check-points. The results showed that GD significantly reduced the extent of necrosis. Noticeable changes were detected in the levels of cyclin D1, cyclin E, p27 and PCNA when compared to those induced by thioacetamide. Thus GD pre-treatment reduced TA-induced liver injury and accelerated the postnecrotic liver regeneration. These results demonstrate that Kupffer cells are involved in TA-induced liver and also in the postnecrotic proliferative liver states.

  14. The regulation effect of STAT 5 signaling pathway on the cell cycle progression of irradiated KG-1 cells

    International Nuclear Information System (INIS)

    Guo Dehuang; Dong Bo; Luo Qingliang; Wen Gengyun; Mao Bingzhi

    2000-01-01

    The author investigated the role of the JAK/STAT signaling pathway regulating cell cycle progression in the irradiated KG-1 cells. By permanent transfecting the cells with DN-STAT 5 cDNA to block the JAK/STAT signaling pathway and then transient transfecting with cyclin D 1 or cyclin B 1 cDNA, the effects of cyclin D 1 protein and cyclin B 1 protein on the cell cycle progression were examined. Results showed that after irradiation with 8Gy 60 Co rays, the irradiated KG-1 cells transfected with only DN-STAT 5 cDNA can not recover form the G 1 arrest, even though GM-CSF was added. Meanwhile, the cells transfected with both the DN-STAT 5 cDNA and cyclin D 1 cDNA or cyclin B 1 cDNA can recover from the G 1 arrest or the G 2 arrest to a great extent. Thus, it was proved indirectly that the JAK/STAT signaling pathway activated by GM-CSF regulated the cell cycle progression through cyclin D 1 and cyclin B 1 protein

  15. Keith's MAGIC: Cloning and the Cell Cycle.

    Science.gov (United States)

    Wells, D N

    2013-10-01

    Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.

  16. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ulf Geisen

    2015-07-01

    Full Text Available Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1. Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application.

  17. Molecular Cogs: Interplay between Circadian Clock and Cell Cycle.

    Science.gov (United States)

    Gaucher, Jonathan; Montellier, Emilie; Sassone-Corsi, Paolo

    2018-05-01

    The cell cycle and the circadian clock operate as biological oscillators whose timed functions are tightly regulated. Accumulating evidence illustrates the presence of molecular links between these two oscillators. This mutual interplay utilizes various coupling mechanisms, such as the use of common regulators. The connection between these two cyclic systems has unique interest in the context of aberrant cell proliferation since both of these oscillators are frequently misregulated in cancer cells. Further studies will provide deeper understanding of the detailed molecular connections between the cell cycle and the circadian clock and may also serve as a basis for the design of innovative therapeutic strategies. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-Wen [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hsieh, Bau-Shan; Cheng, Hsiao-Ling [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hu, Yu-Chen [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chang, Wen-Tsan [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Division of Hepatobiliarypancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan (China); Chang, Kee-Lung, E-mail: Chang.KeeLung@msa.hinet.net [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

    2012-01-15

    Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M

  19. The timing of T cell priming and cycling

    Directory of Open Access Journals (Sweden)

    Reinhard eObst

    2015-11-01

    Full Text Available The proliferation of specific lymphocytes is the central tenet of the clonal selection paradigm. Antigen recognition by T cells triggers a series of events that produces expanded clones of differentiated effector cells. TCR signaling events are detectable within seconds and minutes and are likely to continue for hours and days in vivo. Here, I review the work done on the importance of TCR signals in the later part of the expansion phase of the primary T cell response, primarily regarding the regulation of the cell cycle in CD4+ and CD8+ cells. The results suggest a degree of programming by early signals for effector differentiation, particularly in the CD8+ T cell compartment, with optimal expansion supported by persistent antigen presentation later on. Differences to CD4+ T cell expansion and new avenues towards a molecular understanding of cell cycle regulation in lymphocytes are discussed.

  20. Thermally regenerative hydrogen/oxygen fuel cell power cycles

    Science.gov (United States)

    Morehouse, J. H.

    1986-01-01

    Two innovative thermodynamic power cycles are analytically examined for future engineering feasibility. The power cycles use a hydrogen-oxygen fuel cell for electrical energy production and use the thermal dissociation of water for regeneration of the hydrogen and oxygen. The TDS (thermal dissociation system) uses a thermal energy input at over 2000 K to thermally dissociate the water. The other cycle, the HTE (high temperature electrolyzer) system, dissociates the water using an electrolyzer operating at high temperature (1300 K) which receives its electrical energy from the fuel cell. The primary advantages of these cycles is that they are basically a no moving parts system, thus having the potential for long life and high reliability, and they have the potential for high thermal efficiency. Both cycles are shown to be classical heat engines with ideal efficiency close to Carnot cycle efficiency. The feasibility of constructing actual cycles is investigated by examining process irreversibilities and device efficiencies for the two types of cycles. The results show that while the processes and devices of the 2000 K TDS exceed current technology limits, the high temperature electrolyzer system appears to be a state-of-the-art technology development. The requirements for very high electrolyzer and fuel cell efficiencies are seen as determining the feasbility of the HTE system, and these high efficiency devices are currently being developed. It is concluded that a proof-of-concept HTE system experiment can and should be conducted.

  1. The Dynamical Mechanisms of the Cell Cycle Size Checkpoint

    International Nuclear Information System (INIS)

    Feng Shi-Fu; Yang Ling; Yan Jie; Liu Zeng-Rong

    2012-01-01

    Cell division must be tightly coupled to cell growth in order to maintain cell size, whereas the mechanisms of how initialization of mitosis is regulated by cell size remain to be elucidated. We develop a mathematical model of the cell cycle, which incorporates cell growth to investigate the dynamical properties of the size checkpoint in embryos of Xenopus laevis. We show that the size checkpoint is naturally raised from a saddle-node bifurcation, and in a mutant case, the cell loses its size control ability due to the loss of this saddle-node point

  2. Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration.

    Science.gov (United States)

    Wagner, Ines; Wang, Heng; Weissert, Philipp M; Straube, Werner L; Shevchenko, Anna; Gentzel, Marc; Brito, Goncalo; Tazaki, Akira; Oliveira, Catarina; Sugiura, Takuji; Shevchenko, Andrej; Simon, András; Drechsel, David N; Tanaka, Elly M

    2017-03-27

    Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2018-05-22

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

  4. The cancer-germline antigen SSX2 causes cell cycle arrest and DNA damage in cancer cells

    DEFF Research Database (Denmark)

    Greve, Katrine Buch Vidén; Lindgreen, Jonas; Terp, Mikkel Green

    2011-01-01

    The SSX family of cancer and germline antigens is mainly expressed in the germ cells of healthy individuals as well as wide range of cancers and is therefore potential targets for immunotherapy. However, little is known about the role of SSX proteins in tumorigenesis and normal cell function. Here......, we show that SSX2 is involved in regulation of cancer cell growth. We found that ectopic expression of SSX2 in melanoma and colon cancer cells strongly reduced cell growth and induced apoptosis in vitro. Importantly, in a xenograft mouse model, the growth of tumors derived from SSX2 overexpressing...... melanoma cells was severely reduced compared to those derived from the isogenic parental cell line. Cell cycle analysis showed that SSX2 caused an accumulation of cells arrested in G1. Consistent with this, we observed a marked decrease in cells expressing the proliferation marker Ki67 and concomitantly...

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

    Science.gov (United States)

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

    2017-10-01

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

  6. Dual Pressure versus Hybrid Recuperation in an Integrated Solid Oxide Fuel Cell Cycle – Steam Cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud

    2014-01-01

    A SOFC (solid oxide fuel cell) cycle running on natural gas was integrated with a ST (steam turbine) cycle. The fuel is desulfurized and pre-reformed before entering the SOFC. A burner was used to combust the remaining fuel after the SOFC stacks. The off-gases from the burner were used to produce...... pressure configuration steam cycle combined with SOFC cycle (SOFC-ST) was new and has not been studied previously. In each of the configuration, a hybrid recuperator was used to recovery the remaining energy of the off-gases after the HRSG. Thus, four different plants system setups were compared to each...... other to reveal the most superior concept with respect to plant efficiency and power. It was found that in order to increase the plant efficiency considerably, it was enough to use a single pressure with a hybrid recuperator instead of a dual pressure Rankine cycle....

  7. Cell cycles and proliferation patterns in Haematococcus pluvialis

    Science.gov (United States)

    Zhang, Chunhui; Liu, Jianguo; Zhang, Litao

    2017-09-01

    Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation; far less attention has been paid to cell cycles and proliferation patterns. The purpose of this study was to clarify cell cycles and proliferation patterns in H. pluvialis microscopically using a camera and video recorder system. The complicated life history of H. pluvialis can be divided into two stages: the motile stage and the non-motile stage. All the cells can be classified into forms as follows: motile cell, nonmotile cell, zoospore and aplanospore. The main cell proliferation, both in the motile phase and non-motile phase in H. pluvialis, is by asexual reproduction. Under normal growth conditions, a motile cell usually produces two, sometimes four, and exceptionally eight zoospores. Under unfavorable conditions, the motile cell loses its flagella and transforms into a non-motile cell, and the non-motile cell usually produces 2, 4 or 8 aplanospores, and occasionally 20-32 aplanospores, which further develop into non-motile cells. Under suitable conditions, the non-motile cell is also able to release zoospores. The larger non-motile cells produce more than 16 zoospores, and the smaller ones produce 4 or 8 zoospores. Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase. There is, as yet, no convincing direct evidence for sexual reproduction.

  8. Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

    Science.gov (United States)

    Farioli-Vecchioli, Stefano; Mattera, Andrea; Micheli, Laura; Ceccarelli, Manuela; Leonardi, Luca; Saraulli, Daniele; Costanzi, Marco; Cestari, Vincenzo; Rouault, Jean-Pierre; Tirone, Felice

    2014-07-01

    Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis. © 2014 AlphaMed Press.

  9. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

  10. Plant Characteristics of an Integrated Solid Oxide Fuel Cell Cycle and a Steam Cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud

    2010-01-01

    Plant characteristics of a system containing a solid oxide fuel cell (SOFC) cycle on the top of a Rankine cycle were investigated. Natural gas (NG) was used as the fuel for the plant. A desulfurization reactor removes the sulfur content in the fuel, while a pre-reformer broke down the heavier...... recovery steam generator (HRSG). The remaining energy of the off-gases was recycled back to the topping cycle for further utilization. Several parameter studies were carried out to investigate the sensitivity of the suggested plant. It was shown that the operation temperature of the desulfurization unit...

  11. Role of insulin-like growth factor-1 (IGF-1) in regulating cell cycle progression

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Qi-lin; Yang, Tian-lun [Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan (China); Yin, Ji-ye [Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan (China); Peng, Zhen-yu [Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan (China); Yu, Min [Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan (China); Liu, Zhao-qian, E-mail: liuzhaoqian63@126.com [Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan (China); Chen, Fang-ping, E-mail: xychenfp@public.cs.hn.Cn [Department of Haematology, Xiangya Hospital, Central South University, Changsha 410008, Hunan (China)

    2009-11-06

    Aims: Insulin-like growth factor-1 (IGF-1) is a polypeptide protein hormone, similar in molecular structure to insulin, which plays an important role in cell migration, cell cycle progression, cell survival and proliferation. In this study, we investigated the possible mechanisms of IGF-1 mediated cell cycle redistribution and apoptosis of vascular endothelial cells. Method: Human umbilical vein endothelial cells (HUVECs) were pretreated with 0.1, 0.5, or 2.5 {mu}g/mL of IGF-1 for 30 min before the addition of Ang II. Cell cycle redistribution and apoptosis were examined by flow cytometry. Expression of Ang II type 1 (AT{sub 1}) mRNA and cyclin E protein were determined by RT-PCR and Western blot, respectively. Results: Ang II (1 {mu}mol/L) induced HUVECs arrested at G{sub 0}/G{sub 1}, enhanced the expression level of AT{sub 1} mRNA in a time-dependent manner, reduced the enzymatic activity of nitric oxide synthase (NOS) and nitric oxide (NO) content as well as the expression level of cyclin E protein. However, IGF-1 enhanced NOS activity, NO content, and the expression level of cyclin E protein, and reduced the expression level of AT{sub 1} mRNA. L-NAME significantly counteracted these effects of IGF-1. Conclusions: Our data suggests that IGF-1 can reverse vascular endothelial cells arrested at G{sub 0}/G{sub 1} and apoptosis induced by Ang II, which might be mediated via a NOS-NO signaling pathway and is likely associated with the expression levels of AT1 mRNA and cyclin E proteins.

  12. Role of insulin-like growth factor-1 (IGF-1) in regulating cell cycle progression

    International Nuclear Information System (INIS)

    Ma, Qi-lin; Yang, Tian-lun; Yin, Ji-ye; Peng, Zhen-yu; Yu, Min; Liu, Zhao-qian; Chen, Fang-ping

    2009-01-01

    Aims: Insulin-like growth factor-1 (IGF-1) is a polypeptide protein hormone, similar in molecular structure to insulin, which plays an important role in cell migration, cell cycle progression, cell survival and proliferation. In this study, we investigated the possible mechanisms of IGF-1 mediated cell cycle redistribution and apoptosis of vascular endothelial cells. Method: Human umbilical vein endothelial cells (HUVECs) were pretreated with 0.1, 0.5, or 2.5 μg/mL of IGF-1 for 30 min before the addition of Ang II. Cell cycle redistribution and apoptosis were examined by flow cytometry. Expression of Ang II type 1 (AT 1 ) mRNA and cyclin E protein were determined by RT-PCR and Western blot, respectively. Results: Ang II (1 μmol/L) induced HUVECs arrested at G 0 /G 1 , enhanced the expression level of AT 1 mRNA in a time-dependent manner, reduced the enzymatic activity of nitric oxide synthase (NOS) and nitric oxide (NO) content as well as the expression level of cyclin E protein. However, IGF-1 enhanced NOS activity, NO content, and the expression level of cyclin E protein, and reduced the expression level of AT 1 mRNA. L-NAME significantly counteracted these effects of IGF-1. Conclusions: Our data suggests that IGF-1 can reverse vascular endothelial cells arrested at G 0 /G 1 and apoptosis induced by Ang II, which might be mediated via a NOS-NO signaling pathway and is likely associated with the expression levels of AT1 mRNA and cyclin E proteins.

  13. Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli.

    Science.gov (United States)

    Kato , J; Katayama, T

    2001-08-01

    The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replication.

  14. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

  15. Neurosecretory cells of the amygdaloid complex during estrous cycle.

    Science.gov (United States)

    Akhmadeev, A V; Kalimullina, L B

    2005-02-01

    Ultrastructure of neurosecretory cells of the dorsomedial nucleus of the cerebral amygdaloid complex (one of the main zones of sexual dimorphism) was studied in different phases of the estrous cycle. The characteristics of the "light" and "dark" cells change depending on the concentrations of sex steroids during estrus and metestrus.

  16. [Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

    Science.gov (United States)

    Li, Dayu; Liu, Yun; Yu, Chunbo; Liu, Xiping; Fan, Fang

    2017-12-01

    Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

  17. Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

    Science.gov (United States)

    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.

  18. RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. Pt. 1

    International Nuclear Information System (INIS)

    Skog, S.; Tribukait, B.; Sundius, G.

    1985-01-01

    The effects of roentgen irradiation on the incorporation of 3 H-uridine and 14 C-leucine into RNA and protein and the RNA and protein contents of in vivo growing Ehrlich ascites tumour cells were studied. The results were related to changes in the composition of cells in cell cycle and compared with the synthesis of RNA and protein in cell material from various parts of the cell cycle obtained by means of elutriator centrifuging. The incorporation expressed by the ratio between acid insoluble/acid soluble activity was unchanged for RNA during the observation period up to 24 hours after a dose of 5.0 Gy. The ratio for protein was markedly decreased between 4 and 24 hours. This decrease was partly due to a decrease of the pool size of leucine as studied by changing the amounts of 14 C leucine used. From these studies, the existence of at least two pools, an expandable and a non-expandable fixed pool can be concluded. There were no differences in the decrease of protein-synthesis between cells from the various parts of the cell cycle. The RNA and protein contents of the irradiated cells from various parts of the cell cycle corresponded to those of non-irradiated cells except for G 1 /early S-phase cells at 15 and 24 hours after irradiation. Possible reasons for this discrepancy are discussed. (orig.)

  19. Radiation could induce p53-independent and cell cycle - unrelated apoptosis in 5-fluorouracil radiosensitized head and neck carcinoma cells

    International Nuclear Information System (INIS)

    Didelot, C.; Mirjolet, J.F.; Barberi-Heyob, M.; Ramacci, C.; Merlin, J.L.

    2002-01-01

    The effect of chemoresistance induction in radio sensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G 2 /M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G 2 /M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G 2 /M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis. the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model. (author)

  20. Insect Cells as Hosts for Recombinat Proteins

    OpenAIRE

    Murwani, Retno

    1997-01-01

    Since the development of recombinant baculovirus expression system, insect cell culture has rapidly gain popularity as the method of choice for production of a variety of biologically active proteins. Up to date tens of recombinant protein have been produced by this method commercially or non-commercially and have been widely used for research. This review describes the basic concept of baculovirus expression vector and the use of insect cells as host for recombinant proteins. Examples of the...

  1. Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Morris Robert

    2010-03-01

    Full Text Available Abstract Background Sulforaphane (SFN, an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC. The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC. Results SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose-polymerase (PARP. Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex. Conclusions SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.

  2. Functional analysis of the cell cycle regulator Rca1 in Drosophila melanogaster

    OpenAIRE

    Zielke, Norman

    2007-01-01

    Tight regulation of APC/C activity is essential for cell cycle progression. An important class of negative APC/C regulators are the Rca1/Emi1 family proteins. All members of the Rca1/Emi1 family share a conserved zinc binding region (ZBR) which is essential for their inhibitory activity. The Rca1/Emi1 proteins belong to the class of F-box proteins that are known to act as substrate recognition subunits in SCF-E3-ligase complexes. Emi1 and Rca1 interact in vitro with members of the Skp family ...

  3. Establishment of human papillomavirus infection requires cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Dohun Pyeon

    2009-02-01

    Full Text Available Human papillomaviruses (HPVs are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these

  4. Cell Division, a new open access online forum for and from the cell cycle community

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2006-04-01

    Full Text Available Abstract Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases.

  5. Radiotherapy and chemotherapy after partial synchronization of cell cycle

    International Nuclear Information System (INIS)

    Hermann, H.J.; Ammon, J.; Nuevemann, M.; Zum Winkel, K.; Technische Hochschule Aachen

    1977-01-01

    Apart from densely ionising radiations, radiotherapy and chemotherapy after partial synchronisation of the cell cycle are, at the moment, the only way to improve the efficiency of a treatment of malignant tumours. The new principle is based on the finding that tumour cells are more sensitive to radiation or chemotherapy in a certain metabolic situation. Partial synchronisation of the cell cycle makes it possible to enrich tumour cells in a certain metabolic state. In order to show the efficiency of such a measure, several methods can be used. Recently, impulse cytophotometry has been replacing these methods, since it permits a quick, simple, and individual control of the synchronisation effect. However, there has not been any clinical experiment yet to prove that tumour cells show a maximum sensitivity to radio- and chemotherapy in the G 2 -M-phase. This is why a number of patients with malignant tumours which could not be operated or treated with the usual radiotherapy or polychemotherapy were treated according to this new therapeutic principle. The results obtained in 233 cases encourage the specialists to continue the experiments. The indication of a treatment after partial synchronisation of the cell cycle should be based on the tumour spread as documented according to the TNM-system. Only when these guidelines are followed will it be possible to explain the problems still unsolved in the principle of radiotherapy and chemotherapy after partial synchronisation of the cell cycle and to carry out radio- and chemotherapy with improved efficiency in the future. (orig./MG) [de

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

    Science.gov (United States)

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

    2018-04-01

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

  7. Cell cycle related /sup 125/IUDR-induced-division delay

    International Nuclear Information System (INIS)

    Scheniderman, M.H.; Hofer, K.G.

    1987-01-01

    A series of experiments were run to determine if /sup 125/I-decays, in /sup 125/IUdR labeled DNA, specifically accumulated at 1, 3, 5, 7 and 9 hours after plating labeled mitotic cells caused a change in the rate or time of cell entry into mitosis. To accomplish this, a pool of labeled mitotic cells was selected in mitosis and plated in replicate flasks. /sup 125/I decays were accumulated in groups of cells by cooling (4 0 C) for 2 hours starting at the designated times. After rewarding, colcemid was added to arrest cells in mitosis. The rate of cell progression into mitosis for each cell cycle time of accumulation was determined by scoring the mitotic index of cells sampled as a function of time after addition of the colcemid. The results are summarized: (1) Decays from /sup 125/I in /sup 125/I(UdR) labeled DNA reduced the rate of cell progression into mitosis and delayed the time of initiation of mitosis. (2) The reduced rate of progression and the delayed time of initiation of mitosis were independent of the cell cycle time that /sup 125/I-decays were accumulated. (3) The reduced rate of progression after cell cycle accumulation of /sup 125/I decay was statistically indistinguishable from the corresponding controls. (4) The delayed initiation of mitosis after specific cell cycle accumulation of /sup 125/I- decays was greater than the corresponding control. The relationship of these data to DNA and non-DNA division delay target(s) is emphasized

  8. Achillea millefolium L. hydroethanolic extract inhibits growth of human tumor cell lines by interfering with cell cycle and inducing apoptosis.

    Science.gov (United States)

    Pereira, Joana M; Peixoto, Vanessa; Teixeira, Alexandra; Sousa, Diana; Barros, Lillian; Ferreira, Isabel C F R; Vasconcelos, M Helena

    2018-06-05

    The cell growth inhibitory activity of the hydroethanolic extract of Achillea millefolium was studied in human tumor cell lines (NCI-H460 and HCT-15) and its mechanism of action was investigated. The GI 50 concentration was determined with the sulforhodamine B assay and cell cycle and apoptosis were analyzed by flow cytometry following incubation with PI or Annexin V FITC/PI, respectively. The expression levels of proteins involved in cell cycle and apoptosis were analyzed by Western blot. The extracts were characterized regarding their phenolic composition by LC-DAD-ESI/MS. 3,5-O-Dicaffeoylquinic acid, followed by 5-O-caffeoylquinic acid, were the main phenolic acids, while, luteolin-O-acetylhexoside and apigenin-O-acetylhexoside were the main flavonoids. This extract decreased the growth of the tested cell lines, being more potent in HCT-15 and then in NCI-H460 cells. Two different concentrations of the extract (75 and 100 μg/mL) caused alterations in cell cycle profile and increased apoptosis levels in HCT-15 and NCI-H460 cells. Moreover, the extract caused an increase in p53 and p21 expression in NCI-H460 cells (which have wt p53), and reduced XIAP levels in HCT-15 cells (with mutant p53). This work enhances the importance of A. millefolium as source of bioactive phenolic compounds, particularly of XIAP inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Sukawa, Yumiko; Okamoto, Takashi

    2018-03-01

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

  11. Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    Shan, Xiaoyin; Roberts, Cleresa; Kim, Eun Ji; Brenner, Ariana; Grant, Gregory; Percec, Ivona

    2017-05-01

    Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401. © 2017 AlphaMed Press.

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

    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.

  14. Circadian clock, cell cycle and cancer

    Directory of Open Access Journals (Sweden)

    Cansu Özbayer

    2011-12-01

    Full Text Available There are a few rhythms of our daily lives that we are under the influence. One of them is characterized by predictable changes over a 24-hour timescale called circadian clock. This cellular clock is coordinated by the suprachiasmatic nucleus in the anterior hypothalamus. The clock consist of an autoregulatory transcription-translation feedback loop compose of four genes/proteins; BMAL1, Clock, Cyrptochrome, and Period. BMAL 1 and Clock are transcriptional factors and Period and Cyrptochrome are their targets. Period and Cyrptochrome dimerize in the cytoplasm to enter the nucleus where they inhibit Clock/BMAL activity.It has been demonstrate that circadian clock plays an important role cellular proliferation, DNA damage and repair mechanisms, checkpoints, apoptosis and cancer.

  15. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  16. Angular-dependent light scattering from cancer cells in different phases of the cell cycle.

    Science.gov (United States)

    Lin, Xiaogang; Wan, Nan; Weng, Lingdong; Zhou, Yong

    2017-10-10

    Cancer cells in different phases of the cell cycle result in significant differences in light scattering properties. In order to harvest cancer cells in particular phases of the cell cycle, we cultured cancer cells through the process of synchronization. Flow cytometric analysis was applied to check the results of cell synchronization and prepare for light scattering measurements. Angular-dependent light scattering measurements of cancer cells arrested in the G1, S, and G2 phases have been performed. Based on integral calculations for scattering intensities from 5° to 10° and from 110° to 150°, conclusions have been reached. Clearly, the sizes of the cancer cells in different phases of the cell cycle dominated the forward scatter. Accompanying the increase of cell size with the progression of the cell cycle, the forward scattering intensity also increased. Meanwhile, the DNA content of cancer cells in every phase of the cell cycle is responsible for light scattering at large scatter angles. The higher the DNA content of cancer cells was, the greater the positive effect on the high-scattering intensity. As expected, understanding the relationships between the light scattering from cancer cells and cell cycles will aid in the development of cancer diagnoses. Also, it may assist in the guidance of antineoplastic drugs clinically.

  17. Identification of a novel EGF-sensitive cell cycle checkpoint

    International Nuclear Information System (INIS)

    Walker, Francesca; Zhang Huihua; Burgess, Antony W.

    2007-01-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells

  18. Labeling of lectin receptors during the cell cycle.

    Science.gov (United States)

    Garrido, J

    1976-12-01

    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

  19. Metabolic behavior of cell surface biotinylated proteins

    International Nuclear Information System (INIS)

    Hare, J.F.; Lee, E.

    1989-01-01

    The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins

  20. Effect of magnetic nanoparticles on apoptosis and cell cycle induced by wogonin in Raji cells

    Directory of Open Access Journals (Sweden)

    Wang XM

    2012-02-01

    Full Text Available Lei Wang1,2,*, Haijun Zhang1,2,*, Baoan Chen1,2, Guohua Xia1,2, Shuai Wang1,2, Jian Cheng1,2, Zeye Shao1,2, Chong Gao1,2, Wen Bao1,2, Liang Tian1,2, Yanyan Ren1,2, Peipei Xu1,2, Xiaohui Cai1,2, Ran Liu1,2, Xuemei Wang3 1Department of Hematology and Oncology, Zhongda Hospital, Medical School, 2Faculty of Oncology, Medical School, 3State Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory, Southeast University, Nanjing, China*These authors contributed equally to this workAbstract: Traditional Chinese medicine is gradually becoming a new source of anticancer drugs. One such example is wogonin, which is cytotoxic to various cancer cell lines in vitro. However, due to its low water solubility, wogonin is restricted to clinical administration. Recently, the application of drug-coated magnetic nanoparticles (MNPs to increase water solubility of the drug and to enhance its chemotherapeutic efficiency has attracted much attention. In this study, wogonin was conjugated with the drug delivery system of MNPs by mechanical absorption polymerization to fabricate wogonin-loaded MNPs. It was demonstrated that MNPs could strengthen wogonin-induced cell inhibition, apoptosis, and cell cycle arrest in Raji cells by methylthiazol tetrazolium assay, flow cytometer assay, and nuclear 4',6-diamidino-2-phenylindole staining. Furthermore, the molecular mechanisms of these phenomena were explored by western blot, in which the protein levels of caspase 8 and caspase 3 were increased significantly while those of survivin and cyclin E were decreased significantly in wogonin-MNPs group. These findings suggest that the combination of wogonin and MNPs provides a promising strategy for lymphoma therapy.Keywords: wogonin, magnetic nanoparticles, Raji cell, apoptosis, cell cycle, caspase 8, caspase 3, survivin, cyclin E

  1. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  2. A combined gas cooled nuclear reactor and fuel cell cycle

    Science.gov (United States)

    Palmer, David J.

    Rising oil costs, global warming, national security concerns, economic concerns and escalating energy demands are forcing the engineering communities to explore methods to address these concerns. It is the intention of this thesis to offer a proposal for a novel design of a combined cycle, an advanced nuclear helium reactor/solid oxide fuel cell (SOFC) plant that will help to mitigate some of the above concerns. Moreover, the adoption of this proposal may help to reinvigorate the Nuclear Power industry while providing a practical method to foster the development of a hydrogen economy. Specifically, this thesis concentrates on the importance of the U.S. Nuclear Navy adopting this novel design for its nuclear electric vessels of the future with discussion on efficiency and thermodynamic performance characteristics related to the combined cycle. Thus, the goals and objectives are to develop an innovative combined cycle that provides a solution to the stated concerns and show that it provides superior performance. In order to show performance, it is necessary to develop a rigorous thermodynamic model and computer program to analyze the SOFC in relation with the overall cycle. A large increase in efficiency over the conventional pressurized water reactor cycle is realized. Both sides of the cycle achieve higher efficiencies at partial loads which is extremely important as most naval vessels operate at partial loads as well as the fact that traditional gas turbines operating alone have poor performance at reduced speeds. Furthermore, each side of the cycle provides important benefits to the other side. The high temperature exhaust from the overall exothermic reaction of the fuel cell provides heat for the reheater allowing for an overall increase in power on the nuclear side of the cycle. Likewise, the high temperature helium exiting the nuclear reactor provides a controllable method to stabilize the fuel cell at an optimal temperature band even during transients helping

  3. Thermodynamics of protein destabilization in live cells.

    Science.gov (United States)

    Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

    2015-10-06

    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

  4. CycleBase.org - a comprehensive multi-organism online database of cell-cycle experiments

    DEFF Research Database (Denmark)

    Gauthier, Nicholas Paul; Larsen, Malene Erup; Wernersson, Rasmus

    2007-01-01

    The past decade has seen the publication of a large number of cell-cycle microarray studies and many more are in the pipeline. However, data from these experiments are not easy to access, combine and evaluate. We have developed a centralized database with an easy-to-use interface, Cyclebase...

  5. Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle.

    Science.gov (United States)

    Grabon, Aby; Orłowski, Adam; Tripathi, Ashutosh; Vuorio, Joni; Javanainen, Matti; Róg, Tomasz; Lönnfors, Max; McDermott, Mark I; Siebert, Garland; Somerharju, Pentti; Vattulainen, Ilpo; Bankaitis, Vytas A

    2017-09-01

    Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITPα to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITPα; (iv) the trajectory of PtdIns or PtdCho into and through the lipid-binding pocket is chaperoned by sets of PITPα residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITPα PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  7. Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death

    Directory of Open Access Journals (Sweden)

    Ur Rahman MS

    2017-08-01

    Full Text Available Muhammad Saif Ur Rahman,1 Ling Zhang,2 Lingyan Wu,1 Yuqiong Xie,1 Chunchun Li,1 Jiang Cao1 1Clinical Research Center, 2Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, People’s Republic of China Abstract: Severe side effects are major problems with chemotherapy of gastric cancer (GC. These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB was used with other DNA linker agents mitomycin C (MMC, cisplatin (DDP, or cyclophosphamide (CTX to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC50 by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G0/G1 and G1/S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC

  8. Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

    Energy Technology Data Exchange (ETDEWEB)

    Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

    2010-01-15

    The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

  9. Functional dynamics of cell surface membrane proteins.

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Analogies between geminivirus and oncovirus: Cell cycle regulation ...

    African Journals Online (AJOL)

    Geminiviruses are a large family of plant viruses whose genome is composed of one or two circular and single strand of DNA. They replicate in the cell nucleus being Rep protein, the only viral protein necessary for their replication process. Geminiviruses as same as animal DNA oncoviruses, like SV40, adenovirus and ...

  11. Sulforaphane enhances irradiation effects in terms of perturbed cell cycle progression and increased DNA damage in pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Patrick Naumann

    Full Text Available Sulforaphane (SFN, an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT.In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT.Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4.Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.

  12. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Bäckström, T; Lauritsen, J P

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the ......The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....

  13. Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

    Science.gov (United States)

    Wiman, K G; Zhivotovsky, B

    2017-05-01

    Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation. © 2017 The Association for the Publication of the Journal of Internal Medicine.

  14. Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Wenmeng Wang

    Full Text Available High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC and human aortic endothelial cells (HAEC down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin.

  15. The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

    Science.gov (United States)

    Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

    2007-10-01

    MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

  16. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  17. Default cycle phases determined after modifying discrete DNA sequences in plant cells

    International Nuclear Information System (INIS)

    Sans, J.; Leyton, C.

    1997-01-01

    After bromosubstituting DNA sequences replicated in the first, second, or third part of the S phase, in Allium cepa L. meristematic cells, radiation at 313 nm wavelength under anoxia allowed ascription of different sequences to both the positive and negative regulation of some cycle phase transitions. The present report shows that the radiation forced cells in late G 1 phase to advance into S, while those in G 2 remained in G 2 and cells in prophase returned to G 2 when both sets of sequences involved in the positive and negative controls were bromosubstituted and later irradiated. In this way, not only G 2 but also the S phase behaved as cycle phases where cells accumulated by default when signals of different sign functionally cancelled out. The treatment did not halt the rates of replication or transcription of plant bromosubstituted DNA. The irradiation under hypoxia apparently prevents the binding of regulatory proteins to Br-DNA. (author)

  18. 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.

    Science.gov (United States)

    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

  19. Extracellular matrix collagen alters cell proliferation and cell cycle progression of human uterine leiomyoma smooth muscle cells.

    Science.gov (United States)

    Koohestani, Faezeh; Braundmeier, Andrea G; Mahdian, Arash; Seo, Jane; Bi, JiaJia; Nowak, Romana A

    2013-01-01

    Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

  20. Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

    Science.gov (United States)

    Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

    2007-05-30

    Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

  1. Cell Cycle Regulation by Alternative Polyadenylation of CCND1.

    Science.gov (United States)

    Wang, Qiong; He, Guopei; Hou, Mengmeng; Chen, Liutao; Chen, Shangwu; Xu, Anlong; Fu, Yonggui

    2018-05-01

    Global shortening of 3'UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.

  2. Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kyung-Mi [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Yun, Ji Ho [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Lee, Dong Hwa [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Park, Young Gyun [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Son, Kun Ho [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Kim, Yeong Shik, E-mail: kims@snu.ac.kr [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of)

    2015-04-17

    We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.

  3. Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

    International Nuclear Information System (INIS)

    Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa; Park, Young Gyun; Son, Kun Ho; Nho, Chu Won; Kim, Yeong Shik

    2015-01-01

    We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus

  4. Study of the cell cycle control for human malignant mesothelioma lines. Interferon and radiations effect

    International Nuclear Information System (INIS)

    Vivo, C.

    1999-01-01

    In order to better understand the inhibition mechanisms of the IFN-R-HU on tumoral development, the IFN-R-U effect on MM lines has been studied. Three groups of lines has been distinguished: eight sensitive lines, two intermediate and three resistant. The sensitive lines showed a triple locking of the cell cycle: in phases S, G1 and G2. The study of the cell cycle control points function, realized by the MM lines radiation exposure showed the points function on G1/S and-or on G2/M and the dependence or non dependence of the cycle stop of the protein P53 and P21 W at F1/CIP1. (A.L.B.)

  5. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    International Nuclear Information System (INIS)