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Treated Cells (treated + cell)
Selected AbstractsAcute 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] Microglial glutamate uptake is coupled to glutathione synthesis and glutamate releaseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2006Mikael Persson Abstract The physiological function of microglial glutamate uptake has been debated as it is about 10% of that measured for astrocytes. This study addresses how glutamate, taken up from the extracellular space, is utilized by microglia. It was found that purified rat microglia incubated for 60 min with 3H-glutamate had an increased intracellular accumulation of 3H-glutamate after 12 h incubation with tumour necrosis factor alpha (TNF-,) but not after incubation with lipopolysaccharide (LPS). Furthermore, LPS- but not TNF-,-treated cells showed an increased efflux of 3H-labelled compounds, presumably glutamate through the XC, system and treatment with LPS or TNF-, increased the microglial glutathione concentrations and led to an increased incorporation of 3H-glutamate into glutathione. Depending on the stimuli, 3,6% of the total labelled contents were found in the form of glutathione and 25,35% in the form of glutamate. These results show that microglial glutamate uptake is directly coupled to glutathione synthesis and release of glutamate and/or glutamate metabolites. Additionally, the increased glutathione contents after LPS or TNF-, treatment were able to reduce microglial cell death after H2O2 challenge, showing a potential (self)-protective function for microglial glutamate transporter expression and glutathione synthesis. [source] Homeostatic plasticity induced by chronic block of AMPA/kainate receptors modulates the generation of rhythmic bursting in rat spinal cord organotypic culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2001Micaela Galante Abstract Generation of spontaneous rhythmic activity is a distinct feature of developing spinal networks. We report that rat embryo organotypic spinal cultures contain the basic circuits responsible for pattern generation. In this preparation rhythmic activity can be recorded from ventral interneurons and is developmentally regulated. When chronically grown in the presence of an AMPA/kainate receptor blocker, this circuit expresses long-term plasticity consisting largely of increased frequency of fast synaptic activity and reduction in slow GABAergic events. We examined whether, once this form of homeostatic plasticity is established, the network could still exhibit rhythmicity with properties similar to controls. Control or chronically treated ventral interneurons spontaneously generated (with similar probability) irregular, network-driven bursts over a background of ongoing synaptic activity. In control cultures increasing network excitability by strychnine plus bicuculline, or by raising [K+]o, induced rapid-onset, regular rhythmic bursts. In treated cultures the same pharmacological block of Cl, -mediated transmission or high-K+ application also induced regular patterned activity, although significantly faster and, in the case of high K+, characterized by slow onset due to postsynaptic current summation. Enhancing GABAergic transmission by pentobarbital surprisingly accelerated the high-K+ rhythm of control cells (though depressing background activity), whereas it slowed it down in chronically treated cells. This contrasting effect of pentobarbital suggests that, to preserve bursting ability, chronic slices developed a distinct GABAergic inhibitory control on over-expressed bursting circuits. Conversely, in control slices GABAergic transmission depressed spontaneous activity but it facilitated bursting frequency. Thus, even after homeostatic rearrangement, developing mammalian spinal networks still generate rhythmic activity. [source] Enhancement of Ca2+ -regulated exocytosis by indomethacin in guinea-pig antral mucous cells: arachidonic acid accumulationEXPERIMENTAL PHYSIOLOGY, Issue 1 2006Shoko Fujiwara Ca2+ -regulated exocytosis is enhanced by an autocrine mechanism via the PGE2,cAMP pathway in antral mucous cells of guinea-pigs. The inhibition of the PGE2,cAMP pathway by H-89 (an inhibitor of protein kinase A, PKA) or aspirin (ASA, an inhibitor of cyclo-oxygenase, COX) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin (IDM, an inhibitor of COX), however, decreased the frequency of ACh-stimulated exocytotic events only by 30%. Moreover, IDM increased the frequency of ACh-stimulated exocytotic events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the synthesis of Prostaglandin (PGG/H) and (15R)-15-hydroxy-5,8,11 cis-13-trans-eicosatetraenoic acid (15R-HPETE), while ASA inhibits only the synthesis of PGG/H. Thus, IDM may accumulate arachidonic acid (AA). AACOCF3 or N -(p -amylcinnamoyl) anthranilic acid (ACA; both inhibitors of phospholipase A2, PLA2), which inhibits AA synthesis, decreased the frequency of ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3 -treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF3 - or ASA-treated cells, similar to IDM in ASA- and H-89-treated cells. Moreover, in the presence of AA, IDM did not increase the frequency of ACh-stimulated exocytotic events in ASA-treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA inhibits the AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose,response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nm. In conclusion, IDM modulates the ACh-stimulated exocytosis via AA accumulation in antral mucous cells. [source] Azidothymidine causes functional and structural destruction of mitochondria, glutathione deficiency and HIV-1 promoter sensitizationFEBS JOURNAL, Issue 11 2002Tokio Yamaguchi Mitochondrial functional and structural impairment and generation of oxidative stress have been implicated in aging, various diseases and chemotherapies. This study analyzed azidothymidine (AZT)-caused failures in mitochondrial functions, in redox regulation and activation of the HIV-1 gene expression. We monitored intracellular concentrations of ATP and glutathione (GSH) as the indicators of energy production and redox conditions, respectively, during the time-course experiments with U937 and MOLT4 human lymphoid cells in the presence of AZT (0.05 mg·mL,1) or H2O2 (0.01 mm) for 15,25 days. Mitochondrial DNA integrity and NF-,B-driven HIV-1 promoter activity were also assessed. ATP concentration began to decrease within several days after exposure to AZT or H2O2, and the decrease continued to reach 30,40% of the normal level. However, decline of GSH was detectable after a retention period for at least 5,6 days, and progressed likewise. PCR analyses found that mitochondrial DNA destruction occurred when the ATP and GSH depletion had progressed, detecting a difference in the deletion pattern between AZT and H2O2 -treated cells. The GSH decrease coincided with HIV-1 promoter sensitization detected by enhanced DNA binding ability of NF-,B and induction of the gene expression upon H2O2 -rechallenge. Our results suggest that, in the process of AIDS myopathy development, AZT or oxidative agents directly impair the energy-producing system of mitochondria, causing dysfunction of cellular redox control, which eventually leads to loss of the mitochondrial DNA integrity. The mechanism of cellular redox condition-mediated NF-,B activation is discussed. [source] Effects of methylcyclodextrin on lysosomesFEBS JOURNAL, Issue 5 2001Michel Jadot The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl- l -phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications. [source] Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors,HUMAN MUTATION, Issue 8 2007Marian Todaro Abstract Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction. Hum Mutat 28(8), 816,823, 2007. © 2007 Wiley-Liss, Inc. [source] Hydrogen peroxide induces expression and activation of AMP-activated protein kinase in a dental pulp cell lineINTERNATIONAL ENDODONTIC JOURNAL, Issue 3 2008Y. Fukuyama Abstract Aim, To investigate the effects of hydrogen peroxide on cell viability and expression and activation of AMP-activated protein kinase (AMPK) in rat dental pulp cell line RPC-C2A. Methodology, RPC-C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 °C, in a humidified atmosphere at 5% CO2. Cells were cultured in the presence or absence of H2O2 for up to 60 min at concentrations of from 0.1 to 3.0 mmol L,1. Cell viability was analysed by WST-1 reduction assay. Expression of AMPK subunit isoforms was analysed by Western blotting using antibodies to the catalytic ,1 and regulatory ,1 and ,1 subunit isoforms. The effect of silencing AMPK,1 on cell viability was determined using siRNA. Results, Exposure to H2O2 decreased cell viability in a time- and dose-dependent manner. The catalytic AMPK,1 subunit and its activated form, phospho-AMPK,, increased with exposure to H2O2 in a time- and dose-dependent manner, whereas the regulatory ,1 and ,1 subunits showed no change. Downregulation of AMPK,1 resulted in a reduction in cell viability in H2O2 -treated cells at a concentration of 0.1 mmol L,1 for 30 min incubation, indicating an increased sensitivity to H2O2. Conclusions, Reactive oxygen induced energy fuel gauge enzyme AMPK, expression and its activation by phosphorylation in RPC-C2A cells, suggesting that AMPK is essential for protection against H2O2 -induced nonapoptotic cell death. Therefore, AMPK may be a therapeutic modulation target for treatment of the dentine,pulp complex injured by reactive oxygen. [source] Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of ,-catenin phosphorylationINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Stephen Hiscox Abstract We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell,cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell,cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated ,-catenin, whilst serine/threonine-phosphorylated ,-catenin was decreased. These cells also displayed loss of association between ,-catenin and E-cadherin, increased cytoplasmic and nuclear ,-catenin and elevated transcription of ,-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced ,-catenin tyrosine phosphorylation, increased ,-catenin,E-cadherin association and promoted cell,cell adhesion. In such treated cells, the association of ,-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of ,-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro. © 2005 Wiley-Liss, Inc. [source] Inter- alu PCR detects high frequency of genetic alterations in glioma cells exposed to sub-lethal cisplatinINTERNATIONAL JOURNAL OF CANCER, Issue 4 2005Tapasya Srivastava Abstract Increased genomic instability contributes to higher frequency of secondary drug resistance and neoplastic progression in tumors as well as in cells exposed to sub-lethal concentrations of chemotherapeutic agents. We have used PCR based DNA fingerprinting techniques of randomly amplified polymorphic DNA (RAPD) and inter- alu PCR to study this phenomenon in the tumor genome. The choice of the primer, either random (for RAPD) or specific (inter- alu PCR) can determine the nature of alterations being assessed. We have compared the inter- alu PCR and RAPD profiles of U87MG glioblastoma cells exposed to sequentially increasing low doses of cisplatin for 24 passages to that of untreated controls. Inter- alu PCR, with 2 primers, demonstrated a number of alterations in the treated cells, in the form of loss / gain and changes in the intensity of bands. No changes were observed by RAPD analysis with 5 primers, however, indicating a preferential increase in the alu mediated recombination frequency in the treated cells (p = 1.866 × 10,4). The number of changes observed with respect to the corresponding leucocyte DNA in the inter- alu PCR profile of 26 primary tumors (Grade II = 13; Grade IV = 13), resected before chemotherapy, for the 2 inter- alu primers was very small. We present a novel application of the inter- alu PCR in detecting alterations in long term cultured cells at low dose exposure to a chemotherapeutic agent. Our results suggest that alu mediated recombination may be important in cells exposed to sub-lethal doses of cisplatin but not in the genesis of primary glioma. © 2005 Wiley-Liss, Inc. [source] Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarrayINTERNATIONAL JOURNAL OF CANCER, Issue 2 2004Jun-Ping Zhang Abstract Cantharidin is a natural toxin that has antitumor properties and causes leukocytosis as well as increasing sensitivity of tumor cells resistant to other chemotherapeutic agents. There is limited information, however, on the molecular pharmacological mechanisms of cantharidin on human cancer cells. We have used cDNA microarrays to identify gene expression changes in HL-60 promyeloid leukemia cells exposed to cantharidin. Cantharidin-treated cells not only decreased expression of genes coding for proteins involved in DNA replication (e.g., DNA polymerase delta), DNA repair (e.g., FANCG, ERCC), energy metabolism (e.g., isocitrate dehydrogenase alpha, ADP/ATP translocase), but also decreased expression of genes coding for proteins that have oncogenic activity (e.g., c-myc, GTPase) or show tumor-specific expression (e.g., phosphatidylinositol 3-kinase). In contrast, these treated cells overexpressed several genes that encode intracellular and secreted growth-inhibitory proteins (e.g., BTG2, MCP-3) as well as proapoptotic genes (e.g., ATL-derived PMA-responsive peptide). Our findings suggest that alterations in specific genes functionally related to cell proliferation or apoptosis may be responsible for cantharidin-mediated cytotoxicity. We also found that exposure of HL-60 cells to cantharidin resulted in the decreased expression of multidrug resistance-associated protein genes (e.g., ABCA3, MOAT-B), suggesting that cantharidin may be used as an oncotherapy sensitizer, and the increased expression of genes in modulating cytokine production and inflammatory response (e.g., NFIL-3, N-formylpeptide receptor), which may partly explain the stimulating effects on leukocytosis. Our data provide new insight into the molecular mechanisms of cantharidin. © 2003 Wiley-Liss, Inc. [source] Coumarin A/AA induces apoptosis-like cell death in HeLa cells mediated by the release of apoptosis-inducing factorJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2009Carolina Álvarez-Delgado Abstract It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral-blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis-inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase-3 was not detected. The overall results indicate that coumarin A/AA induces a caspase-independent apoptotic-like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:263,272, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20288 [source] Diarsenic and tetraarsenic oxide inhibit cell cycle progression and bFGF- and VEGF-induced proliferation of human endothelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Sang Hyeok Woo Abstract Arsenic trioxide (As2O3, diarsenic oxide) has recently been reported to induce apoptosis and inhibit the proliferation of various human cancer cells derived from solid tumors as well as hematopoietic malignancies. In this study, the in vitro effects of As2O3 and tetraasrsenic oxide (As4O6) on cell cycle regulation and basic fibroblast growth factor (bFGF)- or vascular endothelial growth factor (VEGF)-stimulated cell proliferation of human umbilical vein endothelial cells (HUVEC) were investigated. Significant dose-dependent inhibition of cell proliferation was observed when HUVEC were treated with either arsenical compound for 48 h, and flow cytometric analysis revealed that these two arsenical compounds induced cell cycle arrest at the G1 and G2/M phases,the increases in cell population at the G1 and G2/M phase were dominantly observed in As2O3 - and As4O6 -treated cells, respectively. In both arsenical compounds-treated cells, the protein levels of cyclin A and CDC25C were significantly reduced in a dose-dependent manner, concomitant to the reduced activities of CDK2- and CDC2-associated kinase. In G1 -synchronized HUVEC, the arsenical compounds prevented the cell cycle progression from G1 to S phase, which was stimulated by bFGF or VEGF, through the inhibition of growth factor-dependent signaling. These results suggest that arsenical compounds inhibit the proliferation of HUVEC via G1 and G2/M phase arrest of the cell cycle. In addition, these inhibitory effects on bFGF- or VEGF-stimulated cell proliferation suggest antiangiogenic potential of these arsenical compounds. © 2005 Wiley-Liss, Inc. [source] Differential control of apoptosis by DJ-1 in prostate benign and cancer cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2004Yaacov Hod Abstract DJ-1 is a conserved protein reported to be involved in diverse cellular processes ranging from cellular transformation, control of protein,RNA interaction, oxidative stress response to control of male infertility, among several others. Mutations in the human gene have been shown to be associated with an autosomal recessive, early onset Parkinson's disease (PARK7). The present study examines the control of DJ-1 expression in prostatic benign hyperplasia (BPH-1) and cancer (PC-3) cell lines in which DJ-1 abundance differs significantly. We show that while BPH-1 cells exhibit low basal level of DJ-1 expression, stress-inducing agents such as H2O2 and mitomycin C markedly increase the intracellular level of the polypeptide. In contrast, DJ-1 expression is relatively high in PC-3 cells, and incubation with the same cytotoxic drugs does not modulate further the level of the polypeptide. In correlation with the expression of DJ-1, both cytotoxic agents activate the apoptotic pathway in the prostatic benign cells but not in PC-3 cells, which are resistant to their action. We further demonstrate that incubation of BPH-1 cells with TNF-related-apoptosis-inducing-ligand/Apo-2L (TRAIL) also enhances DJ-1 expression and that TRAIL and H2O2 act additively to stimulate DJ-1 accumulation but synergistically in the activation of the apoptotic pathway. Time-course analysis of DJ-1 stimulation shows that while DJ-1 level increases without significant lag in TRAIL-treated cells, there is a delay in H2O2 -treated cells, and that the increase in DJ-1 abundance precedes the activation of apoptosis. Unexpectedly, over-expression of DJ-1 de-sensitizes BPH-1 cells to the action of apoptotic-inducing agents. However, RNA-interference-mediated silencing of DJ-1 expression results in sensitization of PC-3 cells to TRAIL action. These results are consistent with a model in which DJ-1 is involved in the control of cell death in prostate cell lines. DJ-1 appears to play a differential role between cells expressing a low but inducible level of DJ-1 (e.g., BPH-1 cells) and those expressing a high but constitutive level of the polypeptide (e.g., PC-3 cells). © 2004 Wiley-Liss, Inc. [source] Kinase suppressor of RAS (KSR) amplifies the differentiation signal provided by low concentrations 1,25-dihydroxyvitamin D3JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Xuening Wang The activity of kinase suppressor of ras (KSR), a kinase or a molecular scaffold upstream from Raf-1, is involved in the MEK/ERK MAP kinase cascade which can signal cell growth, survival, or differentiation, depending on the cellular context. We provide evidence here that KSR is upregulated in HL60 cells undergoing differentiation induced by low (0.3,3 nM) concentrations of 1,25-dihydroxyvitamin D3 (1,25D3), and an antisense oligo (AS), but not a sense oligo, to KSR inhibits this differentiation. The inhibition of differentiation by AS,KSR oligo was less apparent when the concentration of 1,25D3 was increased, suggesting that at the higher concentrations of 1,25D3 KSR is not essential for the signaling of the differentiated phenotype. The reduced differentiation of HL60 cells exposed to AS,KSR was paralleled by reduced phosphorylation of Raf-1 Ser 259, and of p90RSK, used here as read-out for MAPK cascade activity. Conversely, ectopic expression of Flag-tagged wild type KSR potentiated the differentiation-inducing effects of low concentrations of 1,25D3. Additional data suggest that the kinase activity of KSR is required for these effects, as transfection of a kinase inactive KSR construct did not significantly increase the 1,25D3 -induced differentiation. Enzyme assays performed with KSR immunoprecipitated from 1,25D3 -treated cells showed kinase activity when recombinant Raf-1 was used as the substrate, but not when the 1,25D3 -treated cells were pretreated with AS,KSR oligos. Taken together, these data suggest that KSR participates in signaling of monocytic differentiation by augmenting the strength of the signal transmitted through Raf-1 to downstream targets. J. Cell. Physiol. 198: 333,342, 2004© 2003 Wiley-Liss, Inc. [source] The core-aldehyde 9-oxononanoyl cholesterol increases the level of transforming growth factor ,1-specific receptors on promonocytic U937 cell membranesAGING CELL, Issue 2 2009Simona Gargiulo Summary Among the broad variety of compounds generated via oxidative reactions in low-density lipoproteins (LDL) and subsequently found in the atherosclerotic plaque are aldehydes that are still esterified to the parent lipid, termed core aldehydes. The most represented cholesterol core aldehyde in LDL is 9-oxononanoyl cholesterol (9-ONC), an oxidation product of cholesteryl linoleate. 9-ONC, at a concentration detectable in biological material, markedly up-regulates mRNA expression and protein level of both the pro-fibrogenic and pro-apoptotic cytokine transforming growth factor ,1 (TGF-,1) and the TGF-, receptor type I (T,RI) in human U937 promonocytic cells. We also observed increased membrane presentation of TGF-, receptor type II (T,RII). Experiments employing the T,RI inhibitor SB431542, or the TGF, antagonist DANFc chimera, have shown that the effect on T,RI is directly induced by 9-ONC, while T,RII up-regulation seems stimulated by its specific ligand, i.e. TGF,1, over-secreted meanwhile by treated cells. Increased levels of the cytokine and of its specific receptors in 9-ONC-treated cells clearly occurs through stimulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), as demonstrated by ERK1/2 knockdown experiments using mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MEK1 and MEK2) siRNAs, or PD98059, a selective MEK1/2 inhibitor. 9-ONC might thus sustain further vascular remodeling due to atherosclerosis, not simply by stimulating synthesis of the pro-fibrogenic cytokine TGF-,1 in vascular cells, but also and chiefly by enhancing the TGF-,1 autocrine loop, because of the marked up-regulation of the cytokine's specific receptors T,RI and T,RII. [source] Chronic exposure to sub-lethal beta-amyloid (A,) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells*JOURNAL OF NEUROCHEMISTRY, Issue 5 2007Daniel Sirk Abstract Studies on amyloid beta (A,|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in A,-mediated neurotoxicity. We used differentiated PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N,-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal A, on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal A,25,35 (10 ,mol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 ± 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub-lethal A,1,42 inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear-encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in A,-treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen-glucose deprivation and altered mitochondrial morphology. These findings suggest that an A,-mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer's disease. [source] Pyroglutamate-modified amyloid ,-peptides , A,N3(pE) , strongly affect cultured neuron and astrocyte survivalJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Claudio Russo Abstract N-terminally truncated amyloid-, (A,) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [A,N3(pE),40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of A, accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of A,1,40, A,1,42, A,N3(pE),40 and A,N3(pE),42. Our data show that fibre morphology of A, peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. A,N3(pE),40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated A,N3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. A,N3(pE),40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full-length peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance ,-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease. [source] Impaired Mitochondrial Function Results in Increased Tissue Transglutaminase Activity In SituJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Mathieu Lesort Abstract: Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD. [source] Cytotoxic effects of dental resin liquids on primary gingival fibroblasts and periodontal ligament cells in vitroJOURNAL OF ORAL REHABILITATION, Issue 12 2004Y.-L. Lai summary, Cytotoxic effects of resin liquids of three in situ relining dental polymers, AlikeTM, Kooliner, and Tokuso Rebase, and their major components, methyl methacrylate (MMA), isobutyl methacrylate (IBMA), and 1,6-hexanediol dimethacrylate (1,6-HDMA) were investigated. The concentrations of major monomers in these resin liquids were determined by high-performance liquid chromatography. Cellular viability of human gingival fibroblasts (GF) and periodontal ligament (PDL) cells were evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. Moreover, patterns of cell death were analysed using annexin V/propidium iodide staining with flow cytometry. The results indicated that AlikeTM liquid contained 91·3% MMA, Kooliner liquid contained 94·5% IBMA, and Tokuso Rebase liquid contained 65·8% 1,6-HDMA. All materials examined had cytotoxic effects on GF and PDL cells in dose-dependent manners. Tokuso Rebase liquid appeared to be the most cytotoxic among the various resin liquids examined. The effects of Kooliner and Tokuso Rebase liquids may have resulted from IBMA and 1,6-HDMA, respectively. Furthermore, the majority of treated cells died from necrosis; whereas a small portion of cells died from apoptosis. In conclusion, the results demonstrated that these liquid forms of dental polymers and their major monomers cause cytotoxic reactions. The direct relining procedure that cures these materials in situ should be used cautiously. [source] Pulsed electromagnetic fields enhance BMP-2 dependent osteoblastic differentiation of human mesenchymal stem cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 9 2008Z. Schwartz Abstract Mesenchymal stem cells (MSCs) express an osteoblastic phenotype when treated with BMP-2, and BMP-2 is used clinically to induce bone formation although high doses are required. Pulsed electromagnetic fields (PEMF) also promote osteogenesis in vivo, in part through direct action on osteoblasts. We tested the hypothesis that PEMF enhances osteogenesis of MSCs in the presence of an inductive stimulus like BMP-2. Confluent cultures of human MSCs were grown on calcium phosphate disks and were treated with osteogenic media (OM), OM containing 40 ng/mL rhBMP-2, OM,+,PEMF (8 h/day), or OM,+,BMP-2,+,PEMF. MSCs demonstrated minor increases in alkaline phosphatase (ALP) during 24 days in culture and no change in osteocalcin. OM increased ALP and osteocalcin by day 6, but PEMF had no additional effect at any time. BMP-2 was stimulatory over OM, and PEMF,+,BMP-2 synergistically increased ALP and osteocalcin. PEMF also enhanced the effects of BMP-2 on PGE2, latent and active TGF-,1, and osteoprotegerin. Effects of PEMF on BMP-2,treated cells were greatest at days 12 to 20. These results demonstrate that PEMF enhances osteogenic effects of BMP-2 on MSCs cultured on calcium phosphate substrates, suggesting that PEMF will improve MSC response to BMP-2 in vivo in a bone environment. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1250,1255, 2008 [source] C5a modulation of interleukin-1, -induced interleukin-6 production by human osteoblast-like cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2000John M. Pobanz Periodontal bone resorption is controlled by osteoblast products, including interleukin (IL)-6, which are stimulated by other cytokines and complement components in the pro-inflammatory milieu. This study demonstrated that human osteoblast-like osteosarcoma cells (MG-63) responded to human recombinant (hr) C5a by releasing significant amounts of the bone-resorbing cytokine IL-6. C5a-induced release of IL-6 was enhanced 330% when cells were exposed to IL-1, prior to C5a challenge at optimal concentrations (1.0 ,g/ml C5a, 0.1 ng/ml IL-1,). Cells simultaneously challenged with these concentrations of C5a and IL-1, produced a 700% increase in IL-6 release relative to cells challenged with IL-1, alone. Incubation of IL-1,-treated cells with anti-human C5a receptor (C5aR) Ab resulted in a 78% suppression of the C5a-induced release of IL-6, but C5aR neutralization did not affect C5a/IL-1, co-stimulation of IL-6. In addition, neither IL-1, nor C5a significantly altered the other's cell-surface receptor relative to binding affinity or density. These results indicate that while MG-63 cells express functional C5aRs, the synergistic effect of C5a and IL-1, on osteoblast IL-6 production is probably controlled by post-receptor signaling events. C5a agonists and antagonist used to alter critical C5a concentrations may present a new point of therapeutic intervention for the treatment of inflammatory bone resorption such as is found in periodontitis. [source] Melatonin inhibits MPP+ -induced caspase-mediated death pathway and DNA fragmentation factor-45 cleavage in SK-N-SH cultured cellsJOURNAL OF PINEAL RESEARCH, Issue 2 2007Jirapa Chetsawang Abstract:, Neurodegenerative diseases such as Parkinson's disease are illnesses associated with high morbidity and mortality with few, or no effective, options available for their treatment. In addition, the direct cause of selective dopaminergic cell loss in Parkinson's disease has not been clearly understood. Taken together, several studies have demonstrated that melatonin has a neuroprotective effect both in vivo and in vitro. Accordingly, the effects of melatonin on 1-methyl, 4-phenyl, pyridinium ion (MPP+)-treated cultured human neuroblastoma SK-N-SH cell lines were investigated in the present study. The results showed that MPP+ significantly decreased cell viability. By contrast, an induction of phosphorylation of c-Jun, activation of caspase-3 enzyme activity, cleavage of DNA fragmentation factors 45 and DNA fragmentation were observed in MPP+ -treated cells. These changes were diminished by melatonin. These results demonstrate the cellular mechanisms of neuronal cell degeneration induced via c-Jun-N-terminal kinases and caspase-dependent signaling, and the potential role of melatonin on protection of neuronal cell death induced by this neurotoxin. [source] Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infectionJOURNAL OF VIRAL HEPATITIS, Issue 8 2007T. N. Q. Pham Summary., Detection of residual HCV in individuals with SVR after treatment of CHC can be significantly heightened by analyzing ex vivo mitogen-activated T and B lymphocytes and applying sensitive nucleic acid amplification assays. However, it remained unknown if synergistic activation of lymphocytes and monocytes would further augment HCV detection, if viral replication becomes universally upregulated in treated cells, and if examining sequential sera and lymphoid cells would improve detection of occult infection. Using paired sera and lymphoid cells collected 1 year apart from 17 individuals with normal liver enzymes for up to 72 months after SVR, it was found that simultaneous activation of lymphocytes and monocytes enhanced identification of silent HCV infection and revealed that in some cases monocytes were the principal immune cell type where HCV persisted. Testing of serial samples further increased detection of occult infection. Ultimately, by combining the above two approaches, all individuals with SVR were found to be silent carriers of HCV. Clonal sequencing revealed HCV variations in sera and lymphoid cells and evolution of viral genomes confirming ongoing virus replication. Surprisingly, similar to those with CHC, naive lymphoid cells from some individuals carried ,103 HCV copies/,g total RNA. HCV loads in naive lymphoid cells predetermined the outcome of ex vivo stimulation with respect to upregulation or inhibition of HCV replication. HCV RNA levels in occult infection were inversely proportional to the expression of IFN, and IFN-inducible MxA, but not to IFN, or tumour necrosis factor , in naive and mitogen-treated lymphoid cells. [source] Surface alteration of Saccharomyces cerevisiae induced by thymol and eugenolLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2004S. Bennis Abstract Aims:, This study aims to bring some information about the mechanism of the fungicidal action of thymol and eugenol; phenolic major components of thyme and clove essential oils respectively. Saccharomyces cerevisiae was used as yeast model. Methods and Results:, Treatment of yeast cells with these components led to their lysis as shown by the release of substances absorbing at 260 nm. In addition, scanning electron microscope observations revealed that the surface of the treated cells was significantly damaged. Conclusions:, Antifungal activity of thymol and eugenol involve alteration of both membrane and cell wall of the yeast. Significance and Impact of the Study:, This work is a preliminary contribution aiming to develop a new generation of efficient and natural antifungal agents. [source] Tumor necrosis factor-, and interferon-, directly impair epithelial barrier function in cultured mouse cholangiocytesLIVER INTERNATIONAL, Issue 1 2003Hanada Shinichiro Abstract:Background/Aims: In primary biliary cirrhosis (PBC), cytokines from CD4 + T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8 + T lymphocytes. Recently, we reported that immunolocalization of 7H6 , a tight junction (TJ)-associated protein , was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines , tumor necrosis factor-, (TNF-,), interferon-, (IFN-,), interleukin-2 and 4 (IL-2 and 4) , on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA)on cytokine-induced changes in paracellular permeability. Methods: Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and 3H-inulin flux. We also performed immunostaining and immunoblotting for TJ-associated proteins , claudin-1 and -3, occludin, zonula occluden-1 (ZO-1) and 7H6. Results: TNF-, and IFN-,, but neither IL-2 nor IL-4, significantly decreased TER (P < 0.005). 3H-inulin flux studies confirmed IFN-,-induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-,- or IFN-,-treated cells. Ursodeoxycholic acidhas been found to have no inhibitory effect on increased paracellular permeability induced by TNF-, or IFN-,. Conclusion: These findings show that TNF-, and IFN-, disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC. [source] Scanning electron microscopy investigations on bis(2-ethylhexyl)phthalate treated Mycobacterium cellsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2006B. Angelova Abstract Comparative investigation of steroid transforming activity and ultrastructural changes of bis(2-ethylhexyl)phthalate (BEHP, phthalate) treated Mycobacterium sp. NRRL B-3805 cells was carried out. Transformation of ,-sitosterol into androstenedione (AD) and androstadienedione (ADD) was performed in phthalate medium by resting cells preincubated in the organic solvent for a period from 3 to 24 h. It was observed that a preincubation greater than 12 h leads to the development of dense formations on the cells surface, reduction in the cell turgor, disruption in the cell walls, and formation of zones with reduced electron density. The preincubation for 24 h causes deeper changes in the cell ultrastructure but the treated cells retain their steroid transforming activity, allowing up to 80% of the substrate to be converted into AD and ADD. A preincubation of the resting Mycobacterium cells in BEHP for 6 h might be recommended as it leads to an achievement of stoichiometrical transformation of the substrate into AD and ADD and slightly higher initial rate of the reaction performed. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [source] Comparison of broadband UVB, narrowband UVB, broadband UVA and UVA1 on activation of apoptotic pathways in human peripheral blood mononuclear cellsPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 1 2007Chanisada Tuchinda Background/purpose: Ultraviolet (UV) radiation is an important therapy for immune-mediated cutaneous diseases. Activation of early apoptotic pathways may play a role in the clinical effectiveness. Different UV wavelengths have different efficacy for various diseases, but it remains unclear whether the ability to induce apoptosis differs with respect to the wavelength, and whether they induce apoptosis through the same mechanism. The aim of this study is to analyze the effects of different UV wavelengths that are used clinically on normal human peripheral blood mononuclear cells (PBMCs). Methods: PBMCs were treated with UV-light sources broadband UVB, narrowband UVB, broadband UVA and UVA1. Initiation of apoptosis was assessed by flow cytometry by staining,treated cells for activated caspases. Immunoblots were performed to measure for cleaved caspase-3, -8, -9, cytochrome c, Bcl 2-interacting domain and poly-(ADP ribose) polymerase cleavage. Results: We demonstrate that all the UV radiation sources induced caspase activation in a dose-and time-dependent manner. Components of both the extrinsic and intrinsic pathways of apoptosis were activated by all of the UV wavelengths tested, but differed in the level of energy needed for activation. Conclusion: The greater effectiveness of UVB on initiation of apoptotic pathway suggests that apoptosis may play a role in the clinical efficacy of UVB-responsive inflammatory cutaneous diseases. [source] The water extract of Omija protects H9c2 cardiomyoblast cells from hydrogen peroxide through prevention of mitochondrial dysfunction and activation of caspases pathwayPHYTOTHERAPY RESEARCH, Issue 1 2007Channy Park Abstract The water extract of Omija (Omija) has been used traditionally in the treatment of ischemic damage of the heart and brain tissues. However, little is known about the mechanism by which it rescues myocardial cells from oxidative stress. This study was designed to investigate the protective mechanisms of Omija on H2O2 -induced cytotoxicity in H9c2 cardiomyoblast cells. Treatment with H2O2 resulted in the death of H9c2 cells, characterized by apparent apoptotic features, including fragmentation of the nucleus and an increase in the sub-G0/G1 fraction of the cell cycle. However, Omija markedly suppressed the apoptotic characteristics of H9c2 cells induced by H2O2. In addition, Omija suppressed the features of mitochondrial dysfunction, including changes in the mitochondrial membrane potential and cytosolic release of cytochrome c in H2O2 -treated cells. Treatment with Omija further inhibited the catalytic activation of caspase-9 and caspase-3 and induction of Fas by H2O2. Taken together, these data indicate that the water extract of Omija protects H9c2 cardiomyoblast cells from oxidative stress of H2O2 through inhibition of mitochondrial dysfunction and activation of intrinsic caspase cascades, including caspase-3 and caspase-9. Copyright © 2006 John Wiley & Sons, Ltd. [source] Prohibitin regulates TGF-, induced apoptosis as a downstream effector of smad-dependent and -independent signalingTHE PROSTATE, Issue 1 2010Brian Zhu Abstract BACKGROUND Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-, (TGF-,) signaling in prostate cancer cells. This study investigated the involvement of PHB in the apoptosis and survival outcomes of human prostate cancer cell to TGF-,. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-, via Smad-dependent mechanism. METHOD TGF-, activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased cell survival. Calcein-based immunofluorescence studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as an integral component of TGF-, induced apoptosis in prostate cancer cells. RESULTS These finding indicates that induction of TGF-, apoptosis is mediated by Smad-dependent and Smad-independent signaling (MAPK) converging at PHB as a downstream effector regulating inner mitochondrial permeability. Putative PHB associated proteins were identified by subjecting TGF-, treated cells to immunoprecipitation with anti-PHB, and mass spectrometry. A screen for the kinase specific phosphorylation sites of PHB revealed three protein kinase (PKC) binding sites. CONCLUSION Our results demonstrate that TGF-, led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-,, was inhibited by the MEK1 inhibitor, documenting a critical interdependence between the MEK-ERK signaling and prohibitin phosphorylation. These findings suggest a dual role for PHB as a downstream determinant of the cellular response to TGF-, via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival). Prostate 70: 17,26, 2010. © 2009 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||