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Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: identification of phenolic acids with cytotoxic potential.

Vaz, Josiana A.; Almeida, Gabriela M.; Ferreira, Isabel C.F.R.; Martins, Anabela; Vasconcelos, M. Helena
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Português
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Mushrooms are a possible rich source of biologically active compounds with potential for drug discovery. The aim of this work was to gain further insight into the citotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterized regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the Clitocybe alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI50 concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 µg/ml) for 48h caused an increase in the levels of wt p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic...

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

PAIVA, Raquel de Melo Alves; FIGUEIREDO, Raquel de Freitas; ANTONUCCI, Gilmara Ausech; PAIVA, Helder Henrique; BIANCHI, Maria de Lourdes Pires; RODRIGUES, Kelly C.; LUCARINI, Rodrigo; CAETANO, Renato Cesar; PIETRO, Rosemeire Cristina Linhari Rodrigues; MA
Fonte: ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER Publicador: ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
Tipo: Artigo de Revista Científica
Português
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The present article describes an L-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host`s immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 mu g/mL and 50 mu g/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase...

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Nolasco, Sofia; Bellido, Javier; Gonçalves, João; Zabala, Juan Carlos; Soares, Helena
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em /07/2005 Português
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Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

Vucic,V.; Isenovic,E.R.; Adzic,M.; Ruzdijic,S.; Radojcic,M.B.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/2006 Português
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Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner...

Characterization of Structural p53 Mutants Which Show Selective Defects in Apoptosis but Not Cell Cycle Arrest

Ryan, Kevin M.; Vousden, Karen H.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1998 Português
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Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest, functions which have been shown to be separable activities of p53. We have characterized a series of p53 mutants with amino acid substitutions at residue 175 and show that these mutants fall into one of three classes: class I, which is essentially wild type for apoptotic and cell cycle arrest functions; class II, which retains cell cycle arrest activity but is impaired in the induction of apoptosis; and class III, which is defective in both activities. Several residue 175 mutants which retain cell cycle arrest function have been detected in cancers, and we show that these have lost apoptotic function. Furthermore, several class II mutants have been found to be temperature sensitive for apoptotic activity while showing constitutive cell cycle arrest function. Taken together, these mutants comprise an excellent system with which to investigate the biochemical nature of p53-mediated apoptosis, the function of principal importance in tumor suppression. All of the mutants that showed loss of apoptotic function also showed defects in the activation of promoters from the potential apoptotic targets Bax and the insulin-like growth factor-binding protein 3 gene (IGF-BP3)...

NS1- and Minute Virus of Mice-Induced Cell Cycle Arrest: Involvement of p53 and p21cip1

Op De Beeck, Anne; Sobczak-Thepot, Joelle; Sirma, Huseyin; Bourgain, Florence; Brechot, Christian; Caillet-Fauquet, Perrine
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2001 Português
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The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G1, S, and G2, well reproducing the arrest in S and G2 observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21cip1 associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21cip1. By comparison, MVMp infection induced the accumulation of both p53 and p21cip1. We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G2 by using p53 wild-type (+/+) and null (−/−) cells. Furthermore, only the G2 arrest was abrogated in p21cip1 null (−/−) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21cip1 independent, whereas the arrest in G2 depends on both p53 and its downstream effector p21cip1. They also suggest that induction of p21cip1 by the viral protein NS1 arrests cells in G2 through inhibition of cyclin A-dependent kinase activity.

p53-Independent Expression of p21CIP1/WAF1 in Plasmacytic Cells during G2 Cell Cycle Arrest Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

Sato, Tsuyoshi; Koseki, Takeyoshi; Yamato, Kenji; Saiki, Keitarou; Konishi, Kiyoshi; Yoshikawa, Masanosuke; Ishikawa, Isao; Nishihara, Tatsuji
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2002 Português
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The cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans has been shown to induce cell cycle arrest in the G2/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) from A. actinomycetemcomitans induced G2 cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21CIP1/WAF1 or G2 cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21CIP1/WAF1 expression or G2 cell cycle arrest in HS-72 cells. These results suggest that the CDT from A. actinomycetemcomitans induces p21CIP1/WAF1 expression and G2 cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT from A. actinomycetemcomitans, the results of this study indicate that CDT-induced p53 accumulation may not be required for G2 cell cycle arrest and that an increased level of p21CIP1/WAF1 may be important for sustaining G2 cell cycle arrest in several mammalian cells.

The First HxRxG Motif in Simian Immunodeficiency Virus mac239 Vpr Is Crucial for G2/M Cell Cycle Arrest

Mueller, Sandra M.; Lang, Sabine M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2002 Português
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The highly conserved Vpr protein mediates cell cycle arrest, transcriptional transactivation, and nuclear import of the preintegration complex in human immunodeficiency virus type 1. To identify functional domains in simian immunodeficiency virus (SIV) mac239 Vpr, we mutagenized selected motifs within an α-helical region and two C-terminal HxRxG motifs. All Vpr mutants located to the nucleus. Substitution of four amino acids in the α-helical domain did not interfere with cell cycle arrest, while a single substitution abolished cell cycle arrest function. Mutation of the first HxRxG motif to AxAxA also resulted in loss of cell cycle arrest, while mutation of the second motif had no effect. Interestingly, both Vpr mutants impaired in cell cycle arrest function also showed reduced transactivation of the SIV long terminal repeat, suggesting that arrest of cells at G2/M mediates or contributes to transactivation by Vpr.

Hypoxia-Inducible Factor 1α Is Essential for Cell Cycle Arrest during Hypoxia

Goda, Nobuhito; Ryan, Heather E.; Khadivi, Bahram; McNulty, Wayne; Rickert, Robert C.; Johnson, Randall S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2003 Português
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A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1α gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1α influenced p53, we also created double-knockout (HIF-1α null, p53 null) strains and cells. In both cell types, loss of HIF-1α abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1α genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition...

CCAAT/Enhancer Binding Protein α Interacts with ZTA and Mediates ZTA-Induced p21CIP-1 Accumulation and G1 Cell Cycle Arrest during the Epstein-Barr Virus Lytic Cycle

Wu, Frederick Y.; Chen, Honglin; Wang, Shizhen Emily; apRhys, Collette M. J.; Liao, Gangling; Fujimuro, Masahiro; Farrell, Christopher J.; Huang, Jian; Hayward, S. Diane; Hayward, Gary S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2003 Português
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Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G1/S through stabilization of p21CIP-1/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G1/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G1/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPα and p21 and blocked the progression into S phase...

Cell Cycle Arrest in G2/M Promotes Early Steps of Infection by Human Immunodeficiency Virus

Groschel, Bettina; Bushman, Frederic
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2005 Português
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We have identified four small molecules that boost transduction of cells by human immunodeficiency virus (HIV) and investigated their mechanism of action. These molecules include etoposide and camptothecin, which induce DNA damage by inhibiting religation of cleaved topoisomerase-DNA complexes, taxol, which interferes with the function of microtubules, and aphidicolin, which inhibits DNA polymerases. All four compounds arrest the cell cycle at G2/M, though in addition high concentrations of aphidicolin arrest in G1. We find that early events of HIV replication, including synthesis of late reverse transcription products, two-long terminal repeat circles, and integrated proviruses, were increased after treatment of cells with concentrations of each compound that arrested in G2/M. Stimulation was seen for both transformed cell lines (293T and HeLa cells) and primary cells (IMR90 lung fibroblasts). Arrest in G1 with high concentrations of aphidicolin boosted transduction, though not much as with lower concentrations that arrested in G2/M. Arrest of IMR90 cells in G1 by serum starvation and contact inhibition reduced transduction. Previously, the proteasome inhibitor MG132 was reported to increase HIV infection—here we investigated the effects of combinations of the cell cycle inhibitors with MG132 and obtained data suggesting that MG132 may also boost transduction by causing G2/M cell cycle arrest. These data document that cell cycle arrest in G2/M boosts the early steps of HIV infection and suggests methods for increasing transduction with HIV-based vectors.

Zinc Finger Transcription Factor INSM1 Interrupts Cyclin D1 and CDK4 Binding and Induces Cell Cycle Arrest*

Zhang, Tao; Liu, Wei-Dong; Saunee, Nicolle A.; Breslin, Mary B.; Lan, Michael S.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 27/02/2009 Português
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INSM1 is a zinc finger transcription factor that plays an important role in pancreatic β-cell development. To further evaluate its role in cell fate determination, we investigated INSM1 effects on cell cycle function. The cyclin box of cyclin D1 is essential for INSM1 binding. Competitive pull-down and co-immunoprecipitation revealed that INSM1 binding to cyclin D1 interrupts its association with CDK4 and induces hypophosphorylation of the retinoblastoma protein. An inducible Tet-on system was established in Cos-7 and Panc-1 cells. Using serum starvation, we synchronized the cell cycle and subsequently induced cell cycle progression by serum stimulation. Comparison of the INSM1 induction group with the noninduced control group, INSM1 ectopic expression causes cell cycle arrest, whereas the INSM1-mediated cell cycle arrest could be reversed by cyclin D1 and CDK4 overexpression. The proline-rich N-terminal portion of INSM1 is required for cyclin D1 binding. Mutation of proline residues abolished cyclin D1 binding and also diminished its ability to induce cell cycle arrest. Cellular proliferation of Panc-1 cells was inhibited by INSM1 overexpression demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay...

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
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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.

Bocavirus Infection Induces Mitochondrion-Mediated Apoptosis and Cell Cycle Arrest at G2/M Phase▿

Chen, Aaron Yun; Luo, Yong; Cheng, Fang; Sun, Yuning; Qiu, Jianming
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Bocavirus is a newly classified genus of the family Parvovirinae. Infection with Bocavirus minute virus of canines (MVC) produces a strong cytopathic effect in permissive Walter Reed/3873D (WRD) canine cells. We have systematically characterized the MVC infection-produced cytopathic effect in WRD cells, namely, the cell death and cell cycle arrest, and carefully examined how MVC infection induces the cytopathic effect. We found that MVC infection induces an apoptotic cell death characterized by Bax translocalization to the mitochondrial outer membrane, disruption of the mitochondrial outer membrane potential, and caspase activation. Moreover, we observed that the activation of caspases occurred only when the MVC genome was replicating, suggesting that replication of the MVC genome induces apoptosis. MVC infection also induced a gradual cell cycle arrest from the S phase in early infection to the G2/M phase at a later stage, which was confirmed by the upregulation of cyclin B1 and phosphorylation of cdc2. Cell cycle arrest at the G2/M phase was reproduced by transfection of a nonreplicative NS1 knockout mutant of the MVC infectious clone, as well as by inoculation of UV-irradiated MVC. In contrast with other parvoviruses, only expression of the MVC proteins by transfection did not induce apoptosis or cell cycle arrest. Taken together...

Nonstructural Protein σ1s Mediates Reovirus-Induced Cell Cycle Arrest and Apoptosis

Boehme, Karl W.; Hammer, Katharina; Tollefson, William C.; Konopka-Anstadt, Jennifer L.; Kobayashi, Takeshi; Dermody, Terence S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2013 Português
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Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably...

Transcription factor-pathway co-expression analysis reveals cooperation between SP1 and ESR1 on dysregulating cell cycle arrest in non-hyperdiploid multiple myeloma

Wang, Xujun; Yan, Zhenyu; Fulciniti, Mariateresa; Li, Yingxiang; Gkotzamanidou, Maria; Amin, Samir B; Shah, Parantu K; Zhang, Yong; Munshi, Nikhil C; Li, Cheng
Fonte: Harvard University Publicador: Harvard University
Tipo: Artigo de Revista Científica
Português
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Multiple myeloma is a hematological cancer of plasma B-cells and remains incurable. Two major subtypes of myeloma, hyperdiploid (HMM) and non-hyperdiploid myeloma (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis but their dysregulation in myeloma subtypes are less studied. Here we develop a TrF-pathway co-expression analysis to identify altered co-expression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, NF-κB and HOXA9 have significantly lower co-expression with cell cycle arrest in HMM, co-occurring with their over-activation in HMM. In contrast, TrFs ESR1, SP1 and E2F1 have significantly lower co-expression with cell cycle arrest in NHMM. SP1 ChIP targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their over-activation in NHMM disrupts proper regulation of cell cycle arrest. Co-targeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore...

VacA von Helicobacter pylori induziert Zell-Zyklus-Arretierung am Modell der Jurkat-Zelle; VacA from helicobacter pylori induces cell-zyklus-arrest on the model of Jurkat-cell

Plauschin, Joerg
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Helicobacter pylori (H. pylori) kann die Magenschleimhaut des Menschen trotz einer reaktiven Immunantwort chronisch infizieren. Das gramnegative Bakterium sezerniert das Vacuolating cytotoxin A (VacA) und hemmt damit die Proliferation der T Lymphozyten. In die Zellumgebung gelöst diffundiert der Virulenzfaktor in die entzündete Mucosa und arretiert den Zellzyklus der Lymphozyten in der G1-Phase. Am Modell der Jurkat-Zelle wurden die zellfreien Überstände der H.-pylori-Stämme P12 (Wildtyp), P12 Delta(VacA) (knock out null Mutant) und P12 Delta(6-27) gegen eine Kontrolle getestet. Unter Verwendung von BrdU ergab sich folgende Fragen: 1. Hemmt gelöstes VacA Jurkat-Zellen in der G1-Phase? 2. Kann die Zyklusarretierung in Co-Kultivierung mit mitogenen Stimuli oder nach Säureaktivierung potenziert werden? Jurkat-Zellen wurden mit den zellfreien Extrakten der H. pylori Kulturen unter Beifügung von BrdU (c = 20 µM) für 24 Stunden inkubiert. Nach zweimaligem Waschen mit PBS und Fixieren mit 70 % Äthanol ruhten die Proben für 24 Stunden bei – 20 °C. Es folgte die Inkubation mit RNase und kaltem Pepsin, jeweils im Wasserbad, sowie die DNA-Denaturierung mit (2N) HCl bei RT. Anti-BrdU Antikörper der Maus demarkierte de novo synthetisierte Chromosomen und mit FITC konjugierte Anti-Maus Antikörper erlaubten diesbezüglich den indirekten Nachweis. Nach DNA-Färbung mit Propidium Iodid folgte die Auswertung im Zytometer. Dabei konnte die hemmende Wirkung des gelöstem VacA auf den Zellzyklus und zwar nach Säureaktivierung mit einem signifikanten Unterschied zur Kontrolle und zu den Extrakten der isogenen Mutanten deutlich gezeigt werden. Bei gleichzeitiger ConA Stimulierung ohne Säureaktivierung war der Unterschied dagegen nur gering. Unter Stimulierung mit PMA/Ionomycin und Säureaktivierung arretierte VacA signifikant mehr Zellen in der G1-Phase als in der Kontrolle und P12Δ(VacA). Diese Daten reihen sich gut in die Ergebnisse anderer Forschergruppen ein. In Übereinstimmung mit Gebert und Sundrud konnte diese Untersuchung aufzeigen...

Actinobacillus pleuropneumoniae induces SJPL cell cycle arrest in G2/M-phase and inhibits porcine reproductive and respiratory syndrome virus replication

Ferreira Barbosa, Jérémy A.; Labrie, Josée; Beaudry, Francis; Gagnon, Carl; Jacques, Mario
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Artigo de Revista Científica
Português
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Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry and causes important economic losses. No effective antiviral drugs against it are commercially available. We recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the porcine pleuropneumonia causative agent, has an antiviral activity in vitro against PRRSV in SJPL cells. Objectives of this study were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae and (ii) to characterize the active molecules present in the bacterial culture supernatant. Methods: Antibody microarray analysis was used in order to point out cellular pathways modulated by the A. pleuropneumoniae supernatant. Subsequent, flow cytometry analysis and cell cycle inhibitors were used to confirm antibody microarray data and to link them to the antiviral activity of the A. pleuropneumoniae supernatant. Finally, A. pleuropneumoniae supernatant characterization was partially achieved using mass spectrometry. Results: Using antibody microarray, we observed modulations in G2/M-phase cell cycle regulation pathway when SJPL cells were treated with A. pleuropneumoniae culture supernatant. These modulations were confirmed by a cell cycle arrest at the G2/M-phase when cells were treated with the A. pleuropneumoniae culture supernatant. Furthermore...

Kaposi's Sarcoma-Associated Herpesvirus Transactivator Rta Induces Cell Cycle Arrest in G0/G1 Phase by Stabilizing and Promoting Nuclear Localization of p27kip

Kumar, Pankaj; Wood, Charles
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2013 Português
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The Kaposi's sarcoma-associated herpesvirus (KSHV) immediate-early gene, replication, and transcription activator (K-Rta) is a key viral protein that serves as the master regulator for viral lytic replication. In this study, we investigated the role of K-Rta in cell cycle regulation and found that the expression of K-Rta in doxycycline (Dox)-inducible BJAB cells induced cell cycle arrest in G0/G1 phase. Western blot analysis of key cell cycle regulators revealed that K-Rta-mediated cell cycle arrest was associated with a decrease in cyclin A and phosphorylated Rb (pS807/pS811) protein levels, both markers of S phase progression, and an increase in protein levels for p27, a cyclin-dependent kinase inhibitor. Further, we found that K-Rta does not affect the transcription of p27 but regulates p27 at the posttranslational level by inhibiting its proteosomal degradation. Immunofluorescence staining and cell fractionation experiments revealed largely nuclear compartmentalization of p27 in K-Rta-expressing cells, demonstrating that K-Rta not only stabilizes p27 but also modulates its cellular localization. Finally, short hairpin RNA knockdown of p27 significantly abrogates cell cycle arrest in K-Rta-expressing cells, supporting its key role in K-Rta-mediated cell cycle arrest. Our findings are consistent with previous studies which showed that expression of immediate-early genes of several herpesviruses...

Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background;

Cook, Matthew; Munger, Steven C.; Capel, Blanche
Fonte: COMPANY OF BIOLOGISTS LTD Publicador: COMPANY OF BIOLOGISTS LTD
Publicado em //2011 Português
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Human germ cell tumors show a strong sensitivity to genetic background similar to Dnd1(Ter/Ter) mutant mice, where testicular teratomas arise only on the 129/SvJ genetic background. The introduction of the Bax mutation onto mixed background Dnd1(Ter/Ter) mutants, where teratomas do not typically develop, resulted in a high incidence of teratomas. However, when Dnd1(Ter/Ter); Bax(-/-) double mutants were backcrossed to C57BL/6J, no tumors arose. Dnd1(Ter/Ter) germ cells show a strong downregulation of male differentiation genes including Nanos2. In susceptible strains, where teratomas initiate around E15.5-E17.5, many mutant germ cells fail to enter mitotic arrest in G0 and do not downregulate the pluripotency markers NANOG, SOX2 and OCT4. We show that DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27(Kip1) and p21(Cip1). P27(Kip1) and P21(Cip1) protein are both significantly decreased in Dnd1(Ter/Ter) germ cells on all strain backgrounds tested, strongly suggesting that DND1 regulates mitotic arrest in male germ cells through translational regulation of cell cycle genes. Nonetheless, in C57BL/6J mutants, germ cells arrest prior to M-phase of the cell cycle and downregulate NANOG...