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Cycle Progression (cycle + progression)
Kinds of Cycle Progression Selected AbstractsRole of D1 and E Cyclins in Cell Cycle Progression of Human Fibroblasts Adhering to Cementum Attachment Protein,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Takayoshi Yokokoji Abstract Cementum attachment protein (CAP) is a collagenous protein present in the matrix of tooth cementum that mediates preferential attachment of some mesenchymal cell types, and CAP binding capacity is related to mineralizing tissue-forming capacity in culture. We have examined if adhesion to surfaces containing CAP as the only attachment protein permits human fibroblasts to escape G1 arrest and synthesize DNA, and if adhesion to CAP modulates the levels of cyclins D1 and E. Human gingival fibroblasts (HGFs) were serum-starved, trypsinized, and added to plates coated with CAP or bovine serum albumin (BSA). Cells were then exposed to either 10% fetal bovine serum (FBS) or to cementum-derived growth factor (CGF), an insulin-like growth factor I (IGF-I)-like molecule sequestered in tooth cementum, plus epidermal growth factor (EGF). DNA synthesis was measured as [3H]thymidine uptake, and cyclin D1 and E levels were determined by Western analysis. Cyclin E-dependent kinase (Cdk) activity was assessed in terms of H1 kinase activity in immunoprecipitates of cyclin E. Cells adhering to CAP synthesized DNA, whereas on BSA they remained unattached and did not synthesize DNA. Protein levels of cyclin D1 were higher in cells adhering to CAP in the absence and presence of growth factors. Cyclin E levels were not affected by adhesion alone, but they increased in the presence of growth factors. Cyclin E-associated kinase activity was higher in cells adherent on CAP, and it increased further in the presence of growth factors. Our results indicate that adhesion to CAP increases cyclin D1 levels and cyclin E-associated Cdk activity, and that these increases contribute to cell cycle progression. We previously observed that the signaling reactions induced during adhesion are characteristic of the CAP; together these observations indicate that specific matrix components present in the local environment can contribute to recruitment and differentiation of specific cell types for normal homeostasis and wound healing. [source] Requirement for Metalloproteinase-dependent ERK and AKT Activation in UVB-induced G1-S Cell Cycle Progression of Human KeratinocytesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009Weinong Han UVB (280,315 nm) in natural sunlight represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here we demonstrate that low doses of UVB induce keratinocyte proliferation and cell cycle progression of human HaCaT keratinocytes. Different from UVA, UVB irradiation induced extracellular signal-regulated kinase (ERK) and AKT activation and their activation are both required for UVB-induced cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVB exposure and is upstream of ERK/AKT/cyclin D1 pathway activation and cell cycle progression following UVB radiation. Furthermore, metalloproteinase (MP) inhibitor GM6001 blocked UVB-induced ERK and AKT activation, cell cycle progression, and decreased the EGFR phosphorylation, demonstrating that MPs mediate the EGFR/ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB radiation. In addition, ERK or AKT activation is essential for EGFR activation because ERK or AKT inhibitor blocks EGFR activation following UVB radiation, indicating that EGFR/AKT/ERK pathways form a regulatory loop and converge into cell cycle progression following UVB radiation. Identification of these signaling pathways in UVB-induced cell cycle progression of quiescent keratinocytes as a process mimicking tumor promotion in vivo will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer. [source] Asperfuranone from Aspergillus nidulans Inhibits Proliferation of Human Non-Small Cell Lung Cancer A549 Cells via Blocking Cell Cycle Progression and Inducing ApoptosisBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2010Clay C. C. Wang To identity the anti-cancer mechanism of asperfuranone, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21 Waf1/Cip1, Fas/APO-1 receptor and Fas ligand. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest might be due to p53-dependent induction of p21 Waf1/Cip1. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by asperfuranone. Our study reports here for the first time that the induction of p53 and the activity of Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of asperfuranone in A549 cells. [source] Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity,BIOTECHNOLOGY PROGRESS, Issue 5 2000Magnus Doverskog Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50,70% of the cells in the G2/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G1 and in particular into the S phase. Maximum rate of proliferation (,N,max) was reached 24,48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G2/M phase. The following declining ,N could be explained by a slow increase in the G2/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G2/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G2/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase ,N,max, decrease the time to reach ,N,max, and decrease the synchronization of cells in G2/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of ,-galactosidase up to 68,75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G2/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G2/M cell cycle arrest. [source] Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005Linping Wang Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15INK4b), and Gas3/PMP22. The expression of p21 and p15INK4b contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death. [source] Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic developmentDEVELOPMENTAL DYNAMICS, Issue 4 2008Cécile Notarnicola Abstract Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch - 1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development. Developmental Dynamics 237:1165,1171, 2008. © 2008 Wiley-Liss, Inc. [source] Differential expression of CaMK-II genes during early zebrafish embryogenesisDEVELOPMENTAL DYNAMICS, Issue 1 2007Sarah C. Rothschild Abstract CaMK-II is a highly conserved Ca2+/calmodulin-dependent protein kinase expressed throughout the lifespan of all vertebrates. During early development, CaMK-II regulates cell cycle progression and "non-canonical" Wnt-dependent convergent extension. In the zebrafish, Danio rerio, CaMK-II activity rises within 2 hr after fertilization. At the time of somite formation, zygotic expression from six genes (camk2a1, camk2b1, camk2g1, camk2g2, camk2d1, camk2d2) results in a second phase of increased activity. Zebrafish CaMK-II genes are 92,95% identical to their human counterparts in the non-variable regions. During the first three days of development, alternative splicing yields at least 20 splice variants, many of which are unique. Whole-mount in situ hybridization reveals that camk2g1 comprises the majority of maternal expression. All six genes are expressed strongly in ventral regions at the 18-somite stage. Later, camk2a1 is expressed in anterior somites, heart, and then forebrain. Camk2b1 is expressed in somites, mid- and forebrain, gut, retina, and pectoral fins. Camk2g1 appears strongly along the midline and then in brain, gut, and pectoral fins. Camk2g2 is expressed early in the midbrain and trunk and exhibits the earliest retinal expression. Camk2d1 is elevated early at somite boundaries, then epidermal tissue, while camk2d2 is expressed in discrete anterior locations, steadily increasing along either side of the dorsal midline and then throughout the brain, including the retina. These findings reveal a complex pattern of CaMK-II gene expression consistent with pleiotropic roles during development. Developmental Dynamics 236:295,305, 2007. © 2006 Wiley-Liss, Inc. [source] Homocysteine inhibits cardiac neural crest cell formation and morphogenesis in vivoDEVELOPMENTAL DYNAMICS, Issue 1 2004Brent J. Tierney Abstract Elevated homocysteine increases the risk of neurocristopathies. Here, we determined whether elevating homocysteine altered the proliferation or number of chick neural crest cells that form between the midotic and third somite in vivo. Homocysteine increased the number of neural tube cells but decreased neural crest cell number. However, the sum total of cells was not different from controls. In controls, the 5-bromo-2,-deoxyuridine-labeling index was higher in newly formed neural crest cells than in their progenitors, paralleling reports showing these progenitors must pass the restriction point before undergoing epithelial,mesenchymal transition. Homocysteine decreased the labeling index of newly formed neural crest cells, suggesting that it inhibited cell cycle progression of neural crest progenitors or the S-phase entry of newly formed neural crest cells. Homocysteine also inhibited neural crest dispersal and decreased the distance they migrated from the neural tube. These results show neural crest morphogenesis is directly altered by elevated homocysteine in vivo. Developmental Dynamics 229:63,73, 2004. © 2003 Wiley-Liss, Inc. [source] Proteomic analysis of osteogenic differentiation of dental follicle precursor cellsELECTROPHORESIS, Issue 7 2009Christian Morsczeck Abstract Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation. The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp-1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in DFPCs. [source] Acute exposure of human lung cells to 1,3-butadiene diepoxide results in G1 and G2 cell cycle arrestENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2005Michael Schmiederer Abstract 1,3-butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2 -treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2 -treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 ,M BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2 -treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2 -treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2 -treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21cip1 was observed in BDO2 -treated cells, suggesting that the cell cycle arrest was p21cip1 -mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells,,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2005Gregory S. Akerman Abstract Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis. Environ. Mol. Mutagen., 2005. Published 2005 Wiley-Liss, Inc. [source] Absence of p16 and p27 gene rearrangements and mutations in de novo myelodysplastic syndromesEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2005Sotirios G. Papageorgiou Abstract:, Myelodysplastic syndromes (MDS) represent a group of clonal hematopoietic disorders characterized by dyshemopoiesis and frequent evolution to acute leukemia. Tumor suppressor gene inactivation may be involved in MDS pathogenesis. The two families of cyclin-dependent kinase inhibitors (CDKIs) (INK4 family of p15, p16, p18 and p19 and CIP/KIP family of p21, p27 and p57) that negatively regulate cell cycle progression are known tumor suppressor genes. To determine whether genetic alterations of p16 and p27 genes play an important role in MDS pathogenesis, we examined DNA from 51 patients classified as 17 refractory anemias (RA), four refractory anemias with ringed sideroblasts (RARS), 19 refractory anemias with an excess of blasts (RAEB), 5 refractory anemias with excess of blasts in transformation (RAEB-t) and 6 chronic myelomonocytic leukemias (CMML). Southern blot analysis detected no homozygous deletions of p16 and p27. Polymerase chain reaction,single-strand conformation polymorphism (PCR,SSCP) and sequencing did not reveal point mutations for both genes with the exception of two allelic polymorphisms, namely a C , G transition at 447 bp of p16exon3 and a T , A transition at 791 bp of p27exon1 genes. Our results suggest that mutations of p16 and p27 genes resulting in abnormal p16 and p27 proteins do not represent a mechanism of gene inactivation involved in the pathogenesis of MDS. [source] Impaired lymphocyte development and function in Clast5/Stra13/DEC1-transgenic miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2004Mika Seimiya Abstract Clast5/Stra13/DEC1 is a member of the helix-loop-helix family of transcriptional repressors. We have previously shown that Clast5 is rapidly down-regulated upon B,cell activation and its overexpression inhibits cell cycle progression in B,lymphoma cells. In the present study, we show that Clast5 expression is developmentally regulated during B,cell differentiation, being expressed at theprogenitor B,cells, down-regulated at the precursor B,cells, elevated in immature and mature resting B,lymphocytes, and down-regulated again in germinal center B,ells. To investigate the function of Clast5 in regulating lymphocyte development, we have generated transgenic mice expressing Clast5 in B- and T-lineage cells (Clast5-Tg). Clast5-Tg mice grew and bred normally but their spleen and thymus cellularity was reduced compared with control littermates. The development of B,cells in the bone marrow and T,cells in the thymus was impaired, with the expansion of progenitor B and T,cells most strongly affected. The frequency of IL-7-responsive cells in the bone marrow of Clast5-Tg mice was reduced by >80% and their proliferative response to IL-7 was also compromised. Mature B,cells from Clast5-Tg mice were hyporesponsive to antigen receptor cross-linking and exhibited mild reduction in the proliferative response to CD40 ligation or lipopolysaccharide stimulation. Moreover, thedevelopment of germinal center B,cells and antibody production against a T-dependent antigen were reduced in Clast5-Tg mice. These results reveal a critical role for Clast5/Stra13/DEC1 in negatively regulating lymphocyte development and function in vivo. [source] Identification of ventricular-side-enriched molecules regulated in a stage-dependent manner during cerebral cortical developmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006Itsuki Ajioka Abstract Radial glial cells are the main component of the embryonic cortical ventricular zone (VZ), producing deep-layer excitatory neurons in the early stage and upper-layer excitatory neurons in the late stage of development. Previous studies have suggested that the laminar fate of deep-layer neurons might be determined by early-stage-specific secretory or transmembrane molecules (S/TMs) in the VZ. However, the different properties required to produce the different types of neurons in early-stage and late-stage VZ cells are largely unknown. Herein, we investigated the stage-dependent transcriptional profiles of the ventricular side of the mouse cortex, which was manually dissected at embryonic day (E)12, E14 and E16, and identified 3985 ,VZ-enriched' genes, regulated stage-dependently, by GeneChip analysis. These molecules were classified into nine types based on stage-dependent regulation patterns. Prediction programs for the S/TMs revealed 659 ,VZ-enriched' S/TMs. In situ hybridization and real-time PCR analysis for several of these molecules showed results consistent with the statistical analysis of the GeneChip experiments. Moreover, we identified 17 cell cycle-related early-stage and ,VZ-enriched' molecules. These molecules included not only those involved in cell cycle progression, but also essential molecules for DNA double-strand break repair, such as Rad51 and Rpa1. These results suggest that the early stage-VZ cells, which produce both deep- and upper-layer neurons, and the late-stage VZ cells, which produce only upper-layer neurons, are intrinsically different. The gene lists presented here will be useful for the investigation of stage-dependent changes in VZ cells and their regulatory mechanisms in the developing cortex. [source] LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific mannerFEBS JOURNAL, Issue 19 2009Fabienne Schmit Recently, the conserved human LINC/DREAM complex has been described as an important regulator of cell cycle genes. LINC consists of a core module that dynamically associates with E2F transcription factors, p130 and the B-MYB transcription factor in a cell cycle-dependent manner. In this study, we analyzed the evolutionary conserved LIN54 subunit of LINC. We found that LIN54 is required for cell cycle progression. Protein interaction studies demonstrated that a predicted helix,coil,helix motif is required for the interaction of LIN54 with p130 and B-MYB. In addition, we found that the cysteine-rich CXC domain of LIN54 is a novel DNA-binding domain that binds to the cdc2 promoter in a sequence-specific manner. We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region at the transcriptional start site. Gel shift assays suggested that, in quiescent cells, the binding of LIN54 at the cell cycle homology region is stabilized by the binding of E2F4 to the adjacent cell cycle-dependent element. Our data demonstrate that LIN54 is an important and integral subunit of LINC. Structured digital abstract ,,MINT-7239362: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with p130 (uniprotkb:Q08999) by anti tag coimmunoprecipitation (MI:0007) ,,MINT-7239376: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with B-Myb (uniprotkb:P10244) by anti tag coimmunoprecipitation (MI:0007) [source] Biochemical insights into the mechanisms central to the response of mammalian cells to cold stress and subsequent rewarmingFEBS JOURNAL, Issue 1 2009Anne Roobol Mammalian cells cultured in vitro are able to recover from cold stress. However, the mechanisms activated during cold stress and recovery are still being determined. We here report the effects of hypothermia on cellular architecture, cell cycle progression, mRNA stability, protein synthesis and degradation in three mammalian cell lines. The cellular structures examined were, in general, well maintained during mild hypothermia (27,32 °C) but became increasingly disrupted at low temperatures (4,10 °C). The degradation rates of all mRNAs and proteins examined were much reduced at 27 °C, and overall protein synthesis rates were gradually reduced with temperature down to 20 °C. Proteins involved in a range of cellular activities were either upregulated or downregulated at 32 and 27 °C during cold stress and recovery. Many of these proteins were molecular chaperones, but they did not include the inducible heat shock protein Hsp72. Further detailed investigation of specific proteins revealed that the responses to cold stress and recovery are at least partially controlled by modulation of p53, Grp75 and eIF3i levels. Furthermore, under conditions of severe cold stress (4 °C), lipid-containing structures were observed that appeared to be in the process of being secreted from the cell that were not observed at less severe cold stress temperatures. Our findings shed light on the mechanisms involved and activated in mammalian cells upon cold stress and recovery. [source] The single Cdk1-G1 cyclin of Cryptococcus neoformans is not essential for cell cycle progression, but plays important roles in the proper commitment to DNA synthesis and bud emergence in this yeastFEMS YEAST RESEARCH, Issue 5 2010Eric V. Virtudazo Abstract The cell cycle pattern of the pathogenic basidiomycetous yeast Cryptococcus neoformans differs from that of the ascomycetous budding yeast Saccharomyces cerevisiae. To clarify the cell cycle control mechanisms at the molecular level, homologues of cell cycle control genes in C. neoformans were cloned and analyzed. Here, we report on the cloning and characterization of genes coding for CDK1 cyclin homologues, in particular, the C. neoformans G1 cyclin. We have identified three putative CDK1 cyclin homologues and two putative CDK5 (PHO85) cyclin homologues from the genome. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that C. neoformans CLN1 is able to complement S. cerevisiae G1 cyclin deficiency, demonstrating that it is a G1 cyclin homologue. Interestingly, cells deleted of the single Cdk1-G1 cyclin were viable, demonstrating that this gene is not essential. However, it exhibited aberrant budding and cell division and a clear delay in the initiation of DNA synthesis as well as an extensive delay in budding. The fact that the mutant managed to traverse the G1 to M phase may be due to the activities of Pho85-related G1 cyclins. Also, that C. neoformans had only a single Cdk1-G1 cyclin highlighted the importance of keeping in order the commitment to the initiation of DNA synthesis first and then that of budding, as discussed. [source] Optimization of the culturing conditions of human umbilical cord blood-derived endothelial colony-forming cells under xeno-free conditions applying a transcriptomic approachGENES TO CELLS, Issue 7 2010Steffen M. Zeisberger Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and characterized by flow cytometry, capillary formation and responsiveness to cytokines. ECFCs were expanded under standard, FBS-containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. ECFC outgrowth in standard medium was successful in 92% of cord blood units. The karyotype of expanded ECFCs remained normal. Without FBS, ECFC initiation and expansion failed. Modest proliferation, changes in cell morphology and organization and cell death have been observed after passaging. Gene ontology analysis revealed a broad down-regulation of genes involved in cell cycle progression and up-regulation of genes involved in stress response and apoptosis. Interestingly, genes participating in lipid biosynthesis were markedly up-regulated. Detection of several endothelial cell-specific marker genes showed the maintenance of the endothelial cell characteristics during serum-free culture. Although ECFCs maintain their endothelial characteristics during serum-free culturing, they could not be expanded. Additional supply of FBS-free media with lipid concentrates might increase the ECFC survival. [source] Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylationGENES TO CELLS, Issue 7 2009Meixiang Sang Plk3, one of Polo-like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study, we found that Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation. During cisplatin (CDDP)-mediated apoptosis, Plk3 was transcriptionally induced, whereas its protein level was kept at basal level, suggesting that Plk3 might rapidly degrade in response to CDDP. Immunoprecipitation and in vitro pull-down experiments demonstrated that Plk3 interacts with p73. Luciferase reporter assays and RT-PCR experiments revealed that Plk3 inhibits p73-mediated transcriptional activity. Consistent with these results, pro-apoptotic activity of p73 was blocked by Plk3. Additionally, Plk3 decreased the stability of p73. Intriguingly, kinase-deficient Plk3 failed to inhibit p73 function, indicating that kinase activity of Plk3 is required for Plk3-mediated inhibition of p73. Indeed, in vitro kinase reaction showed that NH2 -terminal portion of p73 is phosphorylated by Plk3. In accordance with these observations, knocking down of Plk3 increased the stability of p73 and promoted CDDP-mediated apoptosis in association with up-regulation of p73. Collectively, our present findings suggest that Plk3 plays an important role in the regulation of cell fate determination in response to DNA damage through the inhibition of p73. [source] Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cellsGENES TO CELLS, Issue 3 2007Ryo Ukekawa 5-Bromodeoxyuridine (BrdU) clearly induces a senescence-like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated-proteins converting enzyme 1 (FACE-1) was markedly down-regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild-type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells. [source] Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic ,-tubulin complexesGENES TO CELLS, Issue 4 2006Hirohisa Masuda The ,-tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S. pombe,-tubulin complex. We show here that overproduction of a carboxy-terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of ,-tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic ,-tubulin complexes. Nuclear Alp4C induces a Wee1-dependent G2 delay, reduces the levels of the ,-tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear ,-tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic ,-tubulin complex activity is important for nuclear positioning and cell polarity control during interphase. [source] Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stabilityGENES TO CELLS, Issue 4 2005Tongyun Dang In response to DNA damage or replication block, cells activate a battery of checkpoint signaling cascades to control cell cycle progression and elicit DNA repair in order to maintain genomic stability and integrity. Identified as a homolog of its fission yeast counterpart, human Rad9 was proposed to form a Rad9-Hus1-Rad1 protein complex to mediate checkpoint signals. However, the precise function of Rad9 in the process of checkpoint activation is not fully understood. Using the RNA interference technique, we investigated the role of Rad9 in the genotoxic stress-induced activation of S-phase checkpoint and the maintenance of chromosomal stability. We found that Rad9 knockdown reduced the phosphorylation of Rad17, Chk1 and Smc1 in response to DNA replication block and certain types of DNA damage. Immunofluorescence studies showed that the removal of Rad9 disrupted the foci formation of phosphorylated Chk1, but not ATR. Moreover, Rad9 knockdown resulted in radioresistant DNA synthesis and reduced cell viability under replication stress. Finally, removal of Rad9 by RNAi led to increased accumulation of spontaneous chromosomal aberrations. Taken together, these results suggest a critical and specific role of Rad9 in the activation of S-phase checkpoint and the maintenance of chromosome stability. [source] The second phase activation of protein kinase C , at late G1 is required for DNA synthesis in serum-induced cell cycle progressionGENES TO CELLS, Issue 4 2003Koichi Kitamura Background: Cell lines that stably over-express protein kinase C (PKC) , frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKC, has a negative effect on cell proliferation. However, the mode of PKC, activation, the cell cycle stage requiring PKC, activity, and the exact role of PKC, at that stage remains unknown. Results: Here we show that the treatment of quiescent fibroblasts with serum activates PKC, at two distinct time points, within 10 min after serum treatment, and for a longer duration between 6 and 10 h. This biphasic activation correlates with the phosphorylation of Thr-505 at the activation loop of PKC,. Importantly, an inhibitor of PKC,, rottlerin, suppresses the biphasic activation of PKC,, and suppression of the second phase of PKC, activation is sufficient for the suppression of DNA synthesis. Consistent with this, the transient over-expression of PKC, mutant molecules lacking kinase activity suppresses serum-induced DNA synthesis. These results imply that PKC, plays a positive role in cell cycle progression. While the over-expression of PKC, enhances serum-induced DNA synthesis, this was not observed for PKC,. Similar experiments using a series of PKC,/, chimeras showed that the carboxyl-terminal 51 amino acids of PKC, are responsible for the stimulatory effect. On the other hand, the over-expression of PKC, suppresses cell entry into M-phase, being consistent with the previous studies based on stable over-expressors. Conclusions: We conclude that PKC, plays a role in the late-G1 phase through the positive regulation of cell-cycle progression, in addition to negative regulation of the entry into M-phase. [source] Enhanced Expression of Transcription Factor E2F in Helicobacter pylori -infected Gastric MucosaHELICOBACTER, Issue 3 2002Hajime Isomoto Abstract Objective.Helicobacter pylori is implicated in gastric carcinogenesis through increased gastric epithelial cell turnover. In fact, high proportions of proliferating and apoptotic epithelial cells are found in H. pylori -infected gastric mucosa. E2F, a transcription factor, induces coordinated transactivation of a set of genes involved in cell cycle progression. The aim of this study was to investigate the expression of E2F in H. pylori -infected gastric mucosa and examine the correlation between such expression and gastric epithelial cell proliferation and apoptosis. Methods. Twenty-five patients with H. pylori -associated gastritis (HAG) and 13 control subjects negative for H. pylori were examined. E2F expression was studied in situ by Southwestern histochemistry, a method used to localize transcription factors. Labeled double-stranded oligo-DNA with specific consensus sequence for E2F binding sites was reacted with frozen sections from antral biopsy specimens obtained at endoscopy. Gastric epithelial cell proliferation was assessed by immunostaining of proliferating cell nuclear antigen (PCNA), while apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The percentages of epithelial cells with nuclear staining for PCNA and E2F were expressed as a positivity index (PI). The percentage of TUNEL-positive epithelial cells was defined as apoptotic index. Results. E2F was expressed in the nuclei of gastric epithelial cells within gastric pits. E2F PI in H. pylori -infected gastric mucosa was significantly higher than that in noninfected. Expression of E2F correlated well with PCNA-positive epithelial cells. We also demonstrated colocalization of PCNA with E2F expression in the same epithelial cells. Apoptotic index was also high in H. pylori -infected mucosa, and correlated with E2F PI. Conclusion. Our results demonstrated a significant increase in the expression of E2F in H. pylori -infected mucosa, which correlated with both the percentages of PCNA- and TUNEL-positive cells. Our results suggest that enhanced E2F expression in gastric mucosa may be involved in H. pylori -related gastric carcinogenesis through accelerated cell turnover. [source] Nucleophosmin (NPM1) mutations in adult and childhood acute myeloid leukaemia: towards definition of a new leukaemia entity,HEMATOLOGICAL ONCOLOGY, Issue 4 2009Rachel Rau Abstract Nucleophosmin (NPM) is a ubiquitously expressed chaperone protein that shuttles rapidly between the nucleus and cytoplasm, but predominantly resides in the nucleolus. It plays key roles in ribosome biogenesis, centrosome duplication, genomic stability, cell cycle progression and apoptosis. Somatic mutations in exon 12 of the NPM gene (NPM1) are the most frequent genetic abnormality in adult acute myeloid leukaemia (AML), found in approximately 35% of all cases and up to 60% of patients with normal karyotype (NK) AML. In children, NPM1 mutations are far less frequent, occurring in 8,10% of all AML cases, and in approximately 25% of those with a NK. NPM1 mutations lead to aberrant localization of the NPM protein into the cytoplasm, thus the designation, NPMc+ AML. NPMc+ AML is seen predominantly in patients with a NK and is essentially mutually exclusive of recurrent chromosomal translocations. Patients with NPM1 mutations are twice as likely as those who lack an NPM1 mutation to also have a FMS-like tyrosine kinase (FLT3) internal tandem duplication (ITD) mutation. NPMc+ AML is also characterized by a unique gene expression signature and microRNA signature. NPMc+ AML has important prognostic significance, as NPMc+ AML, in the absence of a coexisting FLT3-ITD mutation, is associated with a favourable outcome. NPM1 mutations have also shown great stability during disease evolution, and therefore represent a possible marker for minimal residual disease detection. Given its distinctive biologic and clinical features and its clear clinical relevance, NPMc+ AML is included as a provisional entity in the 2008 WHO classifications. There is still much to be learned about this genetic alteration, including its exact role in leukaemogenesis, how it interacts with other mutations and why it confers a more favourable prognosis. Further, it represents a potential therapeutic target warranting research aimed at identifying novel small molecules with activity in NPMc+ AML. Copyright © 2009 John Wiley & Sons, Ltd. [source] Hepatitis B virus X protein blunts senescence-like growth arrest of human hepatocellular carcinoma by reducing Notch1 cleavage,HEPATOLOGY, Issue 1 2010Jiejie Xu One of the serious sequelae of chronic hepatitis B virus (HBV) infection is hepatocellular carcinoma (HCC). Among all the proteins encoded by the HBV genome, hepatitis B virus X protein (HBx) is highly associated with the development of HCC. Although Notch1 signaling has been found to exert a tumor-suppressive function during HCC development, the mechanism of interaction between HBx expression and Notch1 signaling needs to be explored. In this study, we report that HBx expression in hepatic and hepatoma cells resulted in decreased endogenous protein levels of Notch1 intracellular domain (ICN1) and messenger RNA levels of its downstream target genes. These effects were due to a reduction of Notch1 cleavage by HBx through the suppression of presenilin1 (Psen1) transcription rather than inhibition of Notch1 transcription or its ligands' expression. Through transient HBx expression, decreased ICN1 resulted in enhanced cell proliferation, induced G1-S cell cycle progression, and blunted cellular senescence in vitro. Furthermore, the effect of blunted senescence-like growth arrest by stable HBx expression through suppression of ICN1 was shown in a nude mouse xenograft transplantation model. The correlation of inhibited Psen1-dependent Notch1 signaling and blunted senescence-like growth arrest was also observed in HBV-associated HCC patient tumor samples. Conclusion: Our results reveal a novel function of HBx in blunting senescence-like growth arrest by decreasing Notch1 signaling, which could be a putative molecular mechanism mediating HBV-associated hepatocarcinogenesis. (HEPATOLOGY 2010;) [source] MicroRNAs control hepatocyte proliferation during liver regeneration,HEPATOLOGY, Issue 5 2010Guisheng Song MicroRNAs (miRNAs) constitute a new class of regulators of gene expression. Among other actions, miRNAs have been shown to control cell proliferation in development and cancer. However, whether miRNAs regulate hepatocyte proliferation during liver regeneration is unknown. We addressed this question by performing 2/3 partial hepatectomy (2/3 PH) on mice with hepatocyte-specific inactivation of DiGeorge syndrome critical region gene 8 (DGCR8), an essential component of the miRNA processing pathway. Hepatocytes of these mice were miRNA-deficient and exhibited a delay in cell cycle progression involving the G1 to S phase transition. Examination of livers of wildtype mice after 2/3 PH revealed differential expression of a subset of miRNAs, notably an induction of miR-21 and repression of miR-378. We further discovered that miR-21 directly inhibits Btg2, a cell cycle inhibitor that prevents activation of forkhead box M1 (FoxM1), which is essential for DNA synthesis in hepatocytes after 2/3 PH. In addition, we found that miR-378 directly inhibits ornithine decarboxylase (Odc1), which is known to promote DNA synthesis in hepatocytes after 2/3 PH. Conclusion: Our results show that miRNAs are critical regulators of hepatocyte proliferation during liver regeneration. Because these miRNAs and target gene interactions are conserved, our findings may also be relevant to human liver regeneration. (HEPATOLOGY 2010) [source] Rapamycin delays tumor development in murine livers by inhibiting proliferation of hepatocytes with DNA damage,HEPATOLOGY, Issue 2 2009Laura Elisa Buitrago-Molina In this study, everolimus (RAD001) was used to determine the role of mammalian target of rapamycin (mTOR) in hepatocarcinogenesis. We show that RAD001 effectively inhibits proliferation of hepatocytes during chronic liver injury. Remarkably, the ability of RAD001 to impair cell cycle progression requires activation of the DNA damage response; loss of p53 significantly attenuates the antiproliferative effects of mTOR inhibition. RAD001 modulates the expression of specific cell cycle,related proteins and the assembly of cyclin,cyclin-dependent kinase complexes to prevent cell cycle progression. Furthermore, RAD001 sustains the apoptosis sensitivity of hepatocytes during chronic liver injury by inhibiting p53-induced p21 expression. Long-term treatment with RAD001 markedly delays DNA damage,induced liver tumor development. Conclusion: We provide evidence that mTOR inhibition has a substantial effect on sequential carcinogenesis and may offer an effective strategy to delay liver tumor development in patients at risk. (HEPATOLOGY 2009;50:500,509.) [source] MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells,HEPATOLOGY, Issue 1 2009Teng Xu Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down-regulation of miR-195 has been observed in various types of cancers. However, the biological function of miR-195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR-195. Our results showed that miR-195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR-195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR-195 blocked G1/S transition, whereas inhibition of miR-195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition-related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR-195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR-195, whereas overexpression of these proteins attenuated miR-195-induced G1 arrest. In addition, miR-195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR-195 may block the G1/S transition by repressing Rb-E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR-195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR-195 in cancer therapy. (HEPATOLOGY 2009.) [source] An MLCK-dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK-p70S6K pathway,HEPATOLOGY, Issue 1 2006Anne Bessard We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant-negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2-dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK-dependent window in late G1 phase of regenerating liver after two-thirds partial hepatectomy. In conclusion, our results underline an MLCK-dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation. (HEPATOLOGY 2006;44:152,163.) [source] | ||||||||||||||||||||||||||||||||||