Página 26 dos resultados de 45945 itens digitais encontrados em 0.055 segundos

‣ Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

SUZUKI, Daniela E.; ARIZA, Carolina B.; PORCIONATTO, Marimelia A.; OKAMOTO, Oswaldo Keith
Fonte: SPRINGER Publicador: SPRINGER
Tipo: Artigo de Revista Científica
Português
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In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) ...

‣ FoxM1 Regulates Transcription of JNK1 to Promote the G1/S Transition and Tumor Cell Invasiveness*

Wang, I-Ching; Chen, Yi-Ju; Hughes, Douglas E.; Ackerson, Timothy; Major, Michael L.; Kalinichenko, Vladimir V.; Costa, Robert H.; Raychaudhuri, Pradip; Tyner, Angela L.; Lau, Lester F.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 25/07/2008 Português
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The Forkhead box M1 (FoxM1) protein is a proliferation-specific transcription factor that plays a key role in controlling both the G1/S and G2/M transitions through the cell cycle and is essential for the development of various cancers. We show here that FoxM1 directly activates the transcription of the c-Jun N-terminal kinase (JNK1) gene in U2OS osteosarcoma cells. Expression of JNK1, which regulates the expression of genes important for the G1/S transition, rescues the G1/S but not the G2/M cell cycle block in FoxM1-deficient cells. Knockdown of either FoxM1 or JNK1 inhibits tumor cell migration, invasion, and anchorage-independent growth. However, expression of JNK1 in FoxM1-depleted cells does not rescue these defects, indicating that JNK1 is a necessary but insufficient downstream mediator of FoxM1 in these processes. Consistent with this interpretation, FoxM1 regulates the expression of the matrix metalloproteinases MMP-2 and MMP-9, which play a role in tumor cell invasion, through JNK1-independent and -dependent mechanisms in U2OS cells, respectively. Taken together, these findings identify JNK1 as a critical transcriptional target of FoxM1 that contributes to FoxM1-regulated cell cycle progression, tumor cell migration...

‣ T Cell Receptor-mediated Activation of CD4+CD44hi T Cells Bypasses Bcl10: AN IMPLICATION OF DIFFERENTIAL NF-κB DEPENDENCE OF NAïVE AND MEMORY T CELLS DURING T CELL RECEPTOR-MEDIATED RESPONSES*

Zeng, Hu; Chen, Yuhong; Yu, Mei; Xue, Liquan; Gao, Xiang; Morris, Stephan W.; Wang, Demin; Wen, Renren
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 05/09/2008 Português
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Previous studies have demonstrated that Bcl10 (B-cell leukemia/lymphoma 10) is essential for T cell receptor-mediated NF-κB activation and subsequent proliferation and interleukin 2 (IL2) production. However, here we demonstrate that, contrary to expectations, Bcl10 is differentially required for T cell activation, including for both proliferation and cytokine production. When CD4+ and CD8+ T cells were divided based on expression levels of CD44, which distinguishes naïve cells (CD44lo) versus those that are antigen-experienced (CD44hi), IL2 production by and proliferation of CD4+CD44lo naïve cells and both subpopulations of CD8+ T cells were clearly Bcl10-dependent, whereas these same functional properties of CD4+CD44hi T cells occurred largely independent of Bcl10. As with the other subpopulations of T cells, CD4+CD44hi T cells did not activate the NF-κB pathway in the absence of Bcl10; nevertheless, these CD4+CD44hi antigen-experienced T cells efficiently secreted IL2 after T cell receptor stimulation. Strikingly, therefore, T cell receptor-mediated IL2 production in these cells is NF-κB-independent. Our studies suggest that antigen-experienced CD4+ T cells differ from their naïve counterparts and from CD8+ T cells in their ability to achieve activation independent of the Bcl10/NF-κB pathway.

‣ Parcs Is a Dual Regulator of Cell Proliferation and Apaf-1 Function*

Sanchez-Olea, Roberto; Ortiz, Sara; Barreto, Odmara; Yang, Qing; Xu, Chi-jie; Zhu, Hong; Yuan, Junying
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 05/09/2008 Português
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Here we identify a novel protein, named Parcs for pro-apoptotic protein required for cell survival, that is involved in both cell cycle progression and apoptosis. Parcs interacted with Apaf-1 by binding to the oligomerization domain of Apaf-1. Apaf-1-mediated activation of caspase-9 and caspase-3 was markedly decreased in a cytosolic fraction isolated from HeLa cells with reduced parcs expression. Interestingly, parcs deficiency blocked cell proliferation in non-tumorigenic cells but not in multiple tumor cell lines. In MCF-10A cells, parcs deficiency led to early G1 arrest. Conditional inactivation of parcs in genetically modified primary mouse embryonic fibroblasts using the Cre-LoxP system also resulted in the inhibition of cell proliferation. We conclude that Parcs may define a molecular checkpoint in the control of cell proliferation for normal cells that is lost in tumor cells.

‣ Assembly of the Yeast Cell Wall: Crh1p AND Crh2p ACT AS TRANSGLYCOSYLASES IN VIVO AND IN VITRO*S⃞

Cabib, Enrico; Farkas, Vladimir; Kosík, Ondrej; Blanco, Noelia; Arroyo, Javier; McPhie, Peter
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 31/10/2008 Português
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The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to β(1–3)glucose branches of β(1–6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches...

‣ Glucose Metabolism Attenuates p53 and Puma-dependent Cell Death upon Growth Factor Deprivation*

Zhao, Yuxing; Coloff, Jonathan L.; Ferguson, Emily C.; Jacobs, Sarah R.; Cui, Kai; Rathmell, Jeffrey C.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 26/12/2008 Português
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Growth factor stimulation and oncogenic transformation lead to increased glucose metabolism that may provide resistance to cell death. We have previously demonstrated that elevated glucose metabolism characteristic of stimulated or cancerous cells can stabilize the anti-apoptotic Bcl-2 family protein Mcl-1 through inhibition of GSK-3. Here we show that the pro-apoptotic Bcl-2 family protein, Puma, is also metabolically regulated. Growth factor deprivation led to the loss of glucose uptake and induction of Puma. Maintenance of glucose uptake after growth factor withdrawal by expression of the glucose transporter, Glut1, however, suppressed Puma up-regulation and attenuated growth factor withdrawal-induced activation of Bax, DNA fragmentation, and cell death. Conversely, glucose deprivation led to Puma induction even in the presence of growth factor. This regulation of Puma expression was a central component in cell death as a consequence of growth factor or glucose deprivation because Puma deficiency suppressed both of these cell death pathways. Puma induction in growth factor or glucose withdrawal was dependent on p53 in cell lines and in activated primary T lymphocytes because p53 deficiency suppressed Puma induction and delayed Bax and caspase activation...

‣ β1 Integrin Cytoplasmic Domain Residues Selectively Modulate Fibronectin Matrix Assembly and Cell Spreading through Talin and Akt-1*

Green, J. Angelo; Berrier, Allison L.; Pankov, Roumen; Yamada, Kenneth M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 20/03/2009 Português
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The integrin β1 cytoplasmic domain (tail) serves as a scaffold for numerous intracellular proteins. The mechanisms by which the tail coordinates these proteins to facilitate extracellular matrix assembly and cell spreading are not clear. This study demonstrates that the β1 cytoplasmic domain can regulate cell spreading on fibronectin and fibronectin matrix assembly through Akt- and talin-dependent mechanisms, respectively. To identify these mechanisms, we characterized GD25 cells expressing the β1 integrin cytoplasmic domain mutants W775A and R760A. Although cell spreading appears normal in R760A mutant-integrin cells compared with wild type, it is inhibited in W775A mutant cells. In contrast, both mutant cell lines show defective fibronectin matrix assembly. Inhibition of cell spreading, but not matrix assembly, in the W775A mutant cells is due to a specific defect in Akt-1 activation. In addition, we find that both W775A and R760A mutant integrins have reduced surface expression of the 9EG7 epitope that correlates with reduced recruitment of talin to β1 integrin cytoplasmic complexes. Down-regulation of talin with small interfering RNA or expression of green fluorescent protein-talin head domain inhibits matrix assembly in β1 wild-type cells...

‣ Presenilin 1 Affects Focal Adhesion Site Formation and Cell Force Generation via c-Src Transcriptional and Posttranslational Regulation*

Waschbüsch, Dieter; Born, Simone; Niediek, Verena; Kirchgessner, Norbert; Tamboli, Irfan Y.; Walter, Jochen; Merkel, Rudolf; Hoffmann, Bernd
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 10/04/2009 Português
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Presenilin 1 and 2 (PS) are critical components of the γ-secretase complex that cleaves type I transmembrane proteins within their transmembrane domains. This process leads to release of proteolytically processed products from cellular membranes and plays an essential role in signal transduction or vital functions as cell adhesion. Here we studied the function of presenilins in cell-matrix interaction of wild-type and PS knock-out mouse embryonic fibroblasts. We found for PS1-/- cells an altered morphology with significantly reduced sizes of focal adhesion sites compared with wild type. Cell force analyses on micropatterned elastomer films revealed PS1-/- cell forces to be reduced by 50%. Pharmacological inhibition confirmed this function of γ-secretase in adhesion site and cell force formation. On the regulatory level, PS1 deficiency was associated with strongly decreased phosphotyrosine levels of focal adhesion site-specific proteins. The reduced tyrosine phosphorylation was caused by a down-regulation of c-Src kinase activity primarily at the level of c-Src transcription. The direct regulatory connection between PS1 and c-Src could be identified with ephrinB2 as PS1 target protein. Overexpression of ephrinB2 cytoplasmic domain resulted in its nuclear translocation with increased levels of c-Src and a full complementation of the PS1-/- adhesion and phosphorylation phenotype. Cleavage of full-length EB2 and subsequent intracellular domain translocation depended on PS1 as these processes were only found in WT cells. Therefore...

‣ The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates Stem Cell Proliferation but Not Pluripotency*

Ma, Tianhua; Wang, Zhe; Guo, Yunqian; Pei, Duanqing
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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Overexpression of Nanog in mouse embryonic stem (ES) cells has been shown to abrogate the requirement of leukemia inhibitory factor for self-renewal in culture. Little is known about the molecular mechanism of Nanog function. Here we describe the role of the tryptophan repeat (WR) domain, one of the two transactivators at its C terminus, in regulating stem cell proliferation as well as pluripotency. We first created a supertransactivator, W2W3×10, by duplicating repeats W2W3 10 times and discovered that it can functionally substitute for wild type WR at sustaining pluripotency, albeit with a significantly slower cell cycle, phenocopying Nanog(9W) with the C-terminal pentapeptide (WNAAP) of WR deleted. ES cells carrying both W2W3×10 and Nanog(9W) have a longer G1 phase, a shorter S phase in cell cycle distribution and progression analysis, and a lower level of pAkt(Ser473) compared with wild type Nanog, suggesting that both mutants impact the cell cycle machinery via the phosphatidylinositol 3-kinase/Akt pathway. Both mutants remain competent in dimerizing with Nanog but cannot form a complex with Nac1 efficiently, suggesting that WNAAP may be involved in Nac1 binding. By tagging Gal4DBD with WNAAP, we demonstrated that this pentapeptide is sufficient to confer Nac1 binding. Furthermore...

‣ Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Koczor, Christopher A.; Shokolenko, Inna N.; Boyd, Amy K.; Balk, Shawn P.; Wilson, Glenn L.; LeDoux, Susan P.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

‣ IIp45 Inhibits Cell Migration through Inhibition of HDAC6*

Wu, Ying; Song, Sonya W.; Sun, Jiyuan; Bruner, Janet M.; Fuller, Gregory N.; Zhang, Wei
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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IIp45 (aka MIIP) is a newly discovered gene whose protein product inhibits cell migration. HDAC6 is a class IIb deacetylase that specifically deacetylates α-tubulin, modulates microtubule dynamics, and promotes cell migration. A yeast two-hybrid assay using IIp45 as bait identified HDAC6 protein as a binding partner of IIp45. This physical interaction of the two functionally antagonistic proteins was confirmed by glutathione S-transferase pulldown assay and co-immunoprecipitation assay in human cells. Serial deletion constructs of HDAC6 were used to characterize the interaction of HDAC6 and IIp45, and this analysis found that the two catalytic domains of HDAC6 protein are required for IIp45 binding. We examined the protein expression patterns of IIp45 and HDAC6 in glioma tissues. Elevated protein levels of HDAC6 were found in high grade glioma samples, in contrast to the decreased protein expression of IIp45. The potential negative regulation of HDAC6 expression by IIp45 was confirmed in cell lines with altered IIp45 expression by constitutive overexpression or small interfering RNA knockdown. Protein turnover study revealed that overexpression of IIp45 significantly reduces the intracellular protein stability of endogenous HDAC6...

‣ The Adipose-derived Stem Cell: Looking Back and Looking Ahead

Zuk, Patricia A.
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em 01/06/2010 Português
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In 2002, researchers at UCLA published a manuscript in Molecular Biology of the Cell describing a novel adult stem cell population isolated from adipose tissue—the adipose-derived stem cell (ASC). Since that time, the ASC has gone on to be one of the most popular adult stem cell populations currently being used in the stem cell field. With multilineage mesodermal potential and possible ectodermal and endodermal potentials also, the ASC could conceivably be an alternate to pluripotent ES cells in both the lab and in the clinic. In this retrospective article, a historical perspective on the ASC is given together with exciting new applications for the stem cell being considered today.

‣ The T-loop Extension of the Tomato Protein Kinase AvrPto-dependent Pto-interacting Protein 3 (Adi3) Directs Nuclear Localization for Suppression of Plant Cell Death*

Ek-Ramos, María J.; Avila, Julian; Cheng, Cheng; Martin, Gregory B.; Devarenne, Timothy P.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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In tomato (Solanum lycopersicum), resistance to Pseudomonas syringae pv. tomato is elicited by the interaction of the host Pto kinase with the pathogen effector protein AvrPto, which leads to various immune responses including localized cell death termed the hypersensitive response. The AGC kinase Adi3 functions to suppress host cell death and interacts with Pto only in the presence of AvrPto. The cell death suppression (CDS) activity of Adi3 requires phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and loss of Adi3 function is associated with the hypersensitive response cell death initiated by the Pto/AvrPto interaction. Here we studied the relationship between Adi3 cellular localization and its CDS activity. Adi3 is a nuclear-localized protein, and this localization is dictated by a nuclear localization signal found in the Adi3 T-loop extension, an ∼80 amino acid insertion into the T-loop, or activation loop, which is phosphorylated for kinase activation. Nuclear localization of Adi3 is required for its CDS activity and loss of nuclear localization causes elimination of Adi3 CDS activity and induction of cell death. This nuclear localization of Adi3 is dependent on Ser-539 phosphorylation by Pdk1 and non-nuclear Adi3 is found in punctate structures throughout the cell. Our data support a model in which Pdk1 phosphorylation of Adi3 directs nuclear localization for CDS and that disruption of Adi3 nuclear localization may be a mechanism for induction of cell death such as that during the Pto/AvrPto interaction.

‣ The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes*♦

Sokabe, Takaaki; Fukumi-Tominaga, Tomoko; Yonemura, Shigenobu; Mizuno, Atsuko; Tominaga, Makoto
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca2+ dynamics. We found that TRPV4 interacts with β-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca2+-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca2+ increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.

‣ Teaching Biology through Statistics: Application of Statistical Methods in Genetics and Zoology Courses

Colon-Berlingeri, Migdalisel; Burrowes, Patricia A.
Fonte: American Society for Cell Biology Publicador: American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
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Incorporation of mathematics into biology curricula is critical to underscore for undergraduate students the relevance of mathematics to most fields of biology and the usefulness of developing quantitative process skills demanded in modern biology. At our institution, we have made significant changes to better integrate mathematics into the undergraduate biology curriculum. The curricular revision included changes in the suggested course sequence, addition of statistics and precalculus as prerequisites to core science courses, and incorporating interdisciplinary (math–biology) learning activities in genetics and zoology courses. In this article, we describe the activities developed for these two courses and the assessment tools used to measure the learning that took place with respect to biology and statistics. We distinguished the effectiveness of these learning opportunities in helping students improve their understanding of the math and statistical concepts addressed and, more importantly, their ability to apply them to solve a biological problem. We also identified areas that need emphasis in both biology and mathematics courses. In light of our observations, we recommend best practices that biology and mathematics academic departments can implement to train undergraduates for the demands of modern biology.

‣ Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations

Lin, Yao-Cheng; Boone, Morgane; Meuris, Leander; Lemmens, Irma; Van Roy, Nadine; Soete, Arne; Reumers, Joke; Moisse, Matthieu; Plaisance, Stéphane; Drmanac, Radoje; Chen, Jason; Speleman, Frank; Lambrechts, Diether; Van de Peer, Yves; Tavernier, Jan; Cal
Fonte: Nature Pub. Group Publicador: Nature Pub. Group
Tipo: Artigo de Revista Científica
Publicado em 03/09/2014 Português
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The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect.

‣ P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Bian, Shu; Sun, Xiaofeng; Bai, Aiping; Zhang, Chunqing; Li, Lingling; Enjoji, Keiichi; Junger, Wolfgang G; Robson, Simon Christopher; Wu, Yan
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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Background: Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms. Methodology/Principal Findings Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death. Conclusions: Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.

‣ Role of chemokine/chemokine receptor axes in the regulation of bone marrow NK cell localization in physiological and pathological conditions

PONZETTA, ANDREA
Fonte: La Sapienza Universidade de Roma Publicador: La Sapienza Universidade de Roma
Tipo: Tese de Doutorado
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Natural Killer (NK) cells represent a lymphocyte population of the innate immunity, involved in the control of viral infections and tumor cells. Among the wide number of functions that NK cells can mediate, the most important are the ability to lyse target cells via the release of cytotoxic granules, and the production of pro-inflammatory mediators, such as cytokines and chemokines. In mouse, according to the expression of the surface markers CD11b and KLRG1, within the NK cell population 4 discrete subsets can be distinguished, which represent different developmental stages and show heterogeneous functional capacities. Moreover, their tissue localization is dissimilar in primary and secondary lymphoid organs, and in non-lymphoid organs: the most immature populations are mainly represented in the bone marrow (BM), thymus and lymph nodes (LN), while the most mature ones preferentially distribute in blood, spleen, liver and lungs. Chemokines are chemotactic molecules of proteic nature, which bind specific GPCR receptors, and direct the migration of several cell types through the formation of a concentration gradient. NK cells express a great variety of chemokine receptors, which regulate their organ distribution and their trafficking to inflamed tissues. The first part of this work dealt with the study of the function of CX3CR1 receptor...

‣ First evidence of skelemin, a myosin-associated protein, in smooth muscle and its involvement in cell adhesion, and, The role of the cell cycle in cell type choice during mammalian development

Schwartz, Jeanette Marie
Fonte: Universidade Rice Publicador: Universidade Rice
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Indirect immunofluorescence in conjunction with Northern and Western blot analysis were used to identify the presence of skelemin, a myosin-associated protein, in smooth muscle. Feline uterus was cryosectioned and triple-stained for skelemin, smooth muscle myosin, and desmin. I observed that skelemin antibodies colocalized with the myosin-II filaments as well as the desmin intermediate filament cytoskeleton. This is the first evidence of a myosin-associated protein within smooth muscle, and it raises the possibility that smooth muscle myosin may be more organized than has been assumed. Antisense oligonucleotide treatment was used to analyze the underexpression of skelemin protein in developing embryoid bodies. Skelemin underexpression was seen to cause loss of cell-cell as well as cell-substrate adhesion. Time lapse video microscopy was used to analyze the role of the cell cycle in cell type choice during mammalian development. Discussed are methods employed to improve resolution for accurate analysis.

‣ p63 promotes cell survival through fatty acid synthase

Di Napoli, Arianna; Seeley, Apryle; Amato, Angela M.; O'Regan, Esther; Sabbisetti, Venkata S; Ghebremichael, Musie Syum; Loda, Massimo; Signoretti, Sabina
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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There is increasing evidence that p63, and specifically ΔNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or ΔN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity...