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Sample records for helahf cells independently

  1. PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway

    SciTech Connect

    Yu Dehua; Fan, Wufang; Liu, Guohong; Nguy, Vivian; Chatterton, Jon E.; Long Shilong; Ke, Ning; Meyhack, Bernd; Bruengger, Adrian; Brachat, Arndt; Wong-Staal, Flossie; Li, Qi-Xiang . E-mail: li@immusol.com

    2006-04-01

    HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.

  2. Focal Adhesion-Independent Cell Migration.

    PubMed

    Paluch, Ewa K; Aspalter, Irene M; Sixt, Michael

    2016-10-06

    Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion-independent migration, and highlight the remaining open questions for the future.

  3. GRID INDEPENDENT FUEL CELL OPERATED SMART HOME

    SciTech Connect

    Dr. Mohammad S. Alam

    2003-12-07

    A fuel cell power plant, which utilizes a smart energy management and control (SEMaC) system, supplying the power need of laboratory based ''home'' has been purchased and installed. The ''home'' consists of two rooms, each approximately 250 sq. ft. Every appliance and power outlet is under the control of a host computer, running the SEMaC software package. It is possible to override the computer, in the event that an appliance or power outage is required. Detailed analysis and simulation of the fuel cell operated smart home has been performed. Two journal papers has been accepted for publication and another journal paper is under review. Three theses have been completed and three additional theses are in progress.

  4. Independence.

    ERIC Educational Resources Information Center

    Stephenson, Margaret E.

    2000-01-01

    Discusses the four planes of development and the periods of creation and crystallization within each plane. Identifies the type of independence that should be achieved by the end of the first two planes of development. Maintains that it is through individual work on the environment that one achieves independence. (KB)

  5. Alternate Fuel Cell Membranes for Energy Independence

    SciTech Connect

    Storey, Robson, F.; Mauritz, Kenneth, A.; Patton, Derek, L.; Savin, Daniel, A.

    2012-12-18

    The overall objective of this project was the development and evaluation of novel hydrocarbon fuel cell (FC) membranes that possess high temperature performance and long term chemical/mechanical durability in proton exchange membrane (PEM) fuel cells (FC). The major research theme was synthesis of aromatic hydrocarbon polymers of the poly(arylene ether sulfone) (PAES) type containing sulfonic acid groups tethered to the backbone via perfluorinated alkylene linkages and in some cases also directly attached to the phenylene groups along the backbone. Other research themes were the use of nitrogen-based heterocyclics instead of acid groups for proton conduction, which provides high temperature, low relative humidity membranes with high mechanical/thermal/chemical stability and pendant moieties that exhibit high proton conductivities in the absence of water, and synthesis of block copolymers consisting of a proton conducting block coupled to poly(perfluorinated propylene oxide) (PFPO) blocks. Accomplishments of the project were as follows: 1) establishment of a vertically integrated program of synthesis, characterization, and evaluation of FC membranes, 2) establishment of benchmark membrane performance data based on Nafion for comparison to experimental membrane performance, 3) development of a new perfluoroalkyl sulfonate monomer, N,N-diisopropylethylammonium 2,2-bis(p-hydroxyphenyl) pentafluoropropanesulfonate (HPPS), 4) synthesis of random and block copolymer membranes from HPPS, 5) synthesis of block copolymer membranes containing high-acid-concentration hydrophilic blocks consisting of HPPS and 3,3'-disulfonate-4,4'-dichlorodiphenylsulfone (sDCDPS), 6) development of synthetic routes to aromatic polymer backbones containing pendent 1H-1,2,3-triazole moieties, 7) development of coupling strategies to create phase-separated block copolymers between hydrophilic sulfonated prepolymers and commodity polymers such as PFPO, 8) establishment of basic performance

  6. Ceramide mediates caspase-independent programmed cell death.

    PubMed

    Thon, Lutz; Möhlig, Heike; Mathieu, Sabine; Lange, Arne; Bulanova, Elena; Winoto-Morbach, Supandi; Schütze, Stefan; Bulfone-Paus, Silvia; Adam, Dieter

    2005-12-01

    Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.

  7. Hematopoietic stem cell-independent B-1a lineage.

    PubMed

    Ghosn, Eliver Eid Bou; Yang, Yang

    2015-12-01

    The accepted dogma has been that a single long-term hematopoietic stem cell (LT-HSC) can reconstitute all components of the immune system. However, our single-cell transfer studies have shown that highly purified LT-HSCs selectively fail to reconstitute B-1a cells in otherwise fully reconstituted hosts (i.e., LT-HSCs fully reconstitute follicular, marginal zone, and B-1b B cells, but not B-1a cells). These results suggest that B-1a cells are a separate B cell lineage that develops independently of classical LT-HSCs. We provide an evolutionary two-pathway development model (HSC independent versus HSC dependent), and suggest that this lineage separation is employed not only by B cells but by all hematopoietic lineages. Collectively, these findings challenge the current notion that LT-HSCs can reconstitute all components of the immune system and raise key questions about human HSC transplantation. We discuss the implications of these findings in light of our recent studies demonstrating the ability of B-1a cells to elicit antigen-specific responses that differ markedly from those mounted by follicular B cells. These findings have implications for vaccine development, in particular vaccines that may elicit the B-1a repertoire.

  8. Human skin cells support thymus-independent T cell development

    PubMed Central

    Clark, Rachael A.; Yamanaka, Kei-ichi; Bai, Mei; Dowgiert, Rebecca; Kupper, Thomas S.

    2005-01-01

    Thymic tissue has previously been considered a requirement for the generation of a functional and diverse population of human T cells. We report that fibroblasts and keratinocytes from human skin arrayed on a synthetic 3-dimensional matrix support the development of functional human T cells from hematopoietic precursor cells in the absence of thymic tissue. Newly generated T cells contained T cell receptor excision circles, possessed a diverse T cell repertoire, and were functionally mature and tolerant to self MHC, indicating successful completion of positive and negative selection. Skin cell cultures expressed the AIRE, Foxn1, and Hoxa3 transcription factors and a panel of autoantigens. Skin and bone marrow biopsies can thus be used to generate de novo functional and diverse T cell populations for potential therapeutic use in immunosuppressed patients. PMID:16224538

  9. Independent controls for neocortical neuron production and histogenetic cell death

    NASA Technical Reports Server (NTRS)

    Verney, C.; Takahashi, T.; Bhide, P. G.; Nowakowski, R. S.; Caviness, V. S. Jr

    2000-01-01

    We estimated the proportion of cells eliminated by histogenetic cell death during the first 2 postnatal weeks in areas 1, 3 and 40 of the mouse parietal neocortex. For each layer and for the subcortical white matter in each neocortical area, the number of dying cells per mm(2) was calculated and the proportionate cell death for each day of the 2-week interval was estimated. The data show that cell death proceeds essentially uniformly across the neocortical areas and layers and that it does not follow either the spatiotemporal gradient of cell cycle progression in the pseudostratified ventricular epithelium of the cerebral wall, the source of neocortical neurons, or the 'inside-out' neocortical neuronogenetic sequence. Therefore, we infer that the control mechanisms of neocortical histogenetic cell death are independent of mechanisms controlling neuronogenesis or neuronal migration but may be associated with the ingrowth, expansion and a system-wide matching of neuronal connectivity. Copyright 2000 S. Karger AG, Basel.

  10. Independent controls for neocortical neuron production and histogenetic cell death

    NASA Technical Reports Server (NTRS)

    Verney, C.; Takahashi, T.; Bhide, P. G.; Nowakowski, R. S.; Caviness, V. S. Jr

    2000-01-01

    We estimated the proportion of cells eliminated by histogenetic cell death during the first 2 postnatal weeks in areas 1, 3 and 40 of the mouse parietal neocortex. For each layer and for the subcortical white matter in each neocortical area, the number of dying cells per mm(2) was calculated and the proportionate cell death for each day of the 2-week interval was estimated. The data show that cell death proceeds essentially uniformly across the neocortical areas and layers and that it does not follow either the spatiotemporal gradient of cell cycle progression in the pseudostratified ventricular epithelium of the cerebral wall, the source of neocortical neurons, or the 'inside-out' neocortical neuronogenetic sequence. Therefore, we infer that the control mechanisms of neocortical histogenetic cell death are independent of mechanisms controlling neuronogenesis or neuronal migration but may be associated with the ingrowth, expansion and a system-wide matching of neuronal connectivity. Copyright 2000 S. Karger AG, Basel.

  11. Na+-independent phosphate transport in Caco2BBE cells

    PubMed Central

    Candeal, Eduardo; Caldas, Yupanqui A.; Guillén, Natalia; Levi, Moshe

    2014-01-01

    Pi transport in epithelia has both Na+-dependent and Na+-independent components, but so far only Na+-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na+-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na+-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO42−, HCO3−, and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved. PMID:25298422

  12. Na+-independent phosphate transport in Caco2BBE cells.

    PubMed

    Candeal, Eduardo; Caldas, Yupanqui A; Guillén, Natalia; Levi, Moshe; Sorribas, Víctor

    2014-12-15

    Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved. Copyright © 2014 the American Physiological Society.

  13. Antizyme (AZ) regulates intestinal cell growth independently of polyamines

    PubMed Central

    Ray, Ramesh M.; Bhattacharya, Sujoy; Bavaria, Mitul N.; Viar, Mary Jane; Johnson, Leonard R.

    2014-01-01

    Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5μM spermidine (SPD), DFMO+ 5μM spermine (SPM), or DFMO+ 10 μM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50%. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines. PMID:24930035

  14. Scaffold-independent Patterning of Cells using Magnetic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Ghosh, Suvojit; Biswas, Moanaro; Elankumaran, Subbiah; Puri, Ishwar

    2013-03-01

    Spatial patterning of cells in vitro relies on direct contact of cells on to solid surfaces. Scaffold independent patterning of cells has never been achieved so far. Patterning of cells has wide applications including stem cell biology, tissue architecture and regenerative medicine besides fundamental biology. Magnetized cells in a suspension can be manipulated using an externally applied magnetic field enabling directed patterning. We magnetized mammalian cells by internalization of superparamagnetic nanoparticles coated with bovine serum albumin (BSA). A magnetic field is then used to arrange cells in a desired pattern on a substrate or in suspension. The control strategy is derived from the self-assembly of magnetic colloids in a liquid considering magnetostatic interactions. The range of achievable structural features promise novel experimental methods investigating the influence of tissue shape and size on cell population dynamics wherein Fickian diffusion of autocrine growth signals are known to play a significant role. By eliminating the need for a scaffold, intercellular adhesion mechanics and the effects of temporally regulated signals can be investigated. The findings can be applied to novel tissue engineering methods.

  15. ADAM12 induces estrogen-independence in breast cancer cells.

    PubMed

    Roy, Roopali; Moses, Marsha A

    2012-02-01

    Antiestrogen therapy has been used successfully to prolong disease-free and overall survival of ER positive breast cancer patients. However, 50% of patients with ER+ tumors fail to respond to such therapy or eventually acquire resistance to endocrine therapy, resulting in tumor progression and mortality. It is imperative, therefore, to understand the mechanisms that lead to hormone refractory breast cancer in order to develop therapeutics that can modulate the resistance to antiestrogen therapy. The protease, ADAM12, can be detected in the urine of breast cancer patients and its levels correlate with disease status, stage, and cancer risk. Within the context of this study, the authors have investigated the role of the two distinct isoforms of ADAM12 in breast tumor cell proliferation and as potential mediators of endocrine resistance. Using stable clones of ADAM12-overexpressing MCF-7 cells, the authors analyzed proliferation rates of these ER+ breast tumor cells both in estrogen-depleted medium and in the presence of the antiestrogens, tamoxifen, and ICI 182,780. Acquired estrogen resistance in these cells was analyzed using phospho-RTK analysis. Upregulation and phosphorylation of proteins were detected via immunoprecipitation and immunoblotting. EGFR and MAPK inhibitors were used to explore the mechanism of acquired estrogen resistance in breast tumor cells. It was observed that overexpression of the two isoforms, transmembrane ADAM12-L, and secreted ADAM12-S, in breast tumor cells promoted estrogen-independent proliferation. In ADAM12-L-expressing cells, estrogen-independence was a direct result of increased EGFR expression and MAPK activation, whereas, the mechanism in ADAM12-S-expressing cells may be enhanced IGF-1R signaling. The importance of the EGFR signaling pathway in the estrogen-independent growth of ADAM12-L expressing cells was highlighted by the effect of EGFR inhibitors AG1478 and PD15035 or MAPK inhibitor U0126, each of which abolished the

  16. Embryonic stem cells as sources of donor-independent platelets.

    PubMed

    Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

    2015-06-01

    The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications.

  17. Embryonic stem cells as sources of donor-independent platelets

    PubMed Central

    Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.

    2015-01-01

    The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications. PMID:25961451

  18. Marine brevetoxin induces IgE-independent mast cell activation.

    PubMed

    Hilderbrand, Susana C; Murrell, Rachel N; Gibson, James E; Brown, Jared M

    2011-02-01

    Brevetoxins (PbTx) are sodium channel neurotoxins produced by the marine dinoflagellate Karenia brevis during red tide blooms. Inhalation of PbTx in normal individuals and individuals with pre-existing airways disease results in adverse airway symptoms including bronchoconstriction. In animal models of allergic inflammation, inhalation of PbTx results in a histamine H₁-mediated bronchoconstriction suggestive of mast cell activation. How mast cells would respond directly to PbTx is unknown. We thus explored the activation of mouse bone marrow-derived mast cells (BMMCs) following exposure to purified PbTx-2. Following in vitro exposure to PbTx-2, we examined cellular viability, mast cell degranulation (β-hexosaminidase release), intracellular Ca²+ and Na+ flux, and the production of inflammatory mediators (IL-6). PbTx-2 induced significant cellular toxicity within 24 h as measured by LDH release and Annexin-V staining. However, within 1 h of exposure, PbTx-2 induced BMMC degranulation and an increase in IL-6 mRNA expression independent of the high-affinity IgE receptor (FcεRI) stimulation. Activation of BMMCs by PbTx-2 was associated with altered intracellular Ca²+ and Na+ levels. Brevenal, a naturally produced compound that antagonizes the activity of PbTx, prevented changes in intracellular Na+ levels but did not alter activation of BMMCs by PbTx-2. These findings demonstrate that PbTx-2 activates mast cells independent of FcεRI providing insight into critical events in the pathogenesis and a potential therapeutic target in brevetoxin-induced airway symptoms.

  19. Independent rate and temporal coding in hippocampal pyramidal cells

    PubMed Central

    Huxter, John; Burgess, Neil; O’Keefe, John

    2009-01-01

    Hippocampal pyramidal cells use temporal 1 as well as rate coding 2 to signal spatial aspects of the animal’s environment or behaviour. The temporal code takes the form of a phase relationship to the concurrent cycle of the hippocampal EEG theta rhythm (Figure 1​; 1). These two codes could each represent a different variable 3,4. However, this requires that rate and phase can vary independently, in contrast to recent suggestions 5,6 that they are tightly coupled: both reflecting the amplitude of the cell’s input. Here we show that the time of firing and firing rate are dissociable and can represent two independent variables, viz, the animal’s location within the place field and its speed of movement through the field, respectively. Independent encoding of location together with actions and stimuli occurring there may help to explain the dual roles of the hippocampus in spatial and episodic memory 7 8 or a more general role in relational/declarative memory9,10. PMID:14574410

  20. Cell volume and membrane stretch independently control K+ channel activity.

    PubMed

    Hammami, Sofia; Willumsen, Niels J; Olsen, Hervør L; Morera, Francisco J; Latorre, Ramón; Klaerke, Dan A

    2009-05-15

    A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of -5.0 +/- 0.1 mmHg the increase amounted to 381 +/- 146% (mean +/- S.E.M., n = 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.

  1. Cell volume and membrane stretch independently control K+ channel activity

    PubMed Central

    Hammami, Sofia; Willumsen, Niels J; Olsen, Hervør L; Morera, Francisco J; Latorre, Ramón; Klaerke, Dan A

    2009-01-01

    A number of potassium channels including members of the KCNQ family and the Ca2+ activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (∼50 μm2 macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of −5.0 ± 0.1 mmHg the increase amounted to 381 ± 146% (mean ±s.e.m., n= 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms. PMID:19289549

  2. Human mast cells costimulate T cells through a CD28-independent interaction.

    PubMed

    Suurmond, Jolien; Dorjée, Annemarie L; Huizinga, Tom W J; Toes, René E M

    2016-05-01

    Mast cells are innate immune cells usually residing in peripheral tissues, where they are likely to activate T-cell responses. Similar to other myeloid immune cells, mast cells can function as antigen-presenting cells. However, little is known about the capacity of human mast cells to costimulate CD4(+) T cells. Here, we studied the T-cell stimulatory potential of human mast cells. Peripheral blood derived mast cells were generated and cocultured with isolated CD4(+) T cells. In the presence of T-cell receptor triggering using anti-CD3, mast cells promoted strong proliferation of T cells, which was two- to fivefold stronger than the "T-cell promoting capacity" of monocytes. The interplay between mast cells and T cells was dependent on cell-cell contact, suggesting that costimulatory molecules on the mast cell surface are responsible for the effect. However, in contrast to monocytes, the T-cell costimulation by mast cells was independent of the classical costimulatory molecule CD28, or that of OX40L, ICOSL, or LIGHT. Our data show that mast cells can costimulate human CD4(+) T cells to induce strong T-cell proliferation, but that therapies aiming at disrupting the interaction of CD28 and B7 molecules do not inhibit mast cell mediated T-cell activation.

  3. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    DOE PAGES

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; ...

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest suchmore » atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.« less

  4. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    PubMed Central

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-01

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly. PMID:26772746

  5. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    SciTech Connect

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.

  6. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

    PubMed Central

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A; Costa, Max

    2016-01-01

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study , we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA – mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. PMID:26780400

  7. Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch.

    PubMed

    Seidel, Hannah S; Kimble, Judith

    2015-11-09

    Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for facultative stem cell quiescence. We find that mitotically dividing germ cells--including germline stem cells--become quiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a block to M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, we demonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance: The signaling pathway required for stem cell maintenance under fed conditions--GLP-1/Notch signaling--becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence can itself maintain stem cells, independent of the signaling pathway otherwise essential for such maintenance.

  8. TNFα protects cardiac mitochondria independently of its cell surface receptors.

    PubMed

    Lacerda, Lydia; McCarthy, Joy; Mungly, Shazia F K; Lynn, Edward G; Sack, Michael N; Opie, Lionel H; Lecour, Sandrine

    2010-11-01

    Our novel proposal is that TNFα exerts a direct effect on mitochondrial respiratory function in the heart, independently of its cell surface receptors. TNFα-induced cardioprotection is known to involve reactive oxygen species (ROS) and sphingolipids. We therefore further propose that this direct mitochondrial effect is mediated via ROS and sphingolipids. The protective concentration of TNFα (0.5 ng/ml) was added to isolated heart mitochondria from black 6 × 129 mice (WT) and double TNF receptor knockout mice (TNFR1&2(-/-)). Respiratory parameters and inner mitochondrial membrane potential were analyzed in the presence/absence of two antioxidants, N-acetyl-L: -cysteine or N-tert-butyl-α-(2-sulfophenyl)nitrone or two antagonists of the sphingolipid pathway, N-oleoylethanolamine (NOE) or imipramine. In WT, TNFα reduced State 3 respiration from 279.3 ± 3 to 119.3 ± 2 (nmol O₂/mg protein/min), increased proton leak from 15.7 ± 0.6% (control) to 36.6 ± 4.4%, and decreased membrane potential by 20.5 ± 3.1% compared to control groups. In TNFR1&2(-/-) mice, TNFα reduced State 3 respiration from 205.2 ± 4 to 75.7 ± 1 (p < 0.05 vs. respective control). In WT mice, both antioxidants added with TNFα restored State 3 respiration to 269.2 ± 2 and 257.6 ± 2, respectively. Imipramine and NOE also restored State 3 respiration to 248.4 ± 2 and 249.0 ± 2, respectively (p < 0.01 vs. TNFα alone). Similarly, both antioxidant and inhibitors of the sphingolipid pathway restored the proton leak to pre-TNF values. TNFα-treated mitochondria or isolated cardiac muscle fibers showed an increase in respiration after anoxia-reoxygenation, but this effect was lost in the presence of an antioxidant or NOE. Similar data were obtained in TNFR1&2(-/-) mice. TNFα exerts a protective effect on respiratory function in isolated mitochondria subjected to an anoxia-reoxygenation insult. This effect appears to be independent of its cell surface receptors, but is likely to be mediated

  9. Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells.

    PubMed

    Okamoto, Mayumi; Miyata, Takaki; Konno, Daijiro; Ueda, Hiroki R; Kasukawa, Takeya; Hashimoto, Mitsuhiro; Matsuzaki, Fumio; Kawaguchi, Ayano

    2016-04-20

    During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

  10. Proepithelin Regulates Prostate Cancer Cell Biology by Promoting Cell Growth, Migration, and Anchorage-Independent Growth

    PubMed Central

    Monami, Giada; Emiliozzi, Velia; Bitto, Alessandro; Lovat, Francesca; Xu, Shi-Qiong; Goldoni, Silvia; Fassan, Matteo; Serrero, Ginette; Gomella, Leonard G.; Baffa, Raffaele; Iozzo, Renato V.; Morrione, Andrea

    2009-01-01

    The growth factor proepithelin has recently emerged as an important regulator of transformation in several physiological and pathological systems. In this study, we determined the biological roles of proepithelin in prostate cancer cells using purified human recombinant proepithelin as well as proepithelin-depletion strategies. Proepithelin promoted the migration of androgen-dependent and -independent human prostate cancer cells; androgen-independent DU145 cells were the more responsive. In these cells, proepithelin additionally stimulated wound closure, invasion, and promotion of cell growth in vitro. These effects required the activation of both the Akt and mitogen-activated protein kinase pathways. We have analyzed proepithelin expression levels in different available prostate cancer microarray studies using the Oncomine database and found a statistically significant increase in proepithelin mRNA expression levels in prostate cancers compared with nonneoplastic controls. Notably, depletion of endogenous proepithelin by siRNA and antisense strategies impaired the ability of DU145 cells to grow and migrate after serum withdrawal and inhibited anchorage-independent growth. Our results provide the first evidence for a role of proepithelin in stimulating the migration, invasion, proliferation, and anchorage-independent growth of prostate cancer cells. This study supports the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and initial progression of prostate cancer. Furthermore, proepithelin may prove to be a useful clinical marker for the diagnosis of prostate tumors. PMID:19179604

  11. Antibody-Independent Function of Human B Cells Contributes to Antifungal T Cell Responses.

    PubMed

    Li, Rui; Rezk, Ayman; Li, Hulun; Gommerman, Jennifer L; Prat, Alexandre; Bar-Or, Amit

    2017-04-15

    Fungal infections (e.g., Candida albicans) can manifest as serious medical illnesses, especially in the elderly and immune-compromised hosts. T cells are important for Candida control. Whether and how B cells are involved in antifungal immunity has been less clear. Although patients with agammaglobulinemia exhibit normal antifungal immunity, increased fungal infections are reported following B cell-depleting therapy, together pointing to Ab-independent roles of B cells in controlling such infections. To test how human B cells may contribute to fungal-associated human T cell responses, we developed a novel Ag-specific human T cell/B cell in vitro coculture system and found that human B cells could induce C. albicans-associated, MHC class II-restricted responses of naive T cells. Activated B cells significantly enhanced C. albicans-mediated Th1 and Th17 T cell responses, which were both strongly induced by CD80/CD86 costimulation. IL-6(+)GM-CSF(+) B cells were the major responding B cell subpopulation to C. albicans and provided efficient costimulatory signals to the T cells. In vivo B cell depletion in humans resulted in reduced C. albicans-associated T responses. Of note, the decreased Th17, but not Th1, responses could be reversed by soluble factors from B cells prior to depletion, in an IL-6-dependent manner. Taken together, our results implicate an Ab-independent cytokine-defined B cell role in human antifungal T cell responses. These findings may be particularly relevant given the prospects of chronic B cell depletion therapy use in lymphoma and autoimmune disease, as patients age and are exposed to serial combination therapies.

  12. Cannabinoid-receptor-independent cell signalling by N-acylethanolamines.

    PubMed Central

    Berdyshev, E V; Schmid, P C; Krebsbach, R J; Hillard, C J; Huang, C; Chen, N; Dong, Z; Schmid, H H

    2001-01-01

    Anandamide and other polyunsaturated N-acylethanolamines (NAEs) exert biological activity by binding to cannabinoid receptors. These receptors are linked to G(i/o) proteins and their activation leads to extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAP kinase) activation, inhibition of cAMP-dependent signalling and complex changes in the expression of various genes. Saturated and monounsaturated NAEs cannot bind to cannabinoid receptors and may thus mediate cell signalling through other targets. Here we report that both saturated/monounsaturated NAEs and anandamide (20:4(n-6) NAE) stimulate cannabinoid-receptor-independent ERK phosphorylation and activator protein-1 (AP-1)-dependent transcriptional activity in mouse epidermal JB6 cells. Using a clone of JB6 P(+) cells with an AP-1 collagen-luciferase reporter construct, we found that 16:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n-6) NAEs stimulated AP-1-dependent transcriptional activity up to 2-fold, with maximal stimulation at approx. 10-15 microM. Higher NAE concentrations had toxic effects mediated by alterations in mitochondrial energy metabolism. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 signalling pathways, because all NAEs stimulated ERK1/ERK2 phosphorylation without having any effect on JNK or p38 kinases. Also, overexpression of dominant negative ERK1/ERK2 kinases completely abolished NAE-induced AP-1 activation. In contrast with 18:1(n-9) NAE and anandamide, the cannabinoid receptor agonist WIN 55,212-2 did not stimulate AP-1 activity and inhibited ERK phosphorylation. The NAE-mediated effects were not attenuated by pertussis toxin and appeared to be NAE-specific, as a close structural analogue, oleyl alcohol, failed to induce ERK phosphorylation. The data support our hypothesis that the major saturated and monounsaturated NAEs are signalling molecules acting through intracellular targets without

  13. TARGETING THE MITOCHONDRIA ACTIVATES TWO INDEPENDENT CELL DEATH PATHWAYS IN THE OVARIAN CANCER STEM CELLS

    PubMed Central

    Alvero, Ayesha B.; Montagna, Michele K.; Holmberg, Jennie C.; Craveiro, Vinicius; Brown, David; Mor, Gil

    2013-01-01

    Cancer stem cells are responsible for tumor initiation and chemo-resistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells (OCSCs) are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the OCSCs are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of OCSCs. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of “cellular starvation” that activates two independent pathways: 1) AMPKα1 pathway leading to mTOR inhibition; and 2) mitochondrial MEK/ERK pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemo-resistant cell population opens a new venue for treating ovarian cancer patients. PMID:21677151

  14. Interleukin 7 independent development of human B cells.

    PubMed Central

    Prieyl, J A; LeBien, T W

    1996-01-01

    Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis. PMID:8816803

  15. Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

    PubMed

    Proff, Julia; Walterskirchen, Christian; Brey, Charlotte; Geyeregger, Rene; Full, Florian; Ensser, Armin; Lehner, Manfred; Holter, Wolfgang

    2016-01-01

    In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins.

  16. Serum stimulation, cell-cell interactions, and extracellular matrix independently influence smooth muscle cell phenotype in vitro.

    PubMed Central

    Kato, S.; Shanley, J. R.; Fox, J. C.

    1996-01-01

    Vascular injury profoundly alters the vessel wall microenvironment, and smooth muscle cells respond with cell cycle re-entry, loss of contractile elements, extracellular matrix remodeling, and altered signaling by endogenous growth factors and their receptors. Environmental cues include stimulation by exogenous mitogens and both cell-cell and cell-matrix interactions. Modeling this process in smooth muscle cells in vitro, these environmental determinants were varied independently and the phenotypic consequences assessed. Mitogenic stimulation with serum promoted the synthesis of collagen and fibronectin and the expression of fibroblast growth factor receptor-1 and suppressed the content of smooth muscle alpha-actin, myosin heavy chain, and basic fibroblast growth factor. Low cell density (reduced cell-cell contact) was also associated with enhanced extracellular matrix protein production, increased fibroblast growth factor receptor-1 expression, and reduced contractile protein and basic fibroblast growth factor content. The influence of serum stimulation and reduced cell-cell contact were independent and additive. Provision of a type I collagen matrix blunted the influence of serum and cell-cell contact on collagen synthesis but had minor effects on other measures of phenotype. Environmental factors thus independently influence smooth muscle cell phenotype, including endogenous growth factor expression and responsiveness, which can in turn influence the microenvironment of the vessel wall after injury. Images Figure 1 Figure 5 Figure 6 PMID:8702006

  17. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    PubMed

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Cap-independent translation by DAP5 controls cell fate decisions in human embryonic stem cells

    PubMed Central

    Yoffe, Yael; David, Maya; Kalaora, Rinat; Povodovski, Lital; Friedlander, Gilgi; Feldmesser, Ester; Ainbinder, Elena; Saada, Ann; Bialik, Shani; Kimchi, Adi

    2016-01-01

    Multiple transcriptional and epigenetic changes drive differentiation of embryonic stem cells (ESCs). This study unveils an additional level of gene expression regulation involving noncanonical, cap-independent translation of a select group of mRNAs. This is driven by death-associated protein 5 (DAP5/eIF4G2/NAT1), a translation initiation factor mediating IRES-dependent translation. We found that the DAP5 knockdown from human ESCs (hESCs) resulted in persistence of pluripotent gene expression, delayed induction of differentiation-associated genes in different cell lineages, and defective embryoid body formation. The latter involved improper cellular organization, lack of cavitation, and enhanced mislocalized apoptosis. RNA sequencing of polysome-associated mRNAs identified candidates with reduced translation efficiency in DAP5-depleted hESCs. These were enriched in mitochondrial proteins involved in oxidative respiration, a pathway essential for differentiation, the significance of which was confirmed by the aberrant mitochondrial morphology and decreased oxidative respiratory activity in DAP5 knockdown cells. Further analysis identified the chromatin modifier HMGN3 as a cap-independent DAP5 translation target whose knockdown resulted in defective differentiation. Thus, DAP5-mediated translation of a specific set of proteins is critical for the transition from pluripotency to differentiation, highlighting the importance of cap-independent translation in stem cell fate decisions. PMID:27664238

  19. Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells

    PubMed Central

    Okamoto, Mayumi; Miyata, Takaki; Konno, Daijiro; Ueda, Hiroki R.; Kasukawa, Takeya; Hashimoto, Mitsuhiro; Matsuzaki, Fumio; Kawaguchi, Ayano

    2016-01-01

    During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode. PMID:27094546

  20. Cytokine dependent and independent iNKT cell activation

    PubMed Central

    Reilly, Emma C.; Wands, Jack R.; Brossay, Laurent

    2010-01-01

    Invariant NKT (iNKT) cells have been extensively studied throughout the last decade due to their ability to polarize and amplify the downstream immune response. Only recently however, have the various mechanisms underlying NKT cell activation begun to unfold. iNKT cells have the ability to respond as innate immune cells with minimal TCR involvement as well as through direct TCR recognition of glycolipid antigens. Additionally, the existence of several subsets of iNKT cells creates the potential for other unique pathways, which are not yet clearly defined. Here we provide an overview of the known mechanisms of invariant NKT cell activation, focusing on cytokine driven pathways and the resulting cytokine responses. PMID:20554220

  1. Ceramide triggers metacaspase-independent mitochondrial cell death in yeast.

    PubMed

    Carmona-Gutierrez, Didac; Reisenbichler, Angela; Heimbucher, Petra; Bauer, Maria A; Braun, Ralf J; Ruckenstuhl, Christoph; Büttner, Sabrina; Eisenberg, Tobias; Rockenfeller, Patrick; Fröhlich, Kai-Uwe; Kroemer, Guido; Madeo, Frank

    2011-11-15

    The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.

  2. Dendritic planarity of Purkinje cells is independent of Reelin signaling.

    PubMed

    Kim, Jinkyung; Park, Tae-Ju; Kwon, Namseop; Lee, Dongmyeong; Kim, Seunghwan; Kohmura, Yoshiki; Ishikawa, Tetsuya; Kim, Kyong-Tai; Curran, Tom; Je, Jung Ho

    2015-07-01

    The dendritic planarity of Purkinje cells is critical for cerebellar circuit formation. In the absence of Crk and CrkL, the Reelin pathway does not function resulting in partial Purkinje cell migration and defective dendritogenesis. However, the relationships among Purkinje cell migration, dendritic development and Reelin signaling have not been clearly delineated. Here, we use synchrotron X-ray microscopy to obtain 3-D images of Golgi-stained Purkinje cell dendrites. Purkinje cells that failed to migrate completely exhibited conical dendrites with abnormal 3-D arborization and reduced dendritic complexity. Furthermore, their spines were fewer in number with a distorted morphology. In contrast, Purkinje cells that migrated successfully displayed planar dendritic and spine morphologies similar to normal cells, despite reduced dendritic complexity. These results indicate that, during cerebellar formation, Purkinje cells migrate into an environment that supports development of dendritic planarity and spine formation. While Reelin signaling is important for the migration process, it does not make a direct major contribution to dendrite formation.

  3. Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch

    PubMed Central

    Seidel, Hannah S; Kimble, Judith

    2015-01-01

    Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for facultative stem cell quiescence. We find that mitotically dividing germ cells—including germline stem cells—become quiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a block to M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, we demonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance: The signaling pathway required for stem cell maintenance under fed conditions—GLP-1/Notch signaling—becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence can itself maintain stem cells, independent of the signaling pathway otherwise essential for such maintenance. DOI: http://dx.doi.org/10.7554/eLife.10832.001 PMID:26551561

  4. Cytolethal distending toxin induces caspase-dependent and -independent cell death in MOLT-4 cells.

    PubMed

    Ohara, Masaru; Hayashi, Tomonori; Kusunoki, Yoichiro; Nakachi, Kei; Fujiwara, Tamaki; Komatsuzawa, Hitoshi; Sugai, Motoyuki

    2008-10-01

    Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.

  5. Apoptosis-Dependent and Apoptosis-Independent Functions of Bim in Prostate Cancer Cells

    DTIC Science & Technology

    2005-03-01

    Independent Functions of Bim in Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Dr. Dean Tang...SUBTITLE 5a. CONTRACT NUMBER Apoptosis-Dependent and Apoptosis-Independent Functions of Bim in Prostate Cancer Cells 5b. GRANT NUMBER DAMD17-03-1...Unlimited 13. SUPPLEMENTARY NOTES Original contains colored plates: ALL DTIC reproductions will be in black and white. 14. ABSTRACT

  6. Human mesenchymal stromal cells suppress T-cell proliferation independent of heme oxygenase-1.

    PubMed

    Patel, Seema R; Copland, Ian B; Garcia, Marco A; Metz, Richard; Galipeau, Jacques

    2015-04-01

    Mesenchymal stromal cells deploy immune suppressive properties amenable for use as cell therapy for inflammatory disorders. It is now recognized that mesenchymal stromal cells necessitate priming with an inflammatory milieu, in particular interferon-γ, to exert augmented immunosuppressive effects. It has been recently suggested that the heme-catabolizing enzyme heme oxygenase-1 is an essential component of the mesenchymal stromal cell-driven immune suppressive response. Because mesenchymal stromal cells upregulate indoleamine 2,3-dioxygenase expression on interferon-γ priming and indoleamine 2,3-dioxygenase requires heme as a cofactor for optimal catabolic function, we investigated the potential antagonism of heme oxygenase-1 activity on indoleamine 2, 3-dioxygenase and the impact on mesenchymal stromal cell immune plasticity. We herein sought to evaluate the molecular genetic effect of cytokine priming on human mesenchymal stromal cell heme oxygenase-1 expression and its functional role in differentially primed mesenchymal stromal cells. Contrary to previous reports, messenger RNA and protein analyses demonstrated that mesenchymal stromal cells derived from normal subjects (n = 6) do not express heme oxygenase-1 at steady state or after interferon-γ, tumor necrosis factor-α, and/or transforming growth factor-β priming. Pharmacological inhibition of heme oxygenase-1 with the use of tin protoporphyrin did not significantly abrogate the ability of mesenchymal stromal cells to suppress T-cell proliferation in vitro. Overall, these results unequivocally demonstrate that under steady state and after cytokine priming, human mesenchymal stromal cells immunoregulate T-cell proliferation independent of heme oxygenase-1.

  7. The antimicrobial peptide cecropin A induces caspase-independent cell death in human promyelocytic leukemia cells.

    PubMed

    Cerón, José María; Contreras-Moreno, Judit; Puertollano, Elena; de Cienfuegos, Gerardo Álvarez; Puertollano, María A; de Pablo, Manuel A

    2010-08-01

    Most antimicrobial peptides have been shown to have antitumoral activity. Cecropin A, a linear 37-residue antimicrobial polypeptide produced by the cecropia moth, has exhibited cytotoxicity in various human cancer cell lines and inhibitory effects on tumor growth. In this study, we investigated the apoptosis induced by cecropin A in the promyelocytic cell line HL-60. Treatment of cells with cecropin A was characterized by loss of viability in a dose-dependent manner, lactate dehydrogenase (LDH) leakage, and modest attenuation of lysosomal integrity measured by neutral red assay. An increase of reactive oxygen species (ROS) generation, DNA fragmentation, and phosphatidylserine externalization were quantified following cecropin A exposure at a concentration of 30 microM, whereas cecropin A-induced apoptosis was independent of caspase family members, because the activity of caspase-8 and -9 were irrelevant. Nevertheless, caspase-3 activity showed a significant increase at concentrations of 20-40 microM, but a considerable reduction at 50 microM. Flow cytometry analysis revealed a dissipation of the mitochondrial transmembrane potential (Deltapsi(m)), and the accumulation of cells at sub-G1 phase measured by FACS analysis of propidium iodide (PI) stained nuclei suggested induction of apoptosis. Morphological changes measured by Hoechst 33342 or acridine orange/ethidium bromide staining showed nuclear condensation, corroborating the apoptotic action of cecropin A. Overall, these data indicate that cecropin A is able to induce apoptosis in HL-60 cells through a signaling mechanism mediated by ROS, but independently of caspase activation.

  8. Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

    PubMed Central

    Hau, Andrew M.; Greenwood, Jeffrey A.; Löhr, Christiane V.; Serrill, Jeffrey D.; Proteau, Philip J.; Ganley, Ian G.; McPhail, Kerry L.; Ishmael, Jane E.

    2013-01-01

    Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and “COMPARE-negative” profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death

  9. Regulatory T-cell vaccination independent of auto-antigen.

    PubMed

    Pascual, David W; Yang, Xinghong; Holderness, Kathryn; Jun, SangMu; Maddaloni, Massimo; Kochetkova, Irina

    2014-03-14

    To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-β and interleukin-10.

  10. Fitness of cell-mediated immunity independent of repertoire diversity.

    PubMed

    AbuAttieh, Mouhammed; Rebrovich, Michelle; Wettstein, Peter J; Vuk-Pavlovic, Zvezdana; Limper, Andrew H; Platt, Jeffrey L; Cascalho, Marilia

    2007-03-01

    Fitness of cell-mediated immunity is thought to depend on TCR diversity; however, this concept has not been tested formally. We tested the concept using JH(-/-) mice that lack B cells and have TCR Vbeta diversity <1% that of wild-type mice and quasimonoclonal (QM) mice with oligoclonal B cells and TCR Vbeta diversity 7% that of wild-type mice. Despite having a TCR repertoire contracted >99% and defective lymphoid organogenesis, JH(-/-) mice rejected H-Y-incompatible skin grafts as rapidly as wild-type mice. JH(-/-) mice exhibited T cell priming by peptide and delayed-type hypersensitivity, although these responses were less than normal owing either to TCR repertoire contraction or defective lymphoid organogenesis. QM mice with TCR diversity contracted >90%, and normal lymphoid organs rejected H-Y incompatible skin grafts as rapidly as wild type mice and exhibited normal T cell priming and normal delayed-type hypersensitivity reactions. QM mice also resisted Pneumocystis murina like wild-type mice. Thus, cell-mediated immunity can function normally despite contractions of TCR diversity >90% and possibly >99%.

  11. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    PubMed Central

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-01

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

  12. Hodgkin's disease cell lines: a model for interleukin-1-independent accessory cell function.

    PubMed

    McKenzie, J L; Egner, W; Calder, V L; Hart, D N

    1992-11-01

    The haemopoietic origins of the Hodgkin's disease (HD)-derived cell lines L428, KM-H2 and HDLM-2 remain controversial. Analysis of T-cell receptor (TcR) and Ig rearrangements cannot resolve this, and lineage promiscuity limits the interpretation of isolated surface antigen expression. Nonetheless the cell marker profile of L428 has similarities with human dendritic cells (DC), and L428 strongly stimulates in the mixed leucocyte reaction (MLR). We therefore undertook an extended immunophenotypic comparison of the HD lines with that recently defined for DC, prior to examining their ability to stimulate allogenic T lymphocytes, and comparing the molecular interactions involved with those of primary MLR stimulatory cells. The immunophenotype of the HD lines failed to establish either a lymphoid or monocytoid derivation. The profile of L428 appeared similar to the human DC. All three lines were potent stimulators in the primary MLR, and each expressed relevant adhesion and signal-transducing molecules important for co-stimulating T lymphocytes. Inhibition studies using monoclonal antibodies indicated similar contributions within HD line-T cell MLR to that documented in human tonsil DC-T cell MLR. The HD lines produced no detectable interleukin-1 (IL-1) by biological or immunological analysis. Moreover they stimulated allogeneic T lymphocytes in the presence of anti-IL-1 antibodies. Thus although IL-1 mRNA can be detected in both HDLM-2 and KM-H2 by polymerase chain reaction, these lines, and L428, share with DC the ability to stimulate allogeneic T lymphocytes in an IL-1-independent manner [corrected]. HD lines, particularly L428, may provide a standardized, reproducible, IL-1-independent model for dissection of the co-stimulatory requirements of the human primary MLR.

  13. Macrophages sustain HIV replication in vivo independently of T cells.

    PubMed

    Honeycutt, Jenna B; Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Madden, Victoria; Sharpe, Garrett; Haase, Ashley T; Eron, Joseph J; Garcia, J Victor

    2016-04-01

    Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

  14. Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction.

    PubMed

    Tezuka, Hiroyuki; Abe, Yukiko; Asano, Jumpei; Sato, Taku; Liu, Jiajia; Iwata, Makoto; Ohteki, Toshiaki

    2011-02-25

    Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in T cell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders.

  15. Identification of cell surface molecules involved in dystroglycan-independent Lassa virus cell entry.

    PubMed

    Shimojima, Masayuki; Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz; Kawaoka, Yoshihiro

    2012-02-01

    Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.

  16. Macrophages sustain HIV replication in vivo independently of T cells

    PubMed Central

    Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Sharpe, Garrett; Haase, Ashley T.; Eron, Joseph J.; Garcia, J. Victor

    2016-01-01

    Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell–only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection. PMID:26950420

  17. Size-independent symmetric division in extraordinarily long cells

    PubMed Central

    Pende, Nika; Leisch, Nikolaus; Gruber-Vodicka, Harald R.; Heindl, Niels R.; Ott, Jörg; den Blaauwen, Tanneke; Bulgheresi, Silvia

    2014-01-01

    Two long-standing paradigms in biology are that cells belonging to the same population exhibit little deviation from their average size and that symmetric cell division is size limited. Here, ultrastructural, morphometric and immunocytochemical analyses reveal that two Gammaproteobacteria attached to the cuticle of the marine nematodes Eubostrichus fertilis and E. dianeae reproduce by constricting a single FtsZ ring at midcell despite being 45 μm and 120 μm long, respectively. In the crescent-shaped bacteria coating E. fertilis, symmetric FtsZ-based fission occurs in cells with lengths spanning one order of magnitude. In the E. dianeae symbiont, formation of a single functional FtsZ ring makes this the longest unicellular organism in which symmetric division has ever been observed. In conclusion, the reproduction modes of two extraordinarily long bacterial cells indicate that size is not the primary trigger of division and that yet unknown mechanisms time the localization of both DNA and the septum. PMID:25221974

  18. Apoptosis-Dependent and Apoptosis-Independent Functions Bim in Prostate Cancer Cells

    DTIC Science & Technology

    2004-03-01

    AD_ Award Number: DAMD17-03-1-0146 TITLE: Apoptosis-Dependent and Apoptosis-Independent Functions of Bim in Prostate Cancer Cells PRINCIPAL...FUNDING NUMBERS Apoptosis-Dependent and Apoptosis-Independent Functions of DAMD17-03-1-0146 Bim in Prostate Cancer Cells 6. A UTHORs) Junwei Liu, M.D...extended cell survival have been implicated in prostate cancer (PCa) development and progression. We recently found that Bim , a BH3-only pro

  19. Tributyltin affects adipogenic cell fate commitment in mesenchymal stem cells by a PPARγ independent mechanism.

    PubMed

    Biemann, Ronald; Fischer, Bernd; Blüher, Matthias; Navarrete Santos, Anne

    2014-05-05

    The food contaminant tributyltin (TBT) is an endocrine disrupting compound (EDC) promoting adipogenic differentiation in vitro and in vivo. Although prenatal TBT exposure has been shown to induce obesity, the underlying mechanisms and the role of the transcription factor PPARγ are not clarified yet. At different stages of adipogenesis, multipotent murine mesenchymal stem cells (MSC), C3H10T1/2, were exposed to TBT and analyzed for adipogenic differentiation, PPARγ promoter activation and PPARγ1, PPARγ2, Pref-1 and SOX9 expression. Depending on the exposure window, TBT promoted subsequent adipogenesis independently and dependently from PPARγ. In undifferentiated MSC, TBT exposure induced a transcriptional PPARγ-independent repression of Pref-1 and SOX9, which are both suppressors of adipogenic cell fate commitment. During hormonal induction TBT additionally enhanced adipogenic differentiation by PPARγ signaling. The impact of TBT on early cell fate development documents a novel mechanistic insight in the development of adipocytes derived from MSC and its susceptibility to EDC.

  20. Connexin 36 and Rod Bipolar Cell Independent Rod Pathways Drive Retinal Ganglion Cells and Optokinetic Reflexes

    PubMed Central

    Cowan, Cameron S.; Abd-El-Barr, Muhammad; van der Heijden, Meike; Lo, Eric M.; Paul, David; Bramblett, Debra E.; Lem, Janis; Simons, David L.; Wu, Samuel M.

    2016-01-01

    Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells. Pharmacological experiments show that rod pathway-driven and Cx36-independent RGC ON responses are also metabotropic glutamate receptor 6-dependent. To validate the RGC findings in awake, behaving animals we measured optokinetic reflexes (OKRs), which are sensitive to changes in ON pathways. Scotopic OKR contrast sensitivity was lost in Trα−/− mice, but indistinguishable from controls in Cx36−/− and rod bipolar cell knockout mice. Mesopic OKRs were also altered in mutant mice: Trα−/− mice had decreased spatial acuity, rod BC knockouts had decreased sensitivity, and Cx36−/− mice had increased sensitivity. These results provide compelling evidence against the complete Cx36 or rod BC dependence of night vision's ON component. Further, the findings suggest the parallel nature of rod pathways provides considerable redundancy to scotopic light sensitivity but distinct contributions to mesopic responses through complicated interactions with cone pathways. PMID:26718442

  1. Pseudomonas aeruginosa forms Biofilms in Acute InfectionIndependent of Cell-to-Cell Signaling

    SciTech Connect

    Schaber, J. Andy; Triffo, W.J.; Suh, Sang J.; Oliver, Jeffrey W.; Hastert, Mary C.; Griswold, John A.; Auer, Manfred; Hamood, Abdul N.; Rumbaugh, Kendra P.

    2006-09-20

    Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 hours of infection in thermally-injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections. P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild type and QS-deficient P. aeruginosa formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independent of QS.

  2. Expression of a hyperactive androgen receptor leads to androgen-independent growth of prostate cancer cells.

    PubMed

    Hsieh, Chen-Lin; Cai, Changmeng; Giwa, Ahmed; Bivins, Aaronica; Chen, Shao-Yong; Sabry, Dina; Govardhan, Kumara; Shemshedini, Lirim

    2008-07-01

    Cellular changes that affect the androgen receptor (AR) can cause prostate cancer to transition from androgen dependent to androgen independent, which is usually lethal. One common change in prostate tumors is overexpression of the AR, which has been shown to lead to androgen-independent growth of prostate cancer cells. This led us to hypothesize that expression of a hyperactive AR would be sufficient for androgen-independent growth of prostate cancer cells. To test this hypothesis, stable lune cancer prostate (LNCaP) cell lines were generated, which express a virion phosphoprotein (VP)16-AR hybrid protein that contains full-length AR fused to the strong viral transcriptional activation domain VP16. This fusion protein elicited as much as a 20-fold stronger transcriptional activity than the natural AR. Stable expression of VP16-AR in LNCaP cells yielded androgen-independent cell proliferation, while under the same growth conditions the parental LNCaP cells exhibited only androgen-dependent growth. These results show that expression of a hyperactive AR is sufficient for androgen-independent growth of prostate cancer cells. To study the molecular basis of this enhanced growth, we measured the expression of soluble guanylyl cyclase-alpha1 (sGCalpha1), a subunit of the sGC, an androgen-regulated gene that has been shown to be involved in prostate cancer cell growth. Interestingly, the expression of sGCalpha1 is androgen independent in VP16-AR-expressing cells, in contrast to its androgen-induced expression in control LNCaP cells. RNA(I)-dependent inhibition of sGCalpha1 expression resulted in significantly reduced proliferation of VP16-AR cells, implicating an important role for sGCalpha1 in the androgen-independent growth of these cells.

  3. Recoverable hybrid enzymatic biofuel cell with molecular oxygen-independence.

    PubMed

    Yu, You; Xu, Miao; Bai, Lu; Han, Lei; Dong, Shaojun

    2016-01-15

    Enzymatic biofuel cells (EBFCs) have drawn great attentions because of its potential in energy conversion. However, designing of highly efficient EBFCs which can adapt to the anaerobic system is still a great challenge. In this study, we propose a novel hybrid enzymatic biofuel cell (HEBFC) which was fabricated by a glucose dehydrogenase modified bioanode and a solid-state silver oxide/silver (Ag2O/Ag) cathode. The as-assembled HEBFC exhibited an open circuit potential of 0.59V and a maximum power output of 0.281mWcm(-2) at 0.34V in air saturated buffer. Especially, due to the introduction of Ag2O/Ag, our HEBFC could also operate under anaerobic condition, while the maximum power output would reach to 0.275mWcm(-2) at 0.34V. Furthermore, our HEBFC had stable cycle operation and could keep high power output for a certain time as the result of the regeneration of Ag2O. Our work provides a new concept to develop EBFCs for efficient energy conversion in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent

    PubMed Central

    Geng, Ying; Kohli, Latika; Klocke, Barbara J.; Roth, Kevin A.

    2010-01-01

    Glioblastoma (GBM) is a high-grade central nervous system malignancy and despite aggressive treatment strategies, GBM patients have a median survival time of just 1 year. Chloroquine (CQ), an antimalarial lysosomotropic agent, has been identified as a potential adjuvant in the treatment regimen of GBMs. However, the mechanism of CQ-induced tumor cell death is poorly defined. We and others have shown that CQ-mediated cell death may be p53-dependent and at least in part due to the intrinsic apoptotic death pathway. Here, we investigated the effects of CQ on 5 established human GBM lines, differing in their p53 gene status. CQ was found to induce a concentration-dependent death in each of these cell lines. Although CQ treatment increased caspase-3–like enzymatic activity in all 5 cell lines, a broad-spectrum caspase inhibitor did not significantly attenuate death. Moreover, CQ caused an accumulation of autophagic vacuoles in all cell lines and was found to affect the levels and subcellular distribution of cathepsin D, suggesting that altered lysosomal function may also play a role in CQ-induced cell death. Thus, CQ can induce p53-independent death in gliomas that do not require caspase-mediated apoptosis. To potentially identify more potent chemotherapeutics, various CQ derivatives and lysosomotropic compounds were tested on the GBM cells. Quinacrine and mefloquine were found to be more potent than CQ in killing GBM cells in vitro and given their superior blood–brain barrier penetration compared with CQ may prove more efficacious as chemotherapeutic agents for GBM patients. PMID:20406898

  5. Cell death-independent activities of the death receptors CD95, TRAILR1, and TRAILR2.

    PubMed

    Siegmund, Daniela; Lang, Isabell; Wajant, Harald

    2017-04-01

    Since their identification more than 20 years ago, the death receptors CD95, TRAILR1, and TRAILR2 have been intensively studied with respect to their cell death-inducing activities. These receptors, however, can also trigger a variety of cell death-independent cellular responses reaching from the activation of proinflammatory gene transcription programs over the stimulation of proliferation and differentiation to induction of cell migration. The cell death-inducing signaling mechanisms of CD95 and the TRAIL death receptors are well understood. In contrast, despite the increasing recognition of the biological and pathophysiological relevance of the cell death-independent activities of CD95, TRAILR1, and TRAILR2, the corresponding signaling mechanisms are less understood and give no fully coherent picture. This review is focused on the cell death-independent activities of CD95 and the TRAIL death receptors and addresses mainly three questions: (a) how are these receptors linked to noncell death pathways at the molecular level, (b) which factors determine the balance of cell death and cell death-independent activities of CD95 and the TRAIL death receptors at the cellular level, and (c) what are the consequences of the cell death-independent functions of these receptors for their role in cancer and inflammatory diseases. © 2016 Federation of European Biochemical Societies.

  6. Evidence for polar positional information independent of cell division and nucleoid occlusion

    PubMed Central

    Janakiraman, Anuradha; Goldberg, Marcia B.

    2004-01-01

    We present evidence that, in Escherichia coli, polar positional information is present at midcell independent of known cell division factors. In filamented cells, IcsA, which is normally polar, localizes at or near potential cell division sites. Because the cell pole is derived from the septum, the sites to which IcsA localizes in filaments correspond to future poles. IcsA localization to these sites is independent of FtsZ, MinCDE, septation, and nucleoid occlusion, indicating that positional information for the future pole is independent of cell division and chromosome positioning. Upon IcsA localization to these sites, septation is inhibited, suggesting that IcsA recognition of this polar positional information may influence cell division. PMID:14715903

  7. Survival Advantage of AMPK Activation to Androgen-Independent Prostate Cancer Cells During Energy Stress

    PubMed Central

    Chhipa, Rishi Raj; Wu, Yue; Mohler, James L.; Ip, Clement

    2016-01-01

    Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells. PMID:20570728

  8. Survival advantage of AMPK activation to androgen-independent prostate cancer cells during energy stress.

    PubMed

    Chhipa, Rishi Raj; Wu, Yue; Mohler, James L; Ip, Clement

    2010-10-01

    Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells. Copyright 2010 Elsevier Inc. All rights reserved.

  9. Oral-nasopharyngeal dendritic cells mediate T cell-independent IgA class switching on B-1 B cells.

    PubMed

    Kataoka, Kosuke; Fujihashi, Keiko; Terao, Yutaka; Gilbert, Rebekah S; Sekine, Shinichi; Kobayashi, Ryoki; Fukuyama, Yoshiko; Kawabata, Shigetada; Fujihashi, Kohtaro

    2011-01-01

    Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cμ circulation transcripts and Iμ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRβ⁻/⁻ mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA⁻ IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI.

  10. The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo.

    PubMed

    Yoshimoto, Momoko

    2015-12-01

    In the developing mouse embryo, there are several waves of hematopoiesis. Primitive and definitive erythromyeloid lineages appear prior to hematopoietic stem cell (HSC) emergence, and these waves are considered to be transient and support embryonic homeostasis until HSC-derived hematopoiesis is established. However, recent evidence strongly suggests that HSC-independent immune cells, such as tissue macrophages and some innate lymphoid cells, develop in the mouse embryo and persist into postnatal life. Innate type B-1 cells have also been reported to emerge from hemogenic endothelial cells in the extraembryonic yolk sac and para-aortic splanchnopleura, and continue to develop in the fetal liver, even in HSC-deficient mouse embryos. Here, this review discusses B-1 cell development in the context of the layered immune system hypothesis of B lymphopoiesis and the emergence of B-1 cells independent of HSCs.

  11. Clonal analysis of childhood acute lymphoblastic leukemia with "cytogenetically independent" cell populations.

    PubMed Central

    Pui, C H; Raskind, W H; Kitchingman, G R; Raimondi, S C; Behm, F G; Murphy, S B; Crist, W M; Fialkow, P J; Williams, D L

    1989-01-01

    Acute lymphoblastic leukemia (ALL) is generally regarded as a clonal disease in which a single abnormal progenitor cell gives rise to neoplastic progeny. Five of 463 cases of childhood ALL with adequately banded leukemic cells were found to have two cytogenetically independent cell populations. In addition, two of the four cases tested had more than two rearranged immunoglobulin genes and (or) T cell receptor genes. To investigate the clonality of these unusual leukemias, we examined the neoplastic cells for X-linked markers extrinsic to the disease. Leukemic cells from each of the three patients heterozygous for an X-linked, restriction fragment length polymorphism showed a single active parental allele, suggesting that both apparently independent cell populations developed from a common progenitor. These cases provide evidence that leukemogenesis involves a multistep process of mutation and suggest that karyotypic abnormalities may be a late event of malignant transformation. Images PMID:2566623

  12. Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity.

    PubMed

    Rouvier, E; Luciani, M F; Golstein, P

    1993-01-01

    Mechanisms of T cell-mediated cytotoxicity remain poorly defined at the molecular level. To investigate some of these mechanisms, we used as target cells, on the one hand, thymocytes from lpr and gld mouse mutants, and on the other hand, L1210 cells transfected or not with the apoptosis-inducing Fas molecule. These independent mutant or transfectant-based approaches both led to the conclusion that Fas was involved in the Ca(2+)-independent component of cytotoxicity mediated by at least two sources of T cells, namely nonantigen-specific in vitro activated hybridoma cells, and antigen-specific in vivo raised peritoneal exudate lymphocytes. Thus, in these cases, T cell-mediated cytotoxicity involved transduction via Fas of the target cell death signal.

  13. AMPK-dependent and independent effects of AICAR and compound C on T-cell responses.

    PubMed

    Rao, Enyu; Zhang, Yuwen; Li, Qiang; Hao, Jiaqing; Egilmez, Nejat K; Suttles, Jill; Li, Bing

    2016-06-07

    As a master metabolic sensor, AMP-activated protein kinase (AMPK) is involved in different fundamental cellular processes. Regulation of AMPK activity either by agonists (e.g., AICAR) or by antagonists (e.g., Compound C) has been widely employed to study the physiological functions of AMPK. However, mounting evidence indicates AMPK-independent effects for these chemicals and how they regulate immune cell functions remains largely unknown. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and Compound Cis mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent rolesof AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these "AMPK regulators".

  14. Berberine inhibits cell growth and mediates caspase-independent cell death in human pancreatic cancer cells.

    PubMed

    Pinto-Garcia, Lina; Efferth, Thomas; Torres, Amada; Hoheisel, Jörg D; Youns, Mahmoud

    2010-08-01

    Pancreatic cancer is one of the most aggressive human malignancies with an increasing incidence worldwide. In addition to the poor survival rates, combinations using gemcitabine as a backbone have failed to show any benefit beyond monotherapy. These facts underscore an urgent need for novel therapeutic options and motivated us to study the effect of berberine on pancreatic cancer cells. Here, we undertook an mRNA-based gene expression profiling study in order to get deeper insight into the molecular targets mediating the growth inhibitory effects of berberine on pancreatic cancer cells compared to normal ones. Twenty-four hours after treatment, berberine showed preferential selectivity toward pancreatic cancer cells compared to normal ones. Moreover, expression profiling and Ingenuity pathway analysis results showed that the cytotoxicity of berberine was accompanied with an activation of BRCA1-mediated DNA damage response, G1/S and G2/M cell cycle checkpoint regulation, and P53 signalling pathways. The activation of these signalling pathways might be explained by the fact that berberine intercalates DNA and induces DNA strand break through inhibition of topoisomerases and induction of DNA lesions.

  15. PORCN moonlights in a Wnt-independent pathway that regulates cancer cell proliferation.

    PubMed

    Covey, Tracy M; Kaur, Simran; Tan Ong, Tina; Proffitt, Kyle D; Wu, Yonghui; Tan, Patrick; Virshup, David M

    2012-01-01

    Porcupine (PORCN) is a membrane-bound O-acyl transferase that is required for the palmitoylation of Wnt proteins, and that is essential in diverse Wnt pathways for Wnt-Wntless (WLS) binding, Wnt secretion, and Wnt signaling activity. We tested if PORCN was required for the proliferation of transformed cells. Knockdown of PORCN by multiple independent siRNAs results in a cell growth defect in a subset of epithelial cancer cell lines. The growth defect is transformation-dependent in human mammary epithelial (HMEC) cells. Additionally, inducible PORCN knockdown by two independent shRNAs markedly reduces the growth of established MDA-MB-231 cancers in orthotopic xenografts in immunodeficient mice. Unexpectedly, the proliferation defect resulting from loss of PORCN occurs in a Wnt-independent manner, as it is rescued by re-expression of catalytically inactive PORCN, and is not seen after RNAi-mediated knockdown of the Wnt carrier protein WLS, nor after treatment with the PORCN inhibitor IWP. Consistent with a role in a Wnt-independent pathway, knockdown of PORCN regulates a distinct set of genes that are not altered by other inhibitors of Wnt signaling. PORCN protein thus appears to moonlight in a novel signaling pathway that is rate-limiting for cancer cell growth and tumorigenesis independent of its enzymatic function in Wnt biosynthesis and secretion.

  16. PRIMA-1(MET) induces death in soft-tissue sarcomas cell independent of p53.

    PubMed

    Grellety, Thomas; Laroche-Clary, Audrey; Chaire, Vanessa; Lagarde, Pauline; Chibon, Frédéric; Neuville, Agnes; Italiano, Antoine

    2015-10-13

    The aim of this study was to explore the efficacy and define mechanisms of action of PRIMA-1(MET) as a TP53 targeted therapy in soft-tissue sarcoma (STS) cells. We investigated effects of PRIMA-1(MET) on apoptosis, cell cycle, and induction of oxidative stress and autophagy in a panel of 6 STS cell lines with different TP53 status. Cell viability reduction by PRIMA-1(MET) was significantly observed in 5 out of 6 STS cell lines. We found that PRIMA-1(MET) was capable to induce cell death not only in STS cells harboring mutated TP53 but also in TP53-null STS cells demonstrating that PRIMA-1(MET) can induce cell death independently of TP53 in STS cells. We identified an important role of reactive oxygen species (ROS), involved in PRIMA-1(MET) toxicity in STS cells leading to a caspase-independent cell death. ROS toxicity was associated with autophagy induction or JNK pathway activation which represented potential mechanisms of cell death induced by PRIMA-1(MET) in STS. PRIMA-1(MET) anti-tumor activity in STS partly results from off-target effects involving ROS toxicity and do not deserve further development as a TP53-targeted therapy in this setting.

  17. MHCII-independent CD4+ T cells protect injured CNS neurons via IL-4.

    PubMed

    Walsh, James T; Hendrix, Sven; Boato, Francesco; Smirnov, Igor; Zheng, Jingjing; Lukens, John R; Gadani, Sachin; Hechler, Daniel; Gölz, Greta; Rosenberger, Karen; Kammertöns, Thomas; Vogt, Johannes; Vogelaar, Christina; Siffrin, Volker; Radjavi, Ali; Fernandez-Castaneda, Anthony; Gaultier, Alban; Gold, Ralf; Kanneganti, Thirumala-Devi; Nitsch, Robert; Zipp, Frauke; Kipnis, Jonathan

    2015-02-01

    A body of experimental evidence suggests that T cells mediate neuroprotection following CNS injury; however, the antigen specificity of these T cells and how they mediate neuroprotection are unknown. Here, we have provided evidence that T cell-mediated neuroprotection after CNS injury can occur independently of major histocompatibility class II (MHCII) signaling to T cell receptors (TCRs). Using two murine models of CNS injury, we determined that damage-associated molecular mediators that originate from injured CNS tissue induce a population of neuroprotective, IL-4-producing T cells in an antigen-independent fashion. Compared with wild-type mice, IL-4-deficient animals had decreased functional recovery following CNS injury; however, transfer of CD4+ T cells from wild-type mice, but not from IL-4-deficient mice, enhanced neuronal survival. Using a culture-based system, we determined that T cell-derived IL-4 protects and induces recovery of injured neurons by activation of neuronal IL-4 receptors, which potentiated neurotrophin signaling via the AKT and MAPK pathways. Together, these findings demonstrate that damage-associated molecules from the injured CNS induce a neuroprotective T cell response that is independent of MHCII/TCR interactions and is MyD88 dependent. Moreover, our results indicate that IL-4 mediates neuroprotection and recovery of the injured CNS and suggest that strategies to enhance IL-4-producing CD4+ T cells have potential to attenuate axonal damage in the course of CNS injury in trauma, inflammation, or neurodegeneration.

  18. Hop/STI1 modulates retinal proliferation and cell death independent of PrPC.

    PubMed

    Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S; Linden, Rafael; Chiarini, Luciana B

    2007-09-21

    Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP(C)). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP(C) dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 (alpha-STI1) blocked both ganglion cell and NBL cell death independent of PrP(C). cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while alpha-STI1 increased proliferation in the developing retina, both independent of PrP(C). We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP(C).

  19. Diquat causes caspase-independent cell death in SH-SY5Y cells by production of ROS independently of mitochondria.

    PubMed

    Nisar, R; Hanson, P S; He, L; Taylor, R W; Blain, P G; Morris, C M

    2015-10-01

    Evidence indicates that Parkinson's disease (PD), in addition to having a genetic aetiology, has an environmental component that contributes to disease onset and progression. The exact nature of any environmental agent contributing to PD is unknown in most cases. Given its similarity to paraquat, an agrochemical removed from registration in the EU for its suspected potential to cause PD, we have investigated the in vitro capacity of the related herbicide Diquat to cause PD-like cell death. Diquat showed greater toxicity towards SH-SY5Y neuroblastoma cells and human midbrain neural cells than paraquat and also MPTP, which was independent of dopamine transporter-mediated uptake. Diquat caused cell death independently of caspase activation, potentially via RIP1 kinase, with only a minor contribution from apoptosis, which was accompanied by enhanced reactive oxygen species production in the absence of major inhibition of complex I of the mitochondrial respiratory chain. No changes in α-synuclein expression were observed following 24-h or 4-week exposure. Diquat may, therefore, kill neural tissue by programmed necrosis rather than apoptosis, reflecting the pathological changes seen following high-level exposure, although its ability to promote PD is unclear.

  20. A new self: MHC-class-I-independent natural-killer-cell self-tolerance.

    PubMed

    Kumar, Vinay; McNerney, Megan E

    2005-05-01

    A fundamental tenet of the immune system is the requirement for lymphocytes to respond to transformed or infected cells while remaining tolerant of normal cells. Natural killer (NK) cells discriminate between self and non-self by monitoring the expression of MHC class I molecules. According to the 'missing-self' hypothesis, cells that express self-MHC class I molecules are protected from NK cells, but those that lack this self-marker are eliminated by NK cells. Recent work has revealed that there is another system of NK-cell inhibition, which is independent of MHC class I molecules. Newly discovered NK-cell inhibitory receptors that have non-MHC-molecule ligands broaden the definition of self as seen by NK cells.

  1. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

    PubMed

    Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

    2006-02-01

    HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

  2. Cell polarity protein CRB3 is an independent favorable prognostic factor for clear cell renal cell carcinoma.

    PubMed

    Mao, Xiaona; Li, Pingping; Ren, Yu; Li, Juan; Zhou, Can; Yang, Jin; Liu, Peijun

    2015-02-01

    Epithelial cells possess apical‑basal polarity and loss of epithelial cell polarity contributes to tumorigenesis and cancer progression. The Crumbs (CRB) polarity protein plays a crucial role in epithelial polarity maintenance, apical membrane formation, and tissue morphogenesis. Although evidence is increasing on involvement of deregulated polarity proteins in cancers, little is currently known about the roles of the CRB (Drosophila), especially the roles of CRB3, a homolog of the CRB, in clear cell renal cell carcinoma (ccRCC). Studies have shown that CRB3 may act as a tumor suppressor in non‑human mammalian cells; the study here was aimed to examine the expression status of CRB3 in ccRCC and the relationships between CRB3 expression and clinicopathologic parameters of ccRCC patients. Our results showed that CRB3 was weakly expressed in ccRCC tissues, but strongly expressed in adjacent normal kidney tissues. Patients with loss of CRB3 expression showed a significantly shorter overall survival (OS) than patients with positive CRB3 expression. Our results suggested that CRB3 may be an independent favorable prognostic factor for patients with ccRCC. We also found that overexpression of CRB3 restrained invasion and migration of 786‑O cells and loss of CRB3 expression promoted invasion and migration of human embryonic kidney 293T (HEK 293T) cells. This finding may explain why the negative CRB3 expression was associated with poor prognosis in human ccRCC. Altogether, our data demonstrated that CRB3 may be used as a new independent favorable prognostic factor for human ccRCC.

  3. Effect of AQP9 Expression in Androgen-Independent Prostate Cancer Cell PC3

    PubMed Central

    Chen, Qiwei; Zhu, Liang; Zheng, Bo; Wang, Jinliang; Song, Xishuang; Zheng, Wei; Wang, Lina; Yang, Deyong; Wang, Jianbo

    2016-01-01

    It is known that aquaporin 9 (AQP9) in the prostate was strictly upregulated by androgen and may represent a novel therapeutic target for several cancers, but whether AQP9 plays a role in the regulation of androgen-independent prostate cancer still remains unclear. In the present study, AQP9 was determined in prostate cancer and adjacent cancer tissues; AQP9-siRNA was applied to silencing AQP9 in androgen-independent prostate cancer cell PC3 cell line. Western blot and flow cytometry analysis were employed to detect changes in related-function of control and AQP9-siRNA groups. The results showed that AQP9 is significantly induced in cancer tissues than that in adjacent cancer tissues. Moreover, knockdown of AQP9 in PC3 androgen-independent prostate cancer cell prostate cancer cells increased inhibition rates of proliferation. In addition, knockdown of AQP9 resulted in a significant decrease in the expression of the Bcl-2 and with a notable increase in the expression of Bax and cleaved caspase 3, indicated that AQP9 knockdown promoted apoptosis in prostate cancer cells. From wound healing assay and matrigel invasion, we suggested that AQP9 expression affects the motility and invasiveness of prostate cancer cells. Moreover, In order to explore the pathway may be involved in AQP9-mediated motility and invasion of prostate cancer cells, the phosphorylation of ERK1/2 was significant suppressed in AQP9 siRNA-transfected cells compared with that in control cells, suggesting that AQP9 is involved in the activation of the ERK pathway in androgen-independent prostate cancer cells. PMID:27187384

  4. Expansion of Th17 Cells by Human Mast Cells Is Driven by Inflammasome-Independent IL-1β.

    PubMed

    Suurmond, Jolien; Habets, Kim L L; Dorjée, Annemarie L; Huizinga, Tom W; Toes, René E M

    2016-12-01

    Mast cells (MC) are most well known for their role in innate immune responses. However, MC are increasingly recognized as important regulators of adaptive immune responses, especially in setting the outcome of T cell responses. In this study we determined the effect of MC on cytokine production by naive and memory human Th cells. CD4(+) T cells were cultured with MC supernatant or control medium, after which cytokine production by T cells was determined. Supernatant of activated MC specifically increased the number of IL-17-producing T cells. This enhancement of Th17 cell number was specifically observed for the memory CD4(+) T cell population and not for the naive CD4(+) T cell population. The effect of MC was inhibited for ∼80% by blocking Abs to IL-1β and the rIL-1R antagonist anakinra. Importantly, secretion of active IL-1β by MC was independent of caspase activity, indicating that Th17 cell expansion by MC occurred through inflammasome-independent IL-1β. Together, these studies reveal a role for human MC in setting the outcome of T cell responses through release of caspase-independent IL-1β, and provide evidence for a novel contribution of MC in boosting the Th17 axis in mucosal immune responses. Copyright © 2016 by The American Association of Immunologists, Inc.

  5. Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor

    PubMed Central

    Wang, Lihong; Liu, Liping; Shi, Yan; Cao, Hanwei; Chaturvedi, Rupesh; Calcutt, M. Wade; Hu, Tianhui; Ren, Xiubao; Wilson, Keith T.; Polk, D. Brent; Yan, Fang

    2012-01-01

    Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apcmin mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth. PMID:22574158

  6. Alpha-Particle Emitting 213Bi-Anti-EGFR Immunoconjugates Eradicate Tumor Cells Independent of Oxygenation

    PubMed Central

    Gaertner, Florian C.; Bruchertseifer, Frank; Morgenstern, Alfred; Essler, Markus; Senekowitsch-Schmidtke, Reingard

    2013-01-01

    Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that cell-bound α-particle emitting 213Bi immunoconjugates kill hypoxic and normoxic CAL33 tumor cells with identical efficiency. For that purpose CAL33 cells were incubated with 213Bi-anti-EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing 213Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml–1.48 MBq/ml) and irradiation with increasing doses of photons (0.5–12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by 213Bi-anti-EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate that α-particle emitting 213Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, 213Bi-radioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors. PMID:23724085

  7. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways.

    PubMed

    Berkowska, Magdalena A; Driessen, Gertjan J A; Bikos, Vasilis; Grosserichter-Wagener, Christina; Stamatopoulos, Kostas; Cerutti, Andrea; He, Bing; Biermann, Katharina; Lange, Johan F; van der Burg, Mirjam; van Dongen, Jacques J M; van Zelm, Menno C

    2011-08-25

    Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27⁻IgG⁺ and CD27⁺IgM⁺ B cells are derived from primary germinal center reactions, and CD27⁺IgA⁺ and CD27⁺IgG⁺ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27⁻IgA⁺ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27⁻IgA⁺ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

  8. Modelling cell lifespan and proliferation: is likelihood to die or to divide independent of age?

    PubMed Central

    Dowling, Mark R; Milutinović, Dejan; Hodgkin, Philip D

    2005-01-01

    In cell lifespan studies the exponential nature of cell survival curves is often interpreted as showing the rate of death is independent of the age of the cells within the population. Here we present an alternative model where cells that die are replaced and the age and lifespan of the population pool is monitored until a steady state is reached. In our model newly generated individual cells are given a determined lifespan drawn from a number of known distributions including the lognormal, which is frequently found in nature. For lognormal lifespans the analytic steady-state survival curve obtained can be well-fit by a single or double exponential, depending on the mean and standard deviation. Thus, experimental evidence for exponential lifespans of one and/or two populations cannot be taken as definitive evidence for time and age independence of cell survival. A related model for a dividing population in steady state is also developed. We propose that the common adoption of age-independent, constant rates of change in biological modelling may be responsible for significant errors, both of interpretation and of mathematical deduction. We suggest that additional mathematical and experimental methods must be used to resolve the relationship between time and behavioural changes by cells that are predominantly unsynchronized. PMID:16849210

  9. Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells.

    PubMed

    Sakuma, Yuji; Matsukuma, Shoichi; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Koizume, Shiro; Sekiguchi, Hironobu; Saito, Haruhiro; Nakayama, Haruhiko; Kameda, Yoichi; Yokose, Tomoyuki; Oguni, Sachiko; Niki, Toshiro; Miyagi, Yohei

    2013-10-01

    Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.

  10. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    PubMed

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  11. Curcumin induces p53-independent necrosis in H1299 cells via a mitochondria-associated pathway.

    PubMed

    Li, Feie; Chen, Xi; Xu, Bing; Zhou, Hua

    2015-11-01

    Curcumin has been shown to have various therapeutic and/or adjuvant therapeutic effects on human cancers, as it inhibits cancer cell proliferation and induces apoptosis through p53-dependent molecular pathways. However, numerous cancer cell types bear a mutant p53 gene, and whether curcumin has any therapeutic effects on p53-deficient/mutant cancer cells has remained elusive. The present study sought to determine whether curcumin exerts any anti-proliferative and cytotoxic effects on the p53-deficient H1299 human lung cancer cell line via a p53-independent mechanism. An MTT assay and flow cytometric analysis indicated that curcumin significantly decreased cell proliferation and induced necrotic cell death. Western blot analysis of the cytosolic and mitochondrial fractions of H1299 cells as well as a fluorometric caspase assay indicated that curcumin-induced necrosis was mitochondria- and caspase-dependent, and resulted in cytochrome c release. Of note, this necrotic cell death was reduced following inhibition of B-cell lymphoma‑2 (Bcl-2)‑associated X protein (Bax) or Bcl‑2 homologous antagonist killer (Bak) as well as overexpression of Bcl-2. In conclusion, the present study suggested that curcumin-induced necrotic cell death was mediated via a p53-independent molecular pathway, which was associated with Bax and Bak translocation, caspase activation and cytochrome c release.

  12. Polyamines regulate cell growth and cellular methylglyoxal in high-glucose medium independently of intracellular glutathione.

    PubMed

    Kwak, Min-Kyu; Lee, Mun-Hyoung; Park, Seong-Jun; Shin, Sang-Min; Liu, Rui; Kang, Sa-Ouk

    2016-03-01

    Polyamines can presumably inhibit protein glycation, when associated with the methylglyoxal inevitably produced during glycolysis. Herein, we hypothesized a nonenzymatic interaction between putrescine and methylglyoxal in putrescine-deficient or -overexpressing Dictyostelium cells in high-glucose medium, which can control methylglyoxal production. Putrescine was essentially required for growth rescue accompanying methylglyoxal detoxification when cells underwent growth defect and cell cycle G1-arrest when supplemented with high glucose. Furthermore, methylglyoxal regulation by putrescine seemed to be a parallel pathway independent of the changes in cellular glutathione content in high-glucose medium. Consequently, we suggest that Dictyostelium cells need polyamines for normal growth and cellular methylglyoxal regulation.

  13. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

    PubMed

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

  14. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma

    PubMed Central

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

  15. Adaptation of mammalian auditory hair cell mechanotransduction is independent of calcium entry.

    PubMed

    Peng, Anthony W; Effertz, Thomas; Ricci, Anthony J

    2013-11-20

    Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca(2+) entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence of adaptation in the mammalian auditory system. Recordings from rat cochlear hair cells demonstrate that altering Ca(2+) entry or internal Ca(2+) buffering has little effect on either adaptation kinetics or steady-state adaptation responses. Two additional findings include a voltage-dependent process and an extracellular Ca(2+) binding site, both modulating the resting open probability independent of adaptation. These data suggest that slow motor adaptation is negligible in mammalian auditory cells and that the remaining adaptation process is independent of calcium entry.

  16. Growth hormone and cell survival in the neural retina: caspase dependence and independence.

    PubMed

    Harvey, Steve; Baudet, Marie-Laure; Sanders, Esmond J

    2006-11-06

    Growth hormone has recently been shown to be expressed in the retinal ganglion cells of embryonic chicks, in which it induces cell survival during neurogenesis. The mechanism of this action has been examined in neural retina explants from 6-day-old and 8-day-old embryos that were incubated for 48 h in 10 M growth hormone, to reduce the number of spontaneous apoptotic cells. This anti-apoptotic action was accompanied by a reduction in caspase-3 expression and, at embryonic day 8, by reduced expression of apoptosis inducing factor-1, which is caspase independent. These actions were specific, as other genes involved in apoptotic signaling (bcl-2, bcl-x, bid and inhibitor of apoptosis protein-1) were unaffected. These results therefore demonstrate caspase-dependent and caspase-independent pathways in growth hormone-induced retinal cell survival.

  17. Enhanced adhesion of osteoblastic cells on polystyrene films by independent control of surface topography and wettability.

    PubMed

    Yang, Seung Yun; Kim, Eung-Sam; Jeon, Gumhye; Choi, Kwan Yong; Kim, Jin Kon

    2013-04-01

    We independently controlled surface topography and wettability of polystyrene (PS) films by CF4 and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF4 plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of ~11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10°, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

    SciTech Connect

    Curry, Merril C.; Peters, Amelia A.; Kenny, Paraic A.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2013-05-10

    Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.

  19. p53- and Caspase-3-Independent Mechanism of Acetaminophen Effect on Human Neural Cells.

    PubMed

    Aleksandrova, A V; Senyavina, N V; Maltseva, D V; Khutornenko, A A; Sakharov, D A

    2016-04-01

    Acetaminophen in a concentration of 5 mM increased the expression of JNK, HIF1A (hypoxiainduced factor), and CASP3, which indicated development of oxidative stress and apoptotic cell death. Acetaminophen in a concentration of 10 mM did not induce expression of HIF1A and CASP3, but reduced expression of chaperone HSP90, which attested to activation of a caspase-3-independent mechanism of cell death. The methods of preventing acetaminophen intoxication are discussed.

  20. Democracy-Independence Trade-Off in Oscillating Dendrites and Its Implications for Grid Cells

    PubMed Central

    Remme, Michiel W.H.; Lengyel, Máté; Gutkin, Boris S.

    2010-01-01

    Summary Dendritic democracy and independence have been characterized for near-instantaneous processing of synaptic inputs. However, a wide class of neuronal computations requires input integration on long timescales. As a paradigmatic example, entorhinal grid fields have been thought to be generated by the democratic summation of independent dendritic oscillations performing direction-selective path integration. We analyzed how multiple dendritic oscillators embedded in the same neuron integrate inputs separately and determine somatic membrane voltage jointly. We found that the interaction of dendritic oscillations leads to phase locking, which sets an upper limit on the timescale for independent input integration. Factors that increase this timescale also decrease the influence that the dendritic oscillations exert on somatic voltage. In entorhinal stellate cells, interdendritic coupling dominates and causes these cells to act as single oscillators. Our results suggest a fundamental trade-off between local and global processing in dendritic trees integrating ongoing signals. PMID:20471355

  1. Democracy-independence trade-off in oscillating dendrites and its implications for grid cells.

    PubMed

    Remme, Michiel W H; Lengyel, Máté; Gutkin, Boris S

    2010-05-13

    Dendritic democracy and independence have been characterized for near-instantaneous processing of synaptic inputs. However, a wide class of neuronal computations requires input integration on long timescales. As a paradigmatic example, entorhinal grid fields have been thought to be generated by the democratic summation of independent dendritic oscillations performing direction-selective path integration. We analyzed how multiple dendritic oscillators embedded in the same neuron integrate inputs separately and determine somatic membrane voltage jointly. We found that the interaction of dendritic oscillations leads to phase locking, which sets an upper limit on the timescale for independent input integration. Factors that increase this timescale also decrease the influence that the dendritic oscillations exert on somatic voltage. In entorhinal stellate cells, interdendritic coupling dominates and causes these cells to act as single oscillators. Our results suggest a fundamental trade-off between local and global processing in dendritic trees integrating ongoing signals.

  2. CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.

    PubMed

    Viganò, Selena; Banga, Riddhima; Bellanger, Florence; Pellaton, Céline; Farina, Alex; Comte, Denis; Harari, Alexandre; Perreau, Matthieu

    2014-09-01

    Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160(+) CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160(+) CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.

  3. HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy.

    PubMed

    Hammer, Markus H; Meyer, Sonja; Brestrich, Gordon; Moosmann, Andreas; Kern, Florian; Tesfa, Lydia; Babel, Nina; Mittenzweig, Alexa; Rooney, Cliona M; Hammerschmidt, Wolfgang; Volk, Hans-Dieter; Reinke, Petra

    2005-07-01

    Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

  4. PRK1/PKN1 controls migration and metastasis of androgen-independent prostate cancer cells

    PubMed Central

    Jilg, Cordula A.; Ketscher, Anett; Metzger, Eric; Hummel, Barbara; Willmann, Dominica; Rüsseler, Vanessa; Drendel, Vanessa; Imhof, Axel; Jung, Manfred; Franz, Henriette; Hölz, Stefanie; Krönig, Malte; Müller, Judith M.; Schüle, Roland

    2014-01-01

    The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Statement of significance Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer. PMID:25504435

  5. Caspase-independent cell death without generation of reactive oxygen species in irradiated MOLT-4 human leukemia cells.

    PubMed

    Yoshida, Kengo; Kubo, Yoshiko; Kusunoki, Yoichiro; Morishita, Yukari; Nagamura, Hiroko; Hayashi, Ikue; Kyoizumi, Seishi; Seyama, Toshio; Nakachi, Kei; Hayashi, Tomonori

    2009-01-01

    To improve our understanding of ionizing radiation effects on immune cells, we investigated steps leading to radiation-induced cell death in MOLT-4, a thymus-derived human leukemia cell. After exposure of MOLT-4 cells to 4 Gy of X-rays, irradiated cells sequentially showed increase in intracellular reactive oxygen species (ROS), decrease in mitochondrial membrane potential, and eventually apoptotic cell death. In the presence of the caspase inhibitor z-VAD-fmk, irradiated cells exhibited necrotic characteristics such as mitochondrial swelling instead of apoptosis. ROS generation was not detected during this necrotic cell death process. These results indicate that radiation-induced apoptosis in MOLT-4 cells requires elevation of intracellular ROS as well as activation of a series of caspases, whereas the cryptic necrosis program--which is independent of intracellular ROS generation and caspase activation--is activated when the apoptosis pathway is blocked.

  6. Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor

    SciTech Connect

    Choi, Sunga; Lim, Mi-Hee; Kim, Ki Mo; Jeon, Byeong Hwa; Song, Won O.; Kim, Tae Woong

    2011-12-15

    Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specific features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer. -- Highlights: Black-Right-Pointing-Pointer We studied the mechanism which cordycepin-induced cell death association with

  7. Lipoic acid induces p53-independent cell death in colorectal cancer cells and potentiates the cytotoxicity of 5-fluorouracil.

    PubMed

    Dörsam, Bastian; Göder, Anja; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-10-01

    Alpha-lipoic acid (LA), which plays a pivotal role in mitochondrial energy metabolism, is an endogenous dithiol compound with an array of antioxidative functions. It has been shown that LA triggers cell death in tumor cell lines, whereas non-transformed cells are hardly affected. In the present study, we analyzed the cytotoxicity of LA on colorectal cancer (CRC) cells differing in their p53 status and investigated a putative synergistic effect with the anticancer drug 5-fluorouracil (5-FU). We show that LA induces a dose-dependent decrease in cell viability, which was independent of the p53 status as attested in isogenic p53-proficient and p53-deficient cell lines. This effect was largely attributable to cell death induction as revealed by Annexin-V/PI staining. LA-treated HCT116 cells underwent caspase-dependent and caspase-independent cell death, which was blocked by the pan-caspase inhibitor zVAD and the RIP-kinase inhibitor Necrostatin-1, respectively. In CaCO-2 and HT29 cells, LA induced caspase-dependent cell demise via activation of caspase-9, caspase-3 and caspase-7 with subsequent PARP-1 cleavage as demonstrated by immunoblot analysis, activity assays and pan-caspase inhibition. Interestingly, LA treatment did neither activate p53 nor induced genotoxic effects as shown by lack of DNA strand breaks and phosphorylation of histone 2AX. Finally, we provide evidence that LA increases the cytotoxic effect induced by the anticancer drug 5-FU as revealed by significantly enhanced cell death rates in HCT116 and CaCO-2 cells. Collectively, these findings demonstrate that LA induces CRC cell death independent of their p53 status and potentiates the cytotoxicity of 5-FU without causing DNA damage on its own, which makes it a candidate for tumor therapy.

  8. Nfil3-independent lineage maintenance and antiviral response of natural killer cells.

    PubMed

    Firth, Matthew A; Madera, Sharline; Beaulieu, Aimee M; Gasteiger, Georg; Castillo, Eliseo F; Schluns, Kimberly S; Kubo, Masato; Rothman, Paul B; Vivier, Eric; Sun, Joseph C

    2013-12-16

    Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3(-/-) mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3(-/-) NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15-dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells.

  9. Nfil3-independent lineage maintenance and antiviral response of natural killer cells

    PubMed Central

    Firth, Matthew A.; Madera, Sharline; Beaulieu, Aimee M.; Gasteiger, Georg; Castillo, Eliseo F.; Schluns, Kimberly S.; Kubo, Masato; Rothman, Paul B.; Vivier, Eric

    2013-01-01

    Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3−/− mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3−/− NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15–dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells. PMID:24277151

  10. SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors.

    PubMed

    De Calisto, Jaime; Wang, Ninghai; Wang, Guoxing; Yigit, Burcu; Engel, Pablo; Terhorst, Cox

    2014-01-01

    Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) plays an essential role in the immune system mediating the function of several members of the SLAM family (SLAMF) of receptors, whose expression is essential for T, NK, and B-cell responses. Additionally, the expression of SAP in double-positive thymocytes is mandatory for natural killer T (NKT) cells and, in mouse, for innate CD8(+) T cell development. To date, only two members of the SLAMF of receptors, Slamf1 and Slamf6, have been shown to positively cooperate during NKT cell differentiation in mouse. However, it is less clear whether other members of this family may also participate in the development of these innate T cells. Here, we show that Slamf[1 + 6](-/-) and Slamf[1 + 5 + 6](-/-) B6 mice have ~70% reduction of NKT cells compared to wild-type B6 mice. Unexpectedly, the proportion of innate CD8(+) T cells slightly increased in the Slamf[1 + 5 + 6](-/-) , but not in the Slamf[1 + 6](-/-) strain, suggesting that Slamf5 may function as a negative regulator of innate CD8(+) T cell development. Accordingly, Slamf5(-/-) B6 mice showed an exclusive expansion of innate CD8(+) T cells, but not NKT cells. Interestingly, the SAP-independent Slamf7(-/-) strain showed an expansion of both splenic innate CD8(+) T cells and thymic NKT cells. On the other hand, and similar to what was recently shown in Slamf3(-/-) BALB/c mice, the proportions of thymic promyelocytic leukemia zinc finger (PLZF(hi)) NKT cells and innate CD8(+) T cells significantly increased in the SAP-independent Slamf8(-/-) BALB/c strain. In summary, these results show that NKT and innate CD8(+) T cell development can be regulated in a SAP-dependent and -independent fashion by SLAMF receptors, in which Slamf1, Slamf6, and Slamf8 affect development of NKT cells, and that Slamf5, Slamf7, and Slamf8 affect the development of innate CD8(+) T cells.

  11. Cell cycle progression following naive T cell activation is independent of Jak3/common gamma-chain cytokine signals.

    PubMed

    Shi, Min; Lin, Tsung H; Appell, Kenneth C; Berg, Leslie J

    2009-10-01

    T cell proliferation following activation is an essential aspect of the adaptive immune response. Multiple factors, such as TCR signaling, costimulation, and signals from cytokines, each contribute to determine the magnitude of T cell expansion. In this report, we examine in detail the role of Jak3/common gamma-chain-dependent cytokines in promoting cell cycle progression and proliferation of naive T cells. Using naive CD4+ T cells from Jak3-deficient mice and wild-type CD4+ T cells treated with a small molecule inhibitor of Jak3, we find that these cytokine signals are not required for proliferation; instead, they are important for the survival of activated T cells. In addition, we show that the percentage of cells entering the cell cycle and the percentage of cells in each round of cell division are comparable between Jak3-deficent and wild-type T cells. Furthermore, cell cycle progression and the regulated expression of key cell cycle proteins are independent of Jak3/common gamma-chain cytokine signals. These findings hold true over a wide range of TCR signal strengths. However, when CD28 costimulatory signals, but not TCR signals, are limiting, Jak3-dependent cytokine signals become necessary for the proliferation of naive T cells. Because CD28 signaling has been found to be dispensable for autoreactive T cell responses, these data suggest the potential for interfering with autoimmune T cell responses by inhibition of Jak3 signaling.

  12. Cyclosporin A enhances neural precursor cell survival in mice through a calcineurin-independent pathway.

    PubMed

    Sachewsky, Nadia; Hunt, Jessica; Cooke, Michael J; Azimi, Ashkan; Zarin, Taraneh; Miu, Carween; Shoichet, Molly S; Morshead, Cindi M

    2014-08-01

    Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neural precursor cells; NPCs) in the adult central nervous system. Administration of CsA in vitro or in vivo promotes the survival of NPCs and expands the pools of NPCs in mice. Moreover, CsA administration is effective in promoting NPC activation, tissue repair and functional recovery in a mouse model of cortical stroke. The mechanism(s) by which CsA mediates this cell survival effect remains unknown. Herein, we examined both calcineurin-dependent and calcineurin-independent pathways through which CsA might mediate NPC survival. To examine calcineurin-dependent pathways, we utilized FK506 (Tacrolimus), an immunosuppressive molecule that inhibits calcineurin, as well as drugs that inhibit cyclophilin A-mediated activation of calcineurin. To evaluate the calcineurin-independent pathway, we utilized NIM811, a non-immunosuppressive CsA analog that functions independently of calcineurin by blocking mitochondrial permeability transition pore formation. We found that only NIM811 can entirely account for the pro-survival effects of CsA on NPCs. Indeed, blocking signaling pathways downstream of calcineurin activation using nNOS mice did not inhibit CsA-mediated cell survival, which supports the proposal that the effects are calcinuerin-independent. In vivo studies revealed that NIM811 administration mimics the pro-survival effects of CsA on NPCs and promotes functional recovery in a model of cortical stroke, identical to the effects seen with CsA administration. We conclude that CsA mediates its effect on NPC survival through calcineurin-independent inhibition of mitochondrial permeability transition pore formation and suggest that this pathway has potential therapeutic benefits for developing NPC-mediated cell replacement strategies. © 2014. Published by The Company of Biologists Ltd.

  13. Assessing light-independent effects of hypericin on cell viability, ultrastructure and metabolism in human glioma and endothelial cells.

    PubMed

    Huntosova, Veronika; Novotova, Marta; Nichtova, Zuzana; Balogova, Lucia; Maslanakova, Maria; Petrovajova, Dana; Stroffekova, Katarina

    2017-04-01

    Cell exposure to light-independent effects of photosensitizers (PS) used in PDT is clinically relevant when PS affect the pro-apoptotic cascade. In many malignant cells, Hypericin (Hyp) has PS displayed light-dependent anti-proliferative and cytotoxic effects with no cytotoxicity in the dark. Recent studies have shown that Hyp also exhibited light-independent cytotoxic effects in a wide range of concentrations. The molecular mechanisms underlying Hyp light-independent (dark) toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and these mechanisms are not understood in detail. Here, we demonstrate that in human glioma and endothelial cells, Hyp displayed light-independent effects at several sub-cellular levels (ultrastructure, mitochondria function and metabolism, and protein synthesis). Taking together previously published and our present results, the findings strongly suggest that Hyp light independent effects: (i) depend on the cell type and metabolism; (ii) underlying molecular mechanisms are due to Hyp interaction with the multiple target molecules including Bcl2 family of proteins. In addition, the findings suggest that Hyp without illumination can be explored as an adjuvant therapeutic drug in combination with chemo- or radiation cancer therapy.

  14. G1/S control of anchorage-independent growth in the fibroblast cell cycle

    PubMed Central

    1991-01-01

    We have developed methodology to identify the block to anchorage- independent growth and position it within the fibroblast cell cycle. Results with NRK fibroblasts show that mitogen stimulation of the G0/G1 transition and G1-associated increases in cell size are minimally affected by loss of cell anchorage. In contrast, the induction of G1/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked. Moreover, we demonstrate that the anchorage-dependent transition maps to late G1 and shortly before activation of the G1/S p34cdc2-like kinase. The G1/S block was also detectable in NIH-3T3 cells. Our results: (a) distinguish control of cell cycle progression by growth factors and anchorage; (b) indicate that anchorage mediates G1/S control in fibroblasts; and (c) identify a physiologic circumstance in which the phenotype of mammalian cell cycle arrest would closely resemble Saccharomyces cerevisiae START. The close correlation between anchorage independence in vitro and tumorigenicity in vivo emphasizes the key regulatory role for G1/S control in mammalian cells. PMID:1955482

  15. AMID mediates adenosine-induced caspase-independent HuH-7 cell apoptosis.

    PubMed

    Yang, Dongqin; Yaguchi, Takahiro; Nagata, Tetsu; Gotoh, Akinobu; Dovat, Sinisa; Song, Chunhua; Nishizaki, Tomoyuki

    2011-01-01

    The mechanism underlying extracellular adenosine-induced caspase-independent apoptosis in HuH-7 human hepatoma cells is not fully understood. The present study investigated the role for apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death (AMID) in the pathway. To see the implication of AMID in adenosine-induced HuH-7 cell apoptosis, real-time reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescent cytochemistry, time-laps GFP monitoring, cell cycle analysis, flow cytometry, Western blotting, cell viability assay, and TUNEL staining were carried out. Adenosine upregulated AMID expression in HuH-7 cells, and translocated AMID from the cytosol into the nucleus. Adenosine induced HuH-7 cell apoptosis, and the effect was further enhanced by overexpressing AMID. Adenosine-induced HuH-7 cell apoptosis, alternatively, was inhibited by knocking-down AMID. The results of the present study provide evidence for AMID as a critical factor for adenosine-induced caspase-independent HuH-7 cell apoptosis. Copyright © 2011 S. Karger AG, Basel.

  16. Glutamine Synthetase Is a Genetic Determinant of Cell Type–Specific Glutamine Independence in Breast Epithelia

    PubMed Central

    Kung, Hsiu-Ni; Marks, Jeffrey R.; Chi, Jen-Tsan

    2011-01-01

    Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type–specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies. PMID:21852960

  17. A DNA Damage-Induced, SOS-Independent Checkpoint Regulates Cell Division in Caulobacter crescentus

    PubMed Central

    Modell, Joshua W.; Kambara, Tracy K.; Perchuk, Barrett S.; Laub, Michael T.

    2014-01-01

    Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage. PMID:25350732

  18. Gremlin-1 Induces BMP-Independent Tumor Cell Proliferation, Migration, and Invasion

    PubMed Central

    Kim, Minsoo; Yoon, Soomin; Lee, Sukmook; Ha, Seon Ah; Kim, Hyun Kee; Kim, Jin Woo; Chung, Junho

    2012-01-01

    Gremlin-1, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2) expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation. PMID:22514712

  19. Gremlin-1 induces BMP-independent tumor cell proliferation, migration, and invasion.

    PubMed

    Kim, Minsoo; Yoon, Soomin; Lee, Sukmook; Ha, Seon Ah; Kim, Hyun Kee; Kim, Jin Woo; Chung, Junho

    2012-01-01

    Gremlin-1, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2) expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.

  20. Alkaline phosphatase determines polyphosphate-induced mineralization in a cell-type independent manner.

    PubMed

    Mikami, Yoshikazu; Tsuda, Hiromasa; Akiyama, Yuko; Honda, Masaki; Shimizu, Noriyoshi; Suzuki, Naoto; Komiyama, Kazuo

    2016-11-01

    Polyphosphate [Poly(P)] has positive effects on osteoblast mineralization; however, the underlying mechanism remains unclear. In addition, it is unknown whether Poly(P) promotes mineralization in soft tissues. We investigated this by using various cells. Poly(P) concentrations of 1 and 0.5 mg/mL yielded high levels of mineralization in ROS17/2.8 osteoblast cells. Similarly, Poly(P) induced mineralization in cell types expressing alkaline phosphatase (ALP), namely, ATDC5 and MC3T3-E1, but not in CHO, C3H10T1/2, C2C12, and 3T3-L1 cells. Furthermore, forced expression of ALP caused Poly(P)-induced mineralization in CHO cells. These results suggest that ALP determines Poly(P)-induced mineralization in a cell-type independent manner.

  1. Independent control of natural killer cell responsiveness and homeostasis at steady-state by CD11c+ dendritic cells

    PubMed Central

    Luu, Thuy Thanh; Ganesan, Sridharan; Wagner, Arnika Kathleen; Sarhan, Dhifaf; Meinke, Stephan; Garbi, Natalio; Hämmerling, Günter; Alici, Evren; Kärre, Klas; Chambers, Benedict J.; Höglund, Petter; Kadri, Nadir

    2016-01-01

    During infection and inflammation, dendritic cells (DC) provide priming signals for natural killer (NK) cells via mechanisms distinct from their antigen processing and presentation functions. The influence of DC on resting NK cells, i.e. at steady-state, is less well studied. We here demonstrate that as early as 1 day after DC depletion, NK cells in naïve mice downregulated the NKG2D receptor and showed decreased constitutive phosphorylation of AKT and mTOR. Subsequently, apoptotic NK cells appeared in the spleen concomitant with reduced NK cell numbers. At 4 days after the onset of DC depletion, increased NK cell proliferation was seen in the spleen resulting in an accumulation of Ly49 receptor-negative NK cells. In parallel, NK cell responsiveness to ITAM-mediated triggering and cytokine stimulation dropped across maturation stages, suggestive of a functional deficiency independent from the homeostatic effect. A role for IL-15 in maintaining NK cell function was supported by a gene signature analysis of NK cell from DC-depleted mice as well as by in vivo DC transfer experiments. We propose that DC, by means of IL-15 transpresentation, are required to maintain not only homeostasis, but also function, at steady-state. These processes appear to be regulated independently from each other. PMID:27905484

  2. Involvement of Cell Surface Structures in Size-Independent Grazing Resistance of Freshwater Actinobacteria▿ †

    PubMed Central

    Tarao, Mitsunori; Jezbera, Jan; Hahn, Martin W.

    2009-01-01

    We compared the influences of grazing by the bacterivorous nanoflagellate Poterioochromonas sp. strain DS on ultramicrobacterial Actinobacteria affiliated with the Luna-2 cluster and ultramicrobacterial Betaproteobacteria of the species Polynucleobacter cosmopolitanus. These bacteria were almost identical in size (<0.1 μm3) and shape. Predation on a Polynucleobacter strain resulted in a reduction of >86% relative to the initial bacterial cell numbers within 20 days, while in comparable predation experiments with nine actinobacterial strains, no significant decrease of cell numbers by predation was observed over the period of ≥39 days. The differences in predation mortality between the actinobacterial strains and the Polynucleobacter strain clearly demonstrated size-independent grazing resistance for the investigated Actinobacteria. Importantly, this size-independent grazing resistance is shared by all nine investigated Luna-2 strains and thus represents a group-specific trait. We investigated if an S-layer, previously observed in an ultrastructure study, was responsible for the grazing resistance of these strains. Experiments aiming for removal of the S-layer or modification of cell surface proteins of one of the grazing-resistant strains by treatment with lithium chloride, EDTA, or formaldehyde resulted in 4.2- to 5.2-fold higher grazing rates in comparison to the levels for untreated cells. These results indicate the protective role of a proteinaceous cell surface structure in the size-independent grazing resistance of the actinobacterial Luna-2 strains, which can be regarded as a group-specific trait. PMID:19502450

  3. Induction of DNA damage and caspase-independent programmed cell death by vitamin E.

    PubMed

    Constantinou, C; Neophytou, C M; Vraka, P; Hyatt, J A; Papas, K A; Constantinou, A I

    2012-01-01

    Vitamin E comprises 8 functionally unique isoforms and may be a suitable candidate for the adjuvant treatment of prostate cancer. In this study, we examined the ability of 2 vitamin E isoforms [α-tocotrienol (γ-TT) and δ-tocotrienol (δ-TT)] and 4 synthetic derivatives [γ- and δ-tocotrienol succinate (γ-TS, δ-TS), α-tocopheryl polyethylene glycol succinate (TPGS), and α-tocopheryl polyethylene glycol ether (TPGS-e)] of vitamin E to induce cell death in AR- (DU145 and PC-3) and AR+ (LNCaP) prostate cancer cell lines. Our results show that δ-TT and TPGS-e are the most effective isoform and synthetic derivative, respectively, of all compounds examined. Overall, the results of our study suggest that isoforms and synthetic derivatives of vitamin E have the potency to trigger both caspase-dependent and -independent DNA damage and dominant caspase-independent programmed cell death. The capacity of vitamin E to trigger caspase-independent programmed cell death suggests that it may be useful in the chemotherapy of prostate cancer since it may prevent the tumor resistance commonly associated with the use of classical chemotherapeutic agents that trigger caspase-dependent programmed cell death.

  4. Role of CSL-dependent and independent Notch signaling pathways in cell apoptosis.

    PubMed

    Zeng, Chong; Xing, Rui; Liu, Jing; Xing, Feiyue

    2016-01-01

    Apoptosis is a normally biological phenomenon in various organisms, involving complexly molecular mechanisms with a series of signaling processes. Notch signaling is found evolutionarily conserved in many species, playing a critical role in embryonic development, normal tissue homeostasis, angiogenesis and immunoregulation. The focus of this review is on currently novel advances about roles of CSL-dependent and independent Notch signaling pathways in cell apoptosis. The CSL can bind Notch intracellular domain (NIC) to act as a switch in mediating transcriptional activation or inactivation of the Notch signaling pathway downstream genes in the nucleus. It shows that CSL-dependent signaling regulates the cell apoptosis through Hes-1-PTEN-AKT-mTOR signaling, but rather the CSL-independent signaling mediates the cell apoptosis possibly via NIC-mTORC2-AKT-mTOR signaling, providing a new insight into apoptotic mechanisms.

  5. Highly flexible SRAM cells based on novel tri-independent-gate FinFET

    NASA Astrophysics Data System (ADS)

    Liu, Chengsheng; Zheng, Fanglin; Sun, Yabin; Li, Xiaojin; Shi, Yanling

    2017-10-01

    In this paper, a novel tri-independent-gate (TIG) FinFET is proposed for highly flexible SRAM cells design. To mitigate the read-write conflict, two kinds of SRAM cells based on TIG FinFETs are designed, and high tradeoff are obtained between read stability and speed. Both cells can offer multi read operations for frequency requirement with single voltage supply. In the first TIG FinFET SRAM cell, the strength of single-fin access transistor (TIG FinFET) can be flexibly adjusted by selecting five different modes to meet the needs of dynamic frequency design. Compared to the previous double-independent-gate (DIG) FinFET SRAM cell, 12.16% shorter read delay can be achieved with only 1.62% read stability decrement. As for the second TIG FinFET SRAM cell, pass-gate feedback technology is applied and double-fin TIG FinFETs are used as access transistors to solve the severe write-ability degradation. Three modes exist to flexibly adjust read speed and stability, and 68.2% larger write margin and 51.7% shorter write delay are achieved at only the expense of 26.2% increase in leakage power, with the same layout area as conventional FinFET SRAM cell.

  6. Glutamine activates STAT3 to control cancer cell proliferation independently of glutamine metabolism.

    PubMed

    Cacace, Andrea; Sboarina, Martina; Vazeille, Thibaut; Sonveaux, Pierre

    2017-04-01

    Cancer cells can use a variety of metabolic substrates to fulfill the bioenergetic and biosynthetic needs of their oncogenic program. Besides bioenergetics, cancer cell metabolism also directly influences genetic, epigenetic and signaling events associated with tumor progression. Many cancer cells are addicted to glutamine, and this addiction is observed in oxidative as well as in glycolytic cells. Although both oxidative and bioreductive glutamine metabolism can contribute to cancer progression and glutamine can further serve to generate peptides (including glutathione) and proteins, we report that glutamine promotes the proliferation of cancer cells independently of its use as a metabolic fuel or as a precursor of glutathione. Extracellular glutamine activates transcription factor signal transducer and activator of transcription 3 (STAT3), which is necessary and sufficient to mediate the proliferative effects of glutamine on glycolytic and oxidative cancer cells. Glutamine also activates transcription factors hypoxia-inducible factor-1, mammalian target of rapamycin and c-Myc, but these factors do not mediate the effects of glutamine on cancer cell proliferation. Our findings shed a new light on the anticancer effects of l-asparaginase that possesses glutaminase activity and converts glutamine into glutamate extracellularly. Conversely, cancer resistance to treatments that block glutamine metabolism could arise from glutamine-independent STAT3 reactivation.

  7. AT7867 Inhibits Human Colorectal Cancer Cells via AKT-Dependent and AKT-Independent Mechanisms

    PubMed Central

    Yao, Chen; Huang, Ping; Zhang, Yi; Cao, Shibing; Li, Xiangcheng

    2017-01-01

    AKT is often hyper-activated in human colorectal cancers (CRC). This current study evaluated the potential anti-CRC activity by AT7867, a novel AKT and p70S6K1 (S6K1) dual inhibitor. We showed that AT7867 inhibited survival and proliferation of established (HT-29, HCT116 and DLD-1 lines) and primary human CRC cells. Meanwhile, it provoked caspase-dependent apoptosis in the CRC cells. Molecularly, AT7867 blocked AKT-S6K1 activation in CRC cells. Restoring AKT-S6K1 activation, via expression of a constitutively-active AKT1 (“ca-AKT1”), only partially attenuated AT7867-induced HT-29 cell death. Further studies demonstrated that AT7867 inhibited sphingosine kinase 1 (SphK1) activity to promote pro-apoptotic ceramide production in HT-29 cells. Such effects by AT7867 were independent of AKT inhibition. AT7867-indued ceramide production and subsequent HT-29 cell apoptosis were attenuated by co-treatment of sphingosine-1-phosphate (S1P), but were potentiated with the glucosylceramide synthase (GCS) inhibitor PDMP. In vivo, intraperitoneal injection of AT7867 inhibited HT-29 xenograft tumor growth in nude mice. AKT activation was also inhibited in AT7867-treated HT-29 tumors. Together, the preclinical results suggest that AT7867 inhibits CRC cells via AKT-dependent and -independent mechanisms. PMID:28081222

  8. Independent regulation of tumor cell migration by matrix stiffness and confinement.

    PubMed

    Pathak, Amit; Kumar, Sanjay

    2012-06-26

    Tumor invasion and metastasis are strongly regulated by biophysical interactions between tumor cells and the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility has been extensively studied in 2D culture, extension of this concept to 3D cultures that more closely resemble tissue has proven challenging, because perturbations that change matrix stiffness often concurrently change cellular confinement. This coupling is particularly problematic given that matrix-imposed steric barriers can regulate invasion speed independent of mechanics. Here we introduce a matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows independent variation of ECM stiffness and channel width. For a given ECM stiffness, cells confined to narrow channels surprisingly migrate faster than cells in wide channels or on unconstrained 2D surfaces, which we attribute to increased polarization of cell-ECM traction forces. Confinement also enables cells to migrate increasingly rapidly as ECM stiffness rises, in contrast with the biphasic relationship observed on unconfined ECMs. Inhibition of nonmuscle myosin II dissipates this traction polarization and renders the relationship between migration speed and ECM stiffness comparatively insensitive to matrix confinement. We test these hypotheses in silico by devising a multiscale mathematical model that relates cellular force generation to ECM stiffness and geometry, which we show is capable of recapitulating key experimental trends. These studies represent a paradigm for investigating matrix regulation of invasion and demonstrate that matrix confinement alters the relationship between cell migration speed and ECM stiffness.

  9. Independent regulation of tumor cell migration by matrix stiffness and confinement

    PubMed Central

    Pathak, Amit; Kumar, Sanjay

    2012-01-01

    Tumor invasion and metastasis are strongly regulated by biophysical interactions between tumor cells and the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility has been extensively studied in 2D culture, extension of this concept to 3D cultures that more closely resemble tissue has proven challenging, because perturbations that change matrix stiffness often concurrently change cellular confinement. This coupling is particularly problematic given that matrix-imposed steric barriers can regulate invasion speed independent of mechanics. Here we introduce a matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows independent variation of ECM stiffness and channel width. For a given ECM stiffness, cells confined to narrow channels surprisingly migrate faster than cells in wide channels or on unconstrained 2D surfaces, which we attribute to increased polarization of cell-ECM traction forces. Confinement also enables cells to migrate increasingly rapidly as ECM stiffness rises, in contrast with the biphasic relationship observed on unconfined ECMs. Inhibition of nonmuscle myosin II dissipates this traction polarization and renders the relationship between migration speed and ECM stiffness comparatively insensitive to matrix confinement. We test these hypotheses in silico by devising a multiscale mathematical model that relates cellular force generation to ECM stiffness and geometry, which we show is capable of recapitulating key experimental trends. These studies represent a paradigm for investigating matrix regulation of invasion and demonstrate that matrix confinement alters the relationship between cell migration speed and ECM stiffness. PMID:22689955

  10. Bupivacaine induces apoptosis through caspase-dependent and -independent pathways in canine mammary tumor cells.

    PubMed

    Chiu, Yi-Shu; Cheng, Yeong-Hsiang; Lin, Sui-Wen; Chang, Te-Sheng; Liou, Chian-Jiun; Lai, Yu-Shen

    2015-06-01

    Local anesthetics have been reported to induce apoptosis in various cell lines. In this study, we showed that bupivacaine also induced apoptosis in DTK-SME cells, a vimentin(+)/AE1(+)/CK7(+)/HSP27(+), tumorigenic, immortalized, canine mammary tumor cell line. Bupivacaine induced apoptosis in DTK-SME cells in a time- and concentration-dependent manner. Apoptosis-associated morphological changes, including cell shrinkage and rounding, chromatin condensation, and formation of apoptotic bodies, were observed in the bupivacaine-treated DTK-SME cells. Apoptosis was further confirmed with annexin V staining, TUNEL staining, and DNA laddering assays. At the molecular level, the activation of caspases-3, -8, and -9 corresponded well to the degree of DNA fragmentation triggered by bupivacaine. We also demonstrated that the pan-caspase inhibitor, z-VAD-fmk, only partially inhibited the apoptosis induced by bupivacaine. Moreover, treated cells increased expression of endonuclease G, a death effector that acts independently of caspases. Our data suggested that bupivacaine-induced apoptosis occurs through both caspase-dependent and caspase-independent apoptotic pathways.

  11. ERK-dependent and -independent pathways trigger human neural progenitor cell migration

    SciTech Connect

    Moors, Michaela . E-mail: moors@uni-duesseldorf.de; Cline, Jason E. . E-mail: jason.cline@uni-duesseldorf.de; Abel, Josef . E-mail: josef.abel@uni-duesseldorf.de; Fritsche, Ellen . E-mail: ellen.fritsche@uni-duesseldorf.de

    2007-05-15

    Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways.

  12. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

    PubMed Central

    Cheng, Tzuling; Sudderth, Jessica; Yang, Chendong; Mullen, Andrew R.; Jin, Eunsook S.; Matés, José M.; DeBerardinis, Ralph J.

    2011-01-01

    Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how “glutamine-addicted” cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis. PMID:21555572

  13. Polymicrobial sepsis alters Ag-dependent and -independent memory CD8 T cell functions1

    PubMed Central

    Duong, Sean; Condotta, Stephanie A.; Rai, Deepa; Martin, Matthew D.; Griffith, Thomas S.; Badovinac, Vladimir P.

    2014-01-01

    Mortality from sepsis frequently results from secondary infections, and the extent to which sepsis affects pathogen-specific memory CD8 T cell responses remains unknown. Using the cecal-ligation and puncture (CLP) model of polymicrobial sepsis, we observed rapid apoptosis of pre-existing memory CD8 T cells after sepsis induction that led to a loss in CD8 T cell-mediated protection. Ag-sensitivity (functional avidity) and Ag-driven secondary expansion of memory CD8 T cells were decreased after sepsis, further contributing to the observed loss in CD8 T cell-mediated immunity. Moreover, Ag-independent bystander activation of memory CD8 T cells in response to heterologous infection was also significantly impaired early after sepsis induction. The reduced sensitivity of pre-existing memory CD8 T cells to sense inflammation and respond to heterologous infection by IFN-γ production was observed in inbred and outbred hosts and controlled by extrinsic (but not cell intrinsic) factors suggesting that sepsis-induced changes in the environment regulates innate functions of memory CD8 T cells. Taken together, the data in this study revealed a previously unappreciated role of sepsis in shaping the quantity and functionality of infection- or vaccine-induced memory CD8 T cells and will help further define the decline in T cell-mediated immunity during the sepsis-induced phase of immunosuppression. PMID:24646738

  14. Regulatory T cells occupy an isolated niche in the intestine that is antigen independent.

    PubMed

    Korn, Lisa L; Hubbeling, Harper G; Porrett, Paige M; Yang, Qi; Barnett, Lisa G; Laufer, Terri M

    2014-12-11

    Regulatory T cells (Tregs) are CD4(+) T cells that maintain immune homeostasis and prevent autoimmunity. Like all CD4(+) T cells, Tregs require antigen-specific signals via T cell receptor-major histocompatibility complex class II (TCR-MHCII) interactions for their development. However, the requirement for MHCII in Treg homeostasis in tissues such as intestinal lamina propria (LP) is unknown. We examined LP Treg homeostasis in a transgenic mouse model that lacks peripheral TCR-MHCII interactions and generation of extrathymic Tregs (iTregs). Thymically generated Tregs entered the LP of weanlings and proliferated independently of MHCII to fill the compartment. The adult LP was a closed niche; new thymic Tregs were excluded, and Tregs in parabiotic pairs were LP resident. The isolated LP niche was interleukin-2 (IL-2) independent but dependent on commensal bacteria. Thus, an LP Treg niche can be filled, isolated, and maintained independently of antigen signals and iTregs. This niche may represent a tissue-specific mechanism for maintaining immune tolerance.

  15. Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin.

    PubMed

    Reig, Núria; Jiang, Aimin; Couture, Rachael; Sutterwala, Fayyaz S; Ogura, Yasunori; Flavell, Richard A; Mellman, Ira; van der Goot, F Gisou

    2008-05-01

    Anthrax lethal toxin (LT) contributes to the immune evasion strategy of Bacillus anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however, not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knockout mice, we found that depending on the mouse strain, death of bone marrow-derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1-dependent, or by a slow caspase-1-independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1-independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses.

  16. Maturation Modulates Caspase-1 Independent Responses of Dendritic Cells to Anthrax Lethal Toxin

    PubMed Central

    Reig, Núria; Jiang, Aimin; Couture, Rachael; Sutterwala, Fayyaz S.; Ogura, Yasunori; Flavell, Richard A.; Mellman, Ira; van der Goot, F. Gisou

    2010-01-01

    Anthrax lethal toxin (LT) contributes to the immune evasion strategy of B. anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knock-out mice, we found that depending on the mouse strain, death of bone marrow derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1 dependent, or by a slow caspase-1 independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1 independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1 independent responses. PMID:18194483

  17. A spindle-independent cleavage pathway controls germ cell formation in Drosophila

    PubMed Central

    Cinalli, Ryan M.; Lehmann, Ruth

    2013-01-01

    The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. While the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using 4D multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to utilizing core regulators of cleavage, including the small GTPase RhoA (Drosophila Rho) and the Rho associated kinase, ROCK (Drosophila Rok), we show that this pathway requires Germ cell-less (Gcl), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternate form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage. PMID:23728423

  18. SOS-independent coupling between DNA replication and cell division in Escherichia coli.

    PubMed Central

    Jaffé, A; D'Ari, R; Norris, V

    1986-01-01

    Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production. Images PMID:3001034

  19. MHCII-independent CD4+ T cells protect injured CNS neurons via IL-4

    PubMed Central

    Walsh, James T.; Hendrix, Sven; Boato, Francesco; Smirnov, Igor; Zheng, Jingjing; Lukens, John R.; Gadani, Sachin; Hechler, Daniel; Gölz, Greta; Rosenberger, Karen; Kammertöns, Thomas; Vogt, Johannes; Vogelaar, Christina; Siffrin, Volker; Radjavi, Ali; Fernandez-Castaneda, Anthony; Gaultier, Alban; Gold, Ralf; Kanneganti, Thirumala-Devi; Nitsch, Robert; Zipp, Frauke; Kipnis, Jonathan

    2015-01-01

    A body of experimental evidence suggests that T cells mediate neuroprotection following CNS injury; however, the antigen specificity of these T cells and how they mediate neuroprotection are unknown. Here, we have provided evidence that T cell–mediated neuroprotection after CNS injury can occur independently of major histocompatibility class II (MHCII) signaling to T cell receptors (TCRs). Using two murine models of CNS injury, we determined that damage-associated molecular mediators that originate from injured CNS tissue induce a population of neuroprotective, IL-4–producing T cells in an antigen-independent fashion. Compared with wild-type mice, IL-4–deficient animals had decreased functional recovery following CNS injury; however, transfer of CD4+ T cells from wild-type mice, but not from IL-4–deficient mice, enhanced neuronal survival. Using a culture-based system, we determined that T cell–derived IL-4 protects and induces recovery of injured neurons by activation of neuronal IL-4 receptors, which potentiated neurotrophin signaling via the AKT and MAPK pathways. Together, these findings demonstrate that damage-associated molecules from the injured CNS induce a neuroprotective T cell response that is independent of MHCII/TCR interactions and is MyD88 dependent. Moreover, our results indicate that IL-4 mediates neuroprotection and recovery of the injured CNS and suggest that strategies to enhance IL-4–producing CD4+ T cells have potential to attenuate axonal damage in the course of CNS injury in trauma, inflammation, or neurodegeneration. PMID:25607842

  20. Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.

    PubMed

    Bourges, Jean-Louis; Valamanesh, Fatemeh; Torriglia, Alicia; Jeanny, Jean-Claude; Savoldelli, Michèle; Renard, Gilles; BenEzra, David; de Kozak, Yvonne; Behar-Cohen, Francine

    2004-08-15

    To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

  1. L1 cell adhesion molecule as a novel independent poor prognostic factor in gallbladder carcinoma.

    PubMed

    Choi, Song-Yi; Jo, Young Suk; Huang, Song-Mei; Liang, Zhe Long; Min, Jeong-Ki; Hong, Hyo Jeong; Kim, Jin-Man

    2011-10-01

    Gallbladder carcinoma is a lethal malignancy and is hard to cure by current treatment. Thus, identification of molecular prognostic markers to predict gallbladder carcinoma as therapeutic targets is urgently needed. Recent studies have demonstrated that L1 cell adhesion molecule is associated with the prognosis of variable malignancy. Here, we investigated L1 cell adhesion molecule expression in gallbladder carcinoma and its prognostic significance. In this study, we examined L1 cell adhesion molecule expression in tumor specimens from 69 patients with gallbladder carcinoma by immunohistochemistry and analyzed the correlation between L1 cell adhesion molecule expression and clinicopathologic factors or survival. L1 cell adhesion molecule was not expressed in the normal epithelium of the gallbladder but in 63.8% of gallbladder carcinomas, remarkably at the invasive front of the tumors. In addition, L1 cell adhesion molecule expression was significantly associated with high histologic grade, advanced pathologic T stage and clinical stage, and positive venous/lymphatic invasion. Multivariate analyses showed that L1 cell adhesion molecule expression (hazard ratio, 3.503; P = .028) and clinical stage (hazard ratio, 3.091; P = .042) were independent risk factor for disease-free survival. L1 cell adhesion molecule expression in gallbladder carcinoma was significantly correlated with tumor progression and unfavorable clinicopathologic features. L1 cell adhesion molecule expression was an independent poor prognostic factor for disease-free survival in patients with gallbladder carcinoma. Taken together, our findings suggest that L1 cell adhesion molecule expression could be used as a novel prognostic factor for patient survival and might be a potential therapeutic target in gallbladder carcinomas. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Ligand-independent requirements of steroid receptors EcR and USP for cell survival

    PubMed Central

    Mansilla, A; Martín, F A; Martín, D; Ferrús, A

    2016-01-01

    The active form of the Drosophila steroid hormone ecdysone, 20-hydroxyecdysone (20E), binds the heterodimer EcR/USP nuclear receptor to regulate target genes that elicit proliferation, cell death and differentiation during insect development. Although the 20E effects are relatively well known, the physiological relevance of its receptors remains poorly understood. We show here that the prothoracic gland (PG), the major steroid-producing organ of insect larvae, requires EcR and USP to survive in a critical period previous to metamorphosis, and that this requirement is 20E-independent. The cell death induced by the downregulation of these receptors involves the activation of the JNK-encoding basket gene and it can be rescued by upregulating EcR isoforms which are unable to respond to 20E. Also, while PG cell death prevents ecdysone production, blocking hormone synthesis or secretion in normal PG does not lead to cell death, demonstrating further the ecdysone-independent nature of the receptor-deprivation cell death. In contrast to PG cells, wing disc or salivary glands cells do not require these receptors for survival, revealing their cell and developmental time specificity. Exploring the potential use of this feature of steroid receptors in cancer, we assayed tumor overgrowth induced by altered yorkie signaling. This overgrowth is suppressed by EcR downregulation in PG, but not in wing disc, cells. The mechanism of all these cell death features is based on the transcriptional regulation of reaper. These novel and context-dependent functional properties for EcR and USP receptors may help to understand the heterogeneous responses to steroid-based therapies in human pathologies. PMID:26250909

  3. RETRA exerts anticancer activity in Ewing's sarcoma cells independent of their TP53 status.

    PubMed

    Sonnemann, Jürgen; Grauel, Désirée; Blümel, Lisa; Hentschel, Julia; Marx, Christian; Blumrich, Annelie; Focke, Katharina; Becker, Sabine; Wittig, Susan; Schinkel, Sandra; Krämer, Oliver H; Beck, James F

    2015-05-01

    Mutant p53 can exert oncogenic activity by inhibitory interaction with p73. The small-molecule RETRA has been described to disrupt this interaction and to suppress carcinoma cells (Kravchenko et al., 2008). RETRA's anticancer activity was restricted to tumour cells bearing mutant p53; it was not active in p53 negative and in p53 wild-type cells. Here, we explored the responsiveness of Ewing's sarcoma (ES) cells with mutant p53 to RETRA. For comparison, we also tested RETRA in p53 null and in p53 wild-type ES cells. We found RETRA to be effective in the three mutant p53 ES cell lines investigated. Strikingly, however, RETRA was similarly effective in the p53-deficient and in the two p53 wild-type ES cell lines examined. RETRA elicited apoptosis, as assessed by flow cytometric analyses of mitochondrial depolarisation and DNA fragmentation, caspase 3/7 activity assay and PARP-1 cleavage immunodetection, and G2/M cell cycle arrest completely independent of the cellular TP53 status. In contrast, various p53-deficient and -proficient carcinoma, osteosarcoma and leukaemia cells were unresponsive to RETRA. RETRA also induced gene expression of p53 target genes PUMA and p21 in ES cells irrespective of their TP53 status. These in vitro findings provide a rationale for an in vivo exploration of RETRA's potential as an effective therapeutic approach for patients with ES.

  4. Intranasal M cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength.

    PubMed

    Rajapaksa, Thejani E; Bennett, Kaila M; Hamer, Mary; Lytle, Christian; Rodgers, Victor G J; Lo, David D

    2010-07-30

    In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery.

  5. MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism.

    PubMed

    Polański, Radosław; Warburton, Hazel E; Ray-Sinha, Arpita; Devling, Timothy; Pakula, Hubert; Rubbi, Carlos P; Vlatković, Nikolina; Boyd, Mark T

    2010-11-19

    Recent studies connect MDM2 with increased cell motility, invasion and/or metastasis proposing an MDM2-mediated ubiquitylation-dependent mechanism. Interestingly, in renal cell carcinoma (RCC) p53/MDM2 co-expression is associated with reduced survival which is independently linked with metastasis. We therefore investigated whether expression of p53 and/or MDM2 promotes aggressive cell phenotypes. Our data demonstrate that MDM2 promotes increased motility and invasiveness in RCC cells (N.B. similar results are obtained in non-RCC cells). This study shows for the first time both that endogenous MDM2 significantly contributes to cell motility and that this does not depend upon the MDM2 RING-finger, i.e. is independent of ubiquitylation (and NEDDylation). Our data suggest that protein-protein interactions provide a likely mechanistic basis for MDM2-promoted motility which may constitute future therapeutic targets. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Neuronal Transcriptional Repressor REST Suppresses an Atoh7-Independent Program for Initiating Retinal Ganglion Cell Development

    PubMed Central

    Mao, Chai-An; Tsai, Wen-Wei; Cho, Jang-Hyeon; Pan, Ping; Barton, Michelle Craig; Klein, William H.

    2010-01-01

    As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs. PMID:20969844

  7. NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence

    PubMed Central

    Kostadinova, Elena; Chaput, Catherine; Gutbier, Birgitt; Lippmann, Juliane; Sander, Leif E.; Mitchell, Timothy J.; Suttorp, Norbert; Witzenrath, Martin; Opitz, Bastian

    2016-01-01

    Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes. PMID:27476670

  8. The Caenorhabditis elegans pvl-5 gene protects hypodermal cells from ced-3-dependent, ced-4-independent cell death.

    PubMed Central

    Joshi, Pradeep; Eisenmann, David M

    2004-01-01

    Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. Here we describe pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway. PMID:15238520

  9. On How Fas Apoptosis-Independent Pathways Drive T Cell Hyperproliferation and Lymphadenopathy in lpr Mice

    PubMed Central

    Balomenos, Dimitrios; Shokri, Rahman; Daszkiewicz, Lidia; Vázquez-Mateo, Cristina; Martínez-A, Carlos

    2017-01-01

    Fas induces massive apoptosis in T cells after repeated in vitro T cell receptor (TCR) stimulation and is critical for lymphocyte homeostasis in Fas-deficient (lpr) mice. Although the in vitro Fas apoptotic mechanism has been defined, there is a large conceptual gap between this in vitro phenomenon and the pathway that leads to in vivo development of lymphadenopathy and autoimmunity. A striking abnormality in lpr mice is the excessive proliferation of CD4+ and CD8+ T cells, and more so of the double-negative TCR+CD4−CD8−B220+ T cells. The basis of lpr T cell hyperproliferation remains elusive, as it cannot be explained by Fas-deficient apoptosis. T cell-directed p21 overexpression reduces hyperactivation/hyperproliferation of all lpr T cell subtypes and lymphadenopathy in lpr mice. p21 controls expansion of repeatedly stimulated T cells without affecting apoptosis. These results confirm a direct link between hyperactivation/hyperproliferation, autoreactivity, and lymphadenopathy in lpr mice and, with earlier studies, suggest that Fas apoptosis-independent pathways control lpr T cell hyperproliferation. lpr T cell hyperproliferation could be an indirect result of the defective apoptosis of repeatedly stimulated lpr T cells. Nonetheless, in this perspective, we argue for an alternative setting, in which lack of Fas would directly cause lpr T cell hyperactivation/hyperproliferation in vivo. We propose that Fas/Fas ligand (FasL) acts as an activation inhibitor of recurrently stimulated T cells, and that its disruption causes overexpansion of T cells in lpr mice. Research to define the underlying mechanism of this Fas/FasL effect could resolve the phenotype of lpr mice and lead to therapeutics for related human syndromes. PMID:28344578

  10. On How Fas Apoptosis-Independent Pathways Drive T Cell Hyperproliferation and Lymphadenopathy in lpr Mice.

    PubMed

    Balomenos, Dimitrios; Shokri, Rahman; Daszkiewicz, Lidia; Vázquez-Mateo, Cristina; Martínez-A, Carlos

    2017-01-01

    Fas induces massive apoptosis in T cells after repeated in vitro T cell receptor (TCR) stimulation and is critical for lymphocyte homeostasis in Fas-deficient (lpr) mice. Although the in vitro Fas apoptotic mechanism has been defined, there is a large conceptual gap between this in vitro phenomenon and the pathway that leads to in vivo development of lymphadenopathy and autoimmunity. A striking abnormality in lpr mice is the excessive proliferation of CD4(+) and CD8(+) T cells, and more so of the double-negative TCR(+)CD4(-)CD8(-)B220(+) T cells. The basis of lpr T cell hyperproliferation remains elusive, as it cannot be explained by Fas-deficient apoptosis. T cell-directed p21 overexpression reduces hyperactivation/hyperproliferation of all lpr T cell subtypes and lymphadenopathy in lpr mice. p21 controls expansion of repeatedly stimulated T cells without affecting apoptosis. These results confirm a direct link between hyperactivation/hyperproliferation, autoreactivity, and lymphadenopathy in lpr mice and, with earlier studies, suggest that Fas apoptosis-independent pathways control lpr T cell hyperproliferation. lpr T cell hyperproliferation could be an indirect result of the defective apoptosis of repeatedly stimulated lpr T cells. Nonetheless, in this perspective, we argue for an alternative setting, in which lack of Fas would directly cause lpr T cell hyperactivation/hyperproliferation in vivo. We propose that Fas/Fas ligand (FasL) acts as an activation inhibitor of recurrently stimulated T cells, and that its disruption causes overexpansion of T cells in lpr mice. Research to define the underlying mechanism of this Fas/FasL effect could resolve the phenotype of lpr mice and lead to therapeutics for related human syndromes.

  11. RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

    PubMed Central

    1990-01-01

    Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with

  12. Zinc induces apoptosis on cervical carcinoma cells by p53-dependent and -independent pathway.

    PubMed

    Bae, Seog Nyeon; Lee, Keun Ho; Kim, Jin Hwi; Lee, Sung Jong; Park, Lae Ok

    2017-02-26

    There is evidence that the mineral zinc is involved in the apoptotic cell death of various carcinoma cells. In this study, we aim to determine whether zinc in the form of CIZAR induces apoptosis in cervical carcinoma cells by increasing intracellular zinc concentration. CaSki and HeLa cervical carcinoma cells and HPV-16 DNA-transformed keratinocyte (CRL2404) were treated with different concentrations of CIZAR. The cell viability test was carried out, the intracellular level of zinc was determined, and apoptosis was confirmed by flow cytometry after propidium iodide (PI) staining and fluorescence microscopy under DAPI staining. The expression of cell-cycle regulators was analyzed by Western blot, including the knock down of p53 and expression of HPV E6 and E7 genes by RT-PCR. Intracellular zinc accumulation induced the down-regulation of E6/E7 proteins through targeting of the specific transcriptional factors in the upstream regulatory region. p53 was induced after CIZAR treatment and p53-dependent apoptosis did not occur after knock down by p53 siRNA. In cervical carcinoma cells, regardless of HPV-infection, CIZAR induces apoptosis by the activation of the p53-independent pathways through the up-regulation of p21waf1, the down-regulation of c-Myc, and by decreasing the Bcl-2/Bax ratio. CIZAR induces apoptosis not only through the restoration of p53/Rb-dependent pathways in HPV-positive cells, but also through the activation of p53/Rb-independent pathways and the mitochondrial death-signal pathway in cervical carcinoma cells regardless of HPV-infection. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. PPARγ-inactive Δ2-troglitazone independently triggers ER stress and apoptosis in breast cancer cells.

    PubMed

    Colin-Cassin, Christelle; Yao, Xiao; Cerella, Claudia; Chbicheb, Sarra; Kuntz, Sandra; Mazerbourg, Sabine; Boisbrun, Michel; Chapleur, Yves; Diederich, Marc; Flament, Stephane; Grillier-Vuissoz, Isabelle

    2015-05-01

    Our aim was to better understand peroxisome proliferator-activated receptor gamma (PPARγ)-independent pathways involved in anti-cancer effects of thiazolidinediones (TZDs). We focused on Δ2-troglitazone (Δ2-TGZ), a PPARγ inactive TZD that affects breast cancer cell viability. Appearance of TUNEL positive cells, changes in mitochondrial membrane potential, cleavage of poly(ADP-ribose) polymerase (PARP)-1 and caspase-7 revealed that apoptosis occurred in both hormone-dependent MCF7 and hormone-independent MDA-MB-231 breast cancer cells after 24 and 48 h of treatment. A microarray study identified endoplasmic reticulum (ER) stress as an essential cellular function since many genes involved in ER stress were upregulated in MCF7 cells following Δ2-TGZ treatment. Δ2-TGZ-induced ER stress was further confirmed in MCF7 cells by phosphorylation of pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase (PERK) and its target eIF2α after 1.5 h, rapid increase in activating transcription factor (ATF) 3 mRNA levels, splicing of X-box binding protein 1 (XBP1) after 3 h, accumulation of binding immunogloblulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) after 6 h. Immunofluorescence microscopy indicated that CHOP was relocalized to the nucleus of treated cells. Similarly, in MDA-MB-231 cells, overexpression of ATF3, splicing of XBP1, and accumulation of BiP and CHOP were observed following Δ2-TGZ treatment. In MCF7 cells, knock-down of CHOP or the inhibition of c-Jun N-terminal kinase (JNK) did not impair cleavage of PARP-1 and caspase-7. Altogether, our results show that ER stress is an early response of major types of breast cancer cells to Δ2-TGZ, prior to, but not causative of apoptosis. © 2013 Wiley Periodicals, Inc.

  14. EGFR-Dependent Regulation of Matrix-Independent Epithelial Cell Survival. Addendum

    DTIC Science & Technology

    2007-04-01

    C-Sixty Inc.) in a novel in vivo vertebrate systen (zebrafish embryos). This work has been since been published in Clinical Cancer Research...32), and, as shown here, squamous cell carcinomas. In summary, these results establish that deregulated NF-nB signaling exerts powerful oncogenic...independent conditions in liquid media. This technique has proven to be a power - ful tool in studying the effects of loss of extracellular matrix

  15. Programmed cell death of retinal cone bipolar cells is independent of afferent or target control.

    PubMed

    Keeley, Patrick W; Madsen, Nils R; St John, Ace J; Reese, Benjamin E

    2014-10-15

    Programmed cell death contributes to the histogenesis of the nervous system, and is believed to be modulated through the sustaining effects of afferents and targets during the period of synaptogenesis. Cone bipolar cells undergo programmed cell death during development, and we confirm that the numbers of three different types are increased when the pro-apoptotic Bax gene is knocked out. When their cone afferents are selectively eliminated, or when the population of retinal ganglion cells is increased, however, cone bipolar cell number remains unchanged. Programmed cell death of the cone bipolar cell populations, therefore, may be modulated cell-intrinsically rather than via interactions with these synaptic partners. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts.

    PubMed

    Gonçalves, N J N; Bressan, F F; Roballo, K C S; Meirelles, F V; Xavier, P L P; Fukumasu, H; Williams, C; Breen, M; Koh, S; Sper, R; Piedrahita, J; Ambrósio, C E

    2017-04-01

    Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.

  17. Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene.

    PubMed Central

    Del Bufalo, D; Biroccio, A; Soddu, S; Laudonio, N; D'Angelo, C; Sacchi, A; Zupi, G

    1996-01-01

    Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentation. Moreover, we find that apoptosis is preceded by accumulation of the cells in the G0/G1 phase of the cell cycle. Interestingly, lonidamine fails to activate the apoptotic program in the corresponding sensitive parental cell lines (ADR-sensitive MCF-7 WT, and nitrosourea-sensitive LI cells) even after long exposure times. The evaluation of bcl-2 protein expression suggests that this different effect of lonidamine treatment in drug-resistant and -sensitive cell lines might not simply be due to dissimilar expression levels of bcl-2 protein. To determine whether the lonidamine-induced apoptosis is mediated by p53 protein, we used cells lacking endogenous p53 and overexpressing either wild-type p53 or dominant-negative p53 mutant. We find that apoptosis by lonidamine is independent of the p53 gene. PMID:8787680

  18. Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.

    PubMed

    Lambrechts, Dennis; Roeffaers, Maarten; Kerckhofs, Greet; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-09-01

    By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.

  19. Autophagy-independent functions of UVRAG are essential for peripheral naive T-cell homeostasis

    PubMed Central

    Afzal, Samia; Hao, Zhenyue; Itsumi, Momoe; Abouelkheer, Yasser; Brenner, Dirk; Gao, Yunfei; Wakeham, Andrew; Hong, Claire; Li, Wanda Y.; Sylvester, Jennifer; Gilani, Syed O.; Brüstle, Anne; Haight, Jillian; You-Ten, Annick J.; Lin, Gloria H. Y.; Inoue, Satoshi; Mak, Tak W.

    2015-01-01

    UV radiation resistance-associated gene (UVRAG) encodes a tumor suppressor with putative roles in autophagy, endocytic trafficking, and DNA damage repair but its in vivo role in T cells is unknown. Because conditional homozygous deletion of Uvrag in mice results in early embryonic lethality, we generated T-cell–specific UVRAG-deficient mice that lacked UVRAG expression specifically in T cells. This loss of UVRAG led to defects in peripheral homeostasis that could not be explained by the increased sensitivity to cell death and impaired proliferation observed for other autophagy-related gene knockout mice. Instead, UVRAG-deficient T-cells exhibited normal mitochondrial clearance and activation-induced autophagy, suggesting that UVRAG has an autophagy-independent role that is critical for peripheral naive T-cell homeostatic proliferation. In vivo, T-cell–specific loss of UVRAG dampened CD8+ T-cell responses to LCMV infection in mice, delayed viral clearance, and impaired memory T-cell generation. Our data provide novel insights into the control of autophagy in T cells and identify UVRAG as a new regulator of naïve peripheral T-cell homeostasis. PMID:25583492

  20. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    SciTech Connect

    Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  1. Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis.

    PubMed

    Oyallon, Justine; Vanzo, Nathalie; Krzemień, Joanna; Morin-Poulard, Ismaël; Vincent, Alain; Crozatier, Michèle

    2016-01-01

    Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis.

  2. Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis

    PubMed Central

    Krzemień, Joanna; Morin-Poulard, Ismaël; Vincent, Alain; Crozatier, Michèle

    2016-01-01

    Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis. PMID:26866694

  3. Storage media enhance osteoclastogenic potential of human periodontal ligament cells via RANKL-independent signaling.

    PubMed

    Zhan, Xuan; Zhang, Chengfei; Dissanayaka, Waruna L; Cheung, Gary S P; Jin, Lijian; Yang, Yangqi; Yan, Fuhua; Tong, Edith H Y

    2013-02-01

    Hank's balanced salt solution (HBSS) and milk have gained wide acceptance as storage media for avulsed tooth. However, the effect of the media and storage time on the periodontal ligament (PDL) cells involvement in the development of root resorption is still unclear. The purpose of this study was to evaluate whether precultured PDL cells in HBSS, milk, or modified Eagle's medium alpha (α-MEM) would affect osteoclastogenesis. PDL cells were precultured in HBSS, milk, or α-MEM for 1 h or 6 h before being co-cultured with RAW 264.7 cells for an additional 3 days for mRNA analysis and 11 days for osteoclastogenesis assay. Cyclooxygenase-2 (COX-2) mRNA was detected immediately in PDL cells precultured in the three storage media. The expression was up-regulated markedly in all co-cultures when compared with RAW cells alone. As a result of the co-culture, interleukin-1β (IL-1β) expression was detectable in both PDL and RAW cells. TRAP+ multinucleated, osteoclast-like cells developed in all co-cultures; the number of TRAP+ cells was highest (P < 0.05) in the co-cultures that PDL cells precultured in milk for 6 h. The mRNA level of receptor activator of nuclear factor-kappa B ligand (RANKL) was not detected in PDL cells. Osteoprotegerin (OPG) mRNA expression reduced with increased preculture time, but the difference was not significant (P > 0.05). PDL cells kept in the three storage media led to TRAP+ multinucleated, osteoclast-like cells formation via RANKL-independent signaling. The ability to induce osteoclastogenesis may be considered as one of the factors to evaluate the ability of storage medium to maintain PDL viability after tooth avulsion. © 2012 John Wiley & Sons A/S.

  4. BGP-15 inhibits caspase-independent programmed cell death in acetaminophen-induced liver injury

    SciTech Connect

    Nagy, Gabor; Szarka, Andras; Lotz, Gabor; Doczi, Judit; Wunderlich, Livius; Kiss, Andras; Jemnitz, Katalin; Veres, Zsuzsa; Banhegyi, Gabor; Schaff, Zsuzsa; Suemegi, Balazs; Mandl, Jozsef

    2010-02-15

    It has been recently shown that acute acetaminophen toxicity results in endoplasmic reticulum redox stress and an increase in cells with apoptotic phenotype in liver. Since activation of effector caspases was absent, the relevance of caspase-independent mechanisms in acetaminophen-induced programmed cell death was investigated. BGP-15, a drug with known protective actions in conditions involving redox imbalance, has been co-administered with a single sublethal dose of acetaminophen. Proapoptotic events and outcome of the injury were investigated. ER redox alterations and early ER-stress-related signaling events induced by acetaminophen, such as ER glutathione depletion, phosphorylation of eIF2alpha and JNK and induction of the transcription factor GADD153, were not counteracted by co-treatment with BGP-15. However, BGP-15 prevented AIF mitochondria-to-nucleus translocation and mitochondrial depolarization. BGP-15 co-treatment attenuated the rate of acetaminophen-induced cell death as assessed by apoptotic index and enzyme serum release. These results reaffirm that acute acetaminophen toxicity involves oxidative stress-induced caspase-independent cell death. In addition, pharmacological inhibition of AIF translocation may effectively protect against or at least delay acetaminophen-induced programmed cell death.

  5. Engineering interpenetrating network hydrogels as biomimetic cell niche with independently tunable biochemical and mechanical properties.

    PubMed

    Tong, Xinming; Yang, Fan

    2014-02-01

    Hydrogels have been widely used as artificial cell niche to mimic extracellular matrix with tunable properties. However, changing biochemical cues in hydrogels developed-to-date would often induce simultaneous changes in mechanical properties, which do not support mechanistic studies on stem cell-niche interactions. Here we report the development of a PEG-based interpenetrating network (IPN), which is composed of two polymer networks that can independently and simultaneously crosslink to form hydrogels in a cell-friendly manner. The resulting IPN hydrogel allows independently tunable biochemical and mechanical properties, as well as stable and more homogeneous presentation of biochemical ligands in 3D than currently available methods. We demonstrate the potential of our IPN platform for elucidating stem cell-niche interactions by modulating osteogenic differentiation of human adipose-derived stem cells. The versatility of such IPN hydrogels is further demonstrated using three distinct and widely used polymers to form the mechanical network while keeping the biochemical network constant. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Disodium cromoglycate inhibits capsaicin-induced eosinophil infiltration of conjunctiva independent of mast cells.

    PubMed

    Ebihara, Nobuyuki; Nishikawa, Motoaki; Murakami, Akira

    2006-01-01

    To investigate whether disodium cromoglycate (DSCG) inhibits capsaicin-induced eosinophil infiltration of the conjunctiva independent of mast cells. We administered 5 microl of capsaicin solution (10(-5) M) into the conjunctival sacs of mast cell-deficient W/W(v) mice (12 animals) and wild-type mice (12 animals). As controls, the eyes of 12 wild-type and 12 W/W(v) mice were treated with phosphate-buffered saline. Following treatment, the eyelids and eyeballs were removed en bloc at 3, 9, or 24 h, and were histologically examined. The number of infiltrated eosinophils and the expression of vascular cell adhesion molecule-l (VCAM-1) in the conjunctiva were quantified by the staining method of Hansel and immunohistochemical analysis. We also investigated whether treatment by depletion of neuropeptides or by DSCG administration could suppress the capsaicin-induced eosinophil infiltration of the conjunctiva. In both W/W(v) and wild-type mice, eosinophil infiltration of conjunctival tissues was observed 3 h after capsaicin administration. In both strains of mice, the number of infiltrated eosinophils increased over time, with VCAM-1 expression on vascular endothelial cells peaking at 9 h after treatment, and decreasing gradually within 24 h after treatment. In both the neuropeptide-depleted and the DSCG-treated groups, eosinophil infiltration and VCAM-1 expression were suppressed in comparison with the nontreated group. DSCG can directly inhibit neuropeptide-induced eosinophil infiltration of the conjunctiva independent of mast cells.

  7. αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

    PubMed Central

    Benjamin, Jacqueline M.; Kwiatkowski, Adam V.; Yang, Changsong; Korobova, Farida; Pokutta, Sabine; Svitkina, Tatyana

    2010-01-01

    αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion. PMID:20404114

  8. BGP-15 inhibits caspase-independent programmed cell death in acetaminophen-induced liver injury.

    PubMed

    Nagy, Gábor; Szarka, András; Lotz, Gábor; Dóczi, Judit; Wunderlich, Lívius; Kiss, András; Jemnitz, Katalin; Veres, Zsuzsa; Bánhegyi, Gábor; Schaff, Zsuzsa; Sümegi, Balázs; Mandl, József

    2010-02-15

    It has been recently shown that acute acetaminophen toxicity results in endoplasmic reticulum redox stress and an increase in cells with apoptotic phenotype in liver. Since activation of effector caspases was absent, the relevance of caspase-independent mechanisms in acetaminophen-induced programmed cell death was investigated. BGP-15, a drug with known protective actions in conditions involving redox imbalance, has been co-administered with a single sublethal dose of acetaminophen. Proapoptotic events and outcome of the injury were investigated. ER redox alterations and early ER-stress-related signaling events induced by acetaminophen, such as ER glutathione depletion, phosphorylation of eIF2alpha and JNK and induction of the transcription factor GADD153, were not counteracted by co-treatment with BGP-15. However, BGP-15 prevented AIF mitochondria-to-nucleus translocation and mitochondrial depolarization. BGP-15 co-treatment attenuated the rate of acetaminophen-induced cell death as assessed by apoptotic index and enzyme serum release. These results reaffirm that acute acetaminophen toxicity involves oxidative stress-induced caspase-independent cell death. In addition, pharmacological inhibition of AIF translocation may effectively protect against or at least delay acetaminophen-induced programmed cell death. Copyright 2009 Elsevier Inc. All rights reserved.

  9. T-cell stimuli independently sum to regulate an inherited clonal division fate

    PubMed Central

    Marchingo, J. M.; Prevedello, G.; Kan, A.; Heinzel, S.; Hodgkin, P. D.; Duffy, K. R.

    2016-01-01

    In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities. PMID:27869196

  10. Memory CD4+ T cells induce innate responses independently of pathogen.

    PubMed

    Strutt, Tara M; McKinstry, K Kai; Dibble, John P; Winchell, Caylin; Kuang, Yi; Curtis, Jonathan D; Huston, Gail; Dutton, Richard W; Swain, Susan L

    2010-05-01

    Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4(+) T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4(+) T cell-induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (T(H)1) or T(H)17 polarized but are independent of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4(+) T cells induce an early innate response that enhances immune protection against pathogens.

  11. Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells

    PubMed Central

    Sugii, Shigeki; Kida, Yasuyuki; Kawamura, Teruhisa; Suzuki, Jotaro; Vassena, Rita; Yin, Yun-Qiang; Lutz, Margaret K.; Berggren, W. Travis; Izpisúa Belmonte, Juan Carlos; Evans, Ronald M.

    2010-01-01

    Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFβ, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance. PMID:20133714

  12. Fusion-independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells

    PubMed Central

    Grassi, Francesca; Pagani, Francesca; Spinelli, Gabriele; Angelis, Luciana De; Cossu, Giulio; Eusebi, Fabrizio

    2004-01-01

    Mesoangioblasts are vessel-associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)-positive mesoangioblasts co-cultured with differentiating C2C12 myogenic cells, using the patch-clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh-evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT-PCR both in single, mononucleated mesoangioblasts co-cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co-cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co-cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment. PMID:15319417

  13. Caspase-dependent and -independent suppression of apoptosis by monoHER in Doxorubicin treated cells

    PubMed Central

    Bruynzeel, A M E; Abou El Hassan, M A; Torun, E; Bast, A; van der Vijgh, W J F; Kruyt, F A E

    2007-01-01

    Doxorubicin (DOX) is an antitumour agent for different types of cancer, but the dose-related cardiotoxicity limits its clinical use. To prevent this side effect we have developed the flavonoid monohydroxyethylrutoside (monoHER), a promising protective agent, which did not interfere with the antitumour activity of DOX. To obtain more insight in the mechanism underlying the selective protective effects of monoHER, we investigated whether monoHER (1 mM) affects DOX-induced apoptosis in neonatal rat cardiac myocytes (NeRCaMs), human endothelial cells (HUVECs) and the ovarian cancer cell lines A2780 and OVCAR-3. DOX-induced cell death was effectively reduced by monoHER in heart, endothelial and A2780 cells. OVCAR-3 cells were highly resistant to DOX-induced apoptosis. Experiments with the caspase-inhibitor zVAD-fmk showed that DOX-induced apoptosis was caspase-dependent in HUVECs and A2780 cells, whereas caspase-independent mechanisms seem to be important in NeRCaMs. MonoHER suppressed DOX-dependent activation of the mitochondrial apoptotic pathway in normal and A2780 cells as illustrated by p53 accumulation and activation of caspase-9 and -3 cleavage. Thus, monoHER acts by suppressing the activation of molecular mechanisms that mediate either caspase-dependent or -independent cell death. In light of the current work and our previous studies, the use of clinically achievable concentrations of monoHER has no influence on the antitumour activity of DOX whereas higher concentrations as used in the present study could influence the antitumour activity of DOX. PMID:17285121

  14. Lipooligosaccharide-independent alteration of cellular homeostasis in Neisseria meningitidis-infected epithelial cells.

    PubMed

    Bonnah, Robert A; Hoelter, Jenny; Steeghs, Liana; Enns, Caroline A; So, Magdalene; Muckenthaler, Martina U

    2005-06-01

    Neisseria meningitidis (MC) is an important cause of meningitis and septic shock. Primary loose attachment of MC to host epithelial cells is mediated by type IV pili. Lipooligosaccharide (LOS), opacity (Opa) proteins and glycolipid adhesins facilitate subsequent tight attachment. MC infection causes numerous changes in host epithelial cell homeostasis. These include cortical plaque formation, increased expression of proinflammatory cytokines and alterations in host iron homeostasis. Using both biochemical and genetic approaches, we examined the role of LOS in mediating these events. We first examined specific cellular iron homeostasis changes that occur following addition of purified MC LOS to epithelial cells. Using an MC mutant that completely lacks LOS (MC lps tbp), we examined pili-mediated attachment and cortical plaque formation in human endocervical epithelial cells (A431). We also tested whether the lack of LOS alters cellular homeostasis, including changes in the levels of host stress response factors and proinflammatory cytokines. MC lps tbp elicited the formation of cortical plaques in A431 cells. However, the plaques were less pronounced than those formed by the MC parent. Surprisingly, the proinflammatory cytokine TNF(alpha) was upregulated during infection in MC lps tbp-infected cells. Furthermore, alterations in iron homeostasis, including lower transferrin receptor 1 (TfR-1) levels, altered TfR-1 trafficking, an 'iron-starvation' gene expression profile and low iron regulatory protein (IRP) binding activity are independent of LOS. Our results demonstrate that LOS is partially involved in both the attachment to host cells and formation of cortical plaques. However, TNFalpha induction and changes in iron homeostasis observed in MC-infected epithelial cells are independent of LOS.

  15. TNF-α Contributes to Caspase-3 Independent Apoptosis in Neuroblastoma Cells: Role of NFAT

    PubMed Central

    Álvarez, Susana; Blanco, Almudena; Fresno, Manuel; Muñoz-Fernández, Ma Ángeles

    2011-01-01

    There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca2+ independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation. PMID:21298033

  16. Increased Th22 cells are independently associated with Th17 cells in type 1 diabetes.

    PubMed

    Xu, Xinyu; Zheng, Shuai; Yang, Fan; Shi, Yun; Gu, Yong; Chen, Heng; Zhang, Mei; Yang, Tao

    2014-05-01

    Type 1 diabetes (T1D) is perceived as an autoimmune disease caused by T cell-mediated destruction of the insulin-producing pancreatic β cells. However, the number of inflammatory T cells in blood, as well as the relative importance of each cell type is unclear. Forty-two patients with T1D and 30 controls were enrolled. Circulating primary CD4(+) or CD8(+) T cells were quantified with 5-color flow cytometry. Serum IL-22 and IL-17 levels were examined by ELISA. Serum autoantibodies were measured by radio-binding assays, using (35)S-labeled glutamic acid decarboxylase-65 (GAD65), protein tyrosine phosphatase-2 (IA-2), and zinc transporter 8 (ZnT8). Th17-Th22 and Tc1-Tc17 were significantly elevated in patients with T1D compared to control subjects, while there were no significant differences in Th1 cells. The levels of these T cells in different stages of T1D were investigated. Th22 cells showed a positive correlation with Th17 cells in T1D patients. However, we did not find any correlation between IL-17 and IL-22 in sera. Autoantibodies were not significantly different between patients with early T1D and those who have had it for a longer duration. This study indicates that Th22 may contribute to the pathogenesis of T1D. Blockade of Th22 cells might be of clinical profit in T1D patients.

  17. Exposure time independent summary statistics for assessment of drug dependent cell line growth inhibition.

    PubMed

    Falgreen, Steffen; Laursen, Maria Bach; Bødker, Julie Støve; Kjeldsen, Malene Krag; Schmitz, Alexander; Nyegaard, Mette; Johnsen, Hans Erik; Dybkær, Karen; Bøgsted, Martin

    2014-06-05

    In vitro generated dose-response curves of human cancer cell lines are widely used to develop new therapeutics. The curves are summarised by simplified statistics that ignore the conventionally used dose-response curves' dependency on drug exposure time and growth kinetics. This may lead to suboptimal exploitation of data and biased conclusions on the potential of the drug in question. Therefore we set out to improve the dose-response assessments by eliminating the impact of time dependency. First, a mathematical model for drug induced cell growth inhibition was formulated and used to derive novel dose-response curves and improved summary statistics that are independent of time under the proposed model. Next, a statistical analysis workflow for estimating the improved statistics was suggested consisting of 1) nonlinear regression models for estimation of cell counts and doubling times, 2) isotonic regression for modelling the suggested dose-response curves, and 3) resampling based method for assessing variation of the novel summary statistics. We document that conventionally used summary statistics for dose-response experiments depend on time so that fast growing cell lines compared to slowly growing ones are considered overly sensitive. The adequacy of the mathematical model is tested for doxorubicin and found to fit real data to an acceptable degree. Dose-response data from the NCI60 drug screen were used to illustrate the time dependency and demonstrate an adjustment correcting for it. The applicability of the workflow was illustrated by simulation and application on a doxorubicin growth inhibition screen. The simulations show that under the proposed mathematical model the suggested statistical workflow results in unbiased estimates of the time independent summary statistics. Variance estimates of the novel summary statistics are used to conclude that the doxorubicin screen covers a significant diverse range of responses ensuring it is useful for biological

  18. Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

    SciTech Connect

    Kim, Yong Chan; Song, Seok Bean; Lee, Mi Hee; Kang, Kwang Il; Lee, Hayyoung; Paik, Sang-Gi; Kim, Kyoon Eon; Kim, Young Sang . E-mail: young@cnu.ac.kr

    2006-01-20

    Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.

  19. PTEN and TRP53 independently suppress Nanog expression in spermatogonial stem cells.

    PubMed

    Kuijk, Ewart W; van Mil, Alain; Brinkhof, Bas; Penning, Louis C; Colenbrander, Ben; Roelen, Bernard A J

    2010-07-01

    Mammalian spermatogonial stem cells are a special type of adult stem cells because they can contribute to the next generation. Knockout studies have indicated a role for TRP53 and PTEN in insulating male germ cells from pluripotency, but the mechanism by which this is achieved is largely unknown. To get more insight in these processes, an RNAi experiment was performed on the mouse spermatogonial stem cell line GSDG1. Lipofectaminemediated transfection of siRNAs directed against Trp53 and Pten resulted in decreased expression levels as determined by quantitative RT-PCR and immunoblotting. The effects of knockdown were examined by determining the expression levels of genes that are involved in reprogramming and pluripotency of cells, specifically Nanog, Eras, c-Myc, Klf4, Oct4, and Sox2. Additionally, the effects of TRP53 or PTEN knockdown on Plzf and Ddx4 expression were measured, which are highly expressed in spermatogonial stem cells and differentiating male germ cells, respectively. The main finding of this study is that knockdown of Trp53 and Pten independently resulted in significantly higher expression levels of the pluripotency-associated gene Nanog, and we hypothesize that TRP53 and PTEN mediated repression is important for the insulation of male germ cells from pluripotency.

  20. Systematic analysis of tumour cell-extracellular matrix adhesion identifies independent prognostic factors in breast cancer

    PubMed Central

    Wong, Jocelyn P.; Natrajan, Rachael C.; Yuan, Yinyin; Tan, Aik-Choon; Huang, Paul H.

    2016-01-01

    Tumour cell-extracellular matrix (ECM) interactions are fundamental for discrete steps in breast cancer progression. In particular, cancer cell adhesion to ECM proteins present in the microenvironment is critical for accelerating tumour growth and facilitating metastatic spread. To assess the utility of tumour cell-ECM adhesion as a means for discovering prognostic factors in breast cancer survival, here we perform a systematic phenotypic screen and characterise the adhesion properties of a panel of human HER2 amplified breast cancer cell lines across six ECM proteins commonly deregulated in breast cancer. We determine a gene expression signature that defines a subset of cell lines displaying impaired adhesion to laminin. Cells with impaired laminin adhesion showed an enrichment in genes associated with cell motility and molecular pathways linked to cytokine signalling and inflammation. Evaluation of this gene set in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort of 1,964 patients identifies the F12 and STC2 genes as independent prognostic factors for overall survival in breast cancer. Our study demonstrates the potential of in vitro cell adhesion screens as a novel approach for identifying prognostic factors for disease outcome. PMID:27556857

  1. DNA-binding independent cell death from a minimal proapoptotic region of E2F-1.

    PubMed

    Bell, L A; O'Prey, J; Ryan, K M

    2006-09-14

    The ability to induce cell cycle progression while evading cell death is a defining characteristic of cancer. Deregulation of E2F is a common event in most human cancers. Paradoxically, this can lead to both cell cycle progression and apoptosis. Although the way in which E2F causes cell cycle progression is well characterized, the pathways by which E2F induces cell death are less well defined. Many of the known mechanisms through which E2F induces apoptosis occur through regulation of E2F target genes. However, mutants of E2F-1 that lack the transactivation domain are still able to induce cell death. To further investigate this activity, we refined a transactivation independent mutant to identify a minimal apoptotic domain. This revealed that only 75 amino acids from within the DNA-binding domain of E2F-1 is sufficient for cell death and that this activity is also present in the DNA-binding domains of E2F-2 and E2F-3. However, analysis of this domain from E2F-1 revealed it does not bind DNA and is consequently unable to transactivate, repress or de-repress E2F target genes. This provocative observation therefore defines a potential new mechanism of death from E2F and opens up new opportunities for inducing cell death in tumours for therapeutic gain.

  2. CAML mediates survival of Myc-induced lymphoma cells independent of tail-anchored protein insertion.

    PubMed

    Shing, Jennifer C; Lindquist, Lonn D; Borgese, Nica; Bram, Richard J

    2017-01-01

    Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions, along with WRB and TRC40, to mediate tail-anchored (TA) protein insertion into the ER membrane. Physiologic roles for CAML include endocytic trafficking, intracellular calcium signaling, and the survival and proliferation of specialized immune cells, recently attributed to its requirement for TA protein insertion. To identify a possible role for CAML in cancer cells, we generated Eμ-Myc transgenic mice that carry a tamoxifen-inducible deletion allele of Caml. In multiple B-cell lymphoma cell lines derived from these mice, homozygous loss of Caml activated apoptosis. Cell death was blocked by Bcl-2/Bcl-xL overexpression; however, rescue from apoptosis was insufficient to restore proliferation. Tumors established from an Eμ-Myc lymphoma cell line completely regressed after tamoxifen administration, suggesting that CAML is also required for these cancer cells to survive and grow in vivo. Cell cycle analyses of Caml-deleted lymphoma cells revealed an arrest in G2/M, accompanied by low expression of the mitotic marker, phospho-histone H3 (Ser10). Surprisingly, lymphoma cell viability did not depend on the domain of CAML required for its interaction with TRC40. Furthermore, a small protein fragment consisting of the C-terminal 111 amino acid residues of CAML, encompassing the WRB-binding domain, was sufficient to rescue growth and survival of Caml-deleted lymphoma cells. Critically, this minimal region of CAML did not restore TA protein insertion in knockout cells. Taken together, these data reveal an essential role for CAML in supporting survival and mitotic progression in Myc-driven lymphomas that is independent of its TA protein insertion function.

  3. CAML mediates survival of Myc-induced lymphoma cells independent of tail-anchored protein insertion

    PubMed Central

    Shing, Jennifer C; Lindquist, Lonn D; Borgese, Nica; Bram, Richard J

    2017-01-01

    Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions, along with WRB and TRC40, to mediate tail-anchored (TA) protein insertion into the ER membrane. Physiologic roles for CAML include endocytic trafficking, intracellular calcium signaling, and the survival and proliferation of specialized immune cells, recently attributed to its requirement for TA protein insertion. To identify a possible role for CAML in cancer cells, we generated Eμ-Myc transgenic mice that carry a tamoxifen-inducible deletion allele of Caml. In multiple B-cell lymphoma cell lines derived from these mice, homozygous loss of Caml activated apoptosis. Cell death was blocked by Bcl-2/Bcl-xL overexpression; however, rescue from apoptosis was insufficient to restore proliferation. Tumors established from an Eμ-Myc lymphoma cell line completely regressed after tamoxifen administration, suggesting that CAML is also required for these cancer cells to survive and grow in vivo. Cell cycle analyses of Caml-deleted lymphoma cells revealed an arrest in G2/M, accompanied by low expression of the mitotic marker, phospho-histone H3 (Ser10). Surprisingly, lymphoma cell viability did not depend on the domain of CAML required for its interaction with TRC40. Furthermore, a small protein fragment consisting of the C-terminal 111 amino acid residues of CAML, encompassing the WRB-binding domain, was sufficient to rescue growth and survival of Caml-deleted lymphoma cells. Critically, this minimal region of CAML did not restore TA protein insertion in knockout cells. Taken together, these data reveal an essential role for CAML in supporting survival and mitotic progression in Myc-driven lymphomas that is independent of its TA protein insertion function. PMID:28580168

  4. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression.

    PubMed

    Theerakitthanakul, Korkiat; Khrueathong, Jeerasak; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression.

  5. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

    PubMed Central

    Theerakitthanakul, Korkiat; Saetang, Jirakrit; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression. PMID:27698927

  6. Marker-independent Method for Isolating Slow-Dividing Cancer Stem Cells in Human Glioblastoma12

    PubMed Central

    Richichi, Cristina; Brescia, Paola; Alberizzi, Valeria; Fornasari, Lorenzo; Pelicci, Giuliana

    2013-01-01

    Glioblastoma (GBM) is a devastating brain tumor with a poor survival outcome. It is generated and propagated by a small subpopulation of rare and hierarchically organized cells that share stem-like features with normal stem cells but, however, appear dysregulated in terms of self-renewal and proliferation and aberrantly differentiate into cells forming the bulk of the disorganized cancer tissues. The complexity and heterogeneity of human GBMs underlie the lack of standardized and effective treatments. This study is based on the assumption that available markers defining cancer stem cells (CSCs) in all GBMs are not conclusive and further work is required to identify the CSC. We implemented a method to isolate CSCs independently from cell surface markers: four patient-derived GBM neurospheres containing stem, progenitors, and differentiated cells were labeled with PKH-26 fluorescent dye that reliably selects for cells that divide at low rate. Through in vitro and in vivo assays, we investigated the growth and self-renewal properties of the two different compartments of high- and slow-dividing cells. Our data demonstrate that only slow-dividing cells retain the ability of a long-lasting self-renewal capacity after serial in vitro passaging, while high-dividing cells eventually exhaust. Moreover, orthotopic transplantation assay revealed that the incidence of tumors generated by the slow-dividing compartment is significantly higher in the four patient-derived GBM neurospheres analyzed. Importantly, slow-dividing cells feature a population made up of homogeneous stem cells that sustain tumor growth and therefore represent a viable target for GBM therapy development. PMID:23814495

  7. Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent

    PubMed Central

    1994-01-01

    Directional cellular locomotion is thought to involve localized intracellular calcium changes and the lateral transport of cell surface molecules. We have examined the roles of both calcium and cell surface glycoprotein redistribution in the directional migration of two murine fibroblastic cell lines, NIH 3T3 and SV101. These cell types exhibit persistent, cathode directed motility when exposed to direct current electric fields. Using time lapse phase contrast microscopy and image analysis, we have determined that electric field-directed locomotion in each cell type is a calcium independent process. Both exhibit cathode directed motility in the absence of extracellular calcium, and electric fields cause no detectable elevations or gradients of cytosolic free calcium. We find evidence suggesting that galvanotaxis in these cells involves the lateral redistribution of plasma membrane glycoproteins. Electric fields cause the lateral migration of plasma membrane concanavalin A receptors toward the cathode in both NIH 3T3 and SV101 fibroblasts. Exposure of directionally migrating cells to Con A inhibits the normal change of cell direction following a reversal of electric field polarity. Additionally, when cells are plated on Con A- coated substrata so that Con A receptors mediate cell-substratum adhesion, cathode-directed locomotion and a cathodal accumulation of Con A receptors are observed. Immunofluorescent labeling of the fibronectin receptor in NIH 3T3 fibroblasts suggests the recruitment of integrins from large clusters to form a more diffuse distribution toward the cathode in field-treated cells. Our results indicate that the mechanism of electric field directed locomotion in NIH 3T3 and SV101 fibroblasts involves the lateral redistribution of plasma membrane glycoproteins involved in cell-substratum adhesion. PMID:7929557

  8. Cell speed is independent of force in a mathematical model of amoeboidal cell motion with random switching terms.

    PubMed

    Dallon, J C; Evans, E J; Grant, Christopher P; Smith, W V

    2013-11-01

    In this paper the motion of a single cell is modeled as a nucleus and multiple integrin based adhesion sites. Numerical simulations and analysis of the model indicate that when the stochastic nature of the adhesion sites is a memoryless and force independent random process, the cell speed is independent of the force these adhesion sites exert on the cell. Furthermore, understanding the dynamics of the attachment and detachment of the adhesion sites is key to predicting cell speed. We introduce a differential equation describing the cell motion and then introduce a conjecture about the expected drift of the cell, the expected average velocity relation conjecture. Using Markov chain theory, we analyze our conjecture in the context of a related (but simpler) model of cell motion, and then numerically compare the results for the simpler model and the full differential equation model. We also heuristically describe the relationship between the simplified and full models as well as provide a discussion of the biological significance of these results. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase.

    PubMed

    Cingolani, Francesca; Simbari, Fabio; Abad, Jose Luis; Casasampere, Mireia; Fabrias, Gemma; Futerman, Anthony H; Casas, Josefina

    2017-08-01

    Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  10. Ultraviolet-induced cell death is independent of DNA replication in rat kangaroo cells.

    PubMed

    Miyaji, E N; Menck, C F

    1995-05-01

    Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained as for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication.

  11. Insulin-Like Growth Factor-1 Controls Type 2 T Cell-Independent B Cell Response

    DTIC Science & Technology

    2005-01-01

    cells with anti-CD43 Ab coupled to magnetic beads and MACS columns ( Miltenyi Biotec) as described previously (34), plated on 96-well plates at 105 cells...CD86 in re- sponse to B cell activation. Cross-linking of IgM, CD40 (Ab-me- diated), or CD14 (with LPS from E. coli) led to increased expres- sion of

  12. cGMP-independent nitric oxide signaling and regulation of the cell cycle

    PubMed Central

    Cui, Xiaolin; Zhang, Jianhua; Ma, Penglin; Myers, Daniela E; Goldberg, Ilana G; Sittler, Kelly J; Barb, Jennifer J; Munson, Peter J; Cintron, Ana del Pilar; McCoy, J Philip; Wang, Shuibang; Danner, Robert L

    2005-01-01

    Background Regulatory functions of nitric oxide (NO•) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO• on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO• donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO•-induced decreases in cAMP. Results NO• regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO•, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO• and Bt2cAMP effects showed that NO• regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO• annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO•, as was E2F1 binding to E2F promoter elements. NO• activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M. Conclusion NO• coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO• may rely substantially on cGMP-independent

  13. Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle.

    PubMed

    Orisaka, Makoto; Mizutani, Tetsuya; Tajima, Kimihisa; Orisaka, Sanae; Shukunami, Ken-ichi; Miyamoto, Kaoru; Kotsuji, Fumikazu

    2006-06-01

    We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle. Copyright 2006 Wiley-Liss, Inc.

  14. Kremen1 and Dickkopf1 control cell survival in a Wnt-independent manner

    PubMed Central

    Causeret, F; Sumia, I; Pierani, A

    2016-01-01

    In multicellular organisms, a tight control of cell death is required to ensure normal development and tissue homeostasis. Improper function of apoptotic or survival pathways can not only affect developmental programs but also favor cancer progression. Here we describe a novel apoptotic signaling pathway involving the transmembrane receptor Kremen1 and its ligand, the Wnt-antagonist Dickkopf1. Using a whole embryo culture system, we first show that Dickkopf1 treatment promotes cell survival in a mouse model exhibiting increased apoptosis in the developing neural plate. Remarkably, this effect was not recapitulated by chemical Wnt inhibition. We then show that Dickkopf1 receptor Kremen1 is a bona fide dependence receptor, triggering cell death unless bound to its ligand. We performed Wnt-activity assays to demonstrate that the pro-apoptotic and anti-Wnt functions mediated by Kremen1 are strictly independent. Furthermore, we combined phylogenetic and mutagenesis approaches to identify a specific motif in the cytoplasmic tail of Kremen1, which is (i) specifically conserved in the lineage of placental mammals and (ii) strictly required for apoptosis induction. Finally, we show that somatic mutations of kremen1 found in human cancers can affect its pro-apoptotic activity, supporting a tumor suppressor function. Our findings thus reveal a new Wnt-independent function for Kremen1 and Dickkopf1 in the regulation of cell survival with potential implications in cancer therapies. PMID:26206087

  15. Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells

    PubMed Central

    Liu, Tong; Brouha, Brook; Grossman, Douglas

    2008-01-01

    The inhibitor of apoptosis (IAP) protein Survivin is expressed in most cancers and is a key factor in maintaining apoptosis resistance. Although several IAPs have been shown to act as direct inhibitors of caspases, the precise antiapoptotic function of Survivin remains controversial. To clarify the mechanism by which Survivin protects cells, we investigated the kinetics of apoptosis and apoptotic events following Survivin inhibition utilizing a melanoma cell line harboring a tetracycline-regulated Survivin dominant-negative mutant (Survivin-T34A). Blocking Survivin resulted in both caspase activation and apoptosis; however, the level of apoptosis was only partially reduced by caspase inhibition. Survivin blockade also resulted in mitochondrial events that preceded caspase activation, including depolarization and release of cytochrome c and Smac/DIABLO. Levels of other IAPs were not altered in Survivin-targeted cells, although modest cleavage of XIAP and Livin was observed. The earliest proapoptotic event observed in Survivin-targeted cells was nuclear translocation of mitochondrial apoptosis-inducing factor (AIF), known to trigger both apoptotic mitochondrial events and caspase-independent DNA fragmentation. These findings suggest that a key anti-apoptotic function of Survivin relates to inhibition of mitochondrial and AIF-dependent apoptotic pathways, and its expression in melanoma and other cancers likely protects against both caspase-independent and -dependent apoptosis. PMID:14712209

  16. A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization.

    PubMed

    Dos-Santos, Carlos M; de Souza, Daniel G; Balsanelli, Eduardo; Cruz, Leonardo Magalhães; de Souza, Emanuel M; Baldani, José I; Schwab, Stefan

    2017-08-01

    Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.

  17. EpCAM-independent capture of circulating tumor cells with a 'universal CTC-chip'.

    PubMed

    Chikaishi, Yasuhiro; Yoneda, Kazue; Ohnaga, Takashi; Tanaka, Fumihiro

    2017-01-01

    Capture of circulating tumor cells (CTCs), which are shed from the primary tumor site and circulate in the blood, remains a technical challenge. CellSearch® is the only clinically approved CTC detection system, but has provided only modest sensitivity in detecting CTCs mainly because epithelial cell adhesion molecule (EpCAM)-negative tumor cells may not be captured. To achieve more sensitive CTC‑capture, we have developed a novel microfluidic platform, a 'CTC-chip' comprised of light-curable resins that has a unique advantage in that any capture antibody is easily conjugated. In the present study, we showed that EpCAM-negative tumor cells as well as EpCAM-positive cells were captured with the novel 'universal CTC-chip' as follows: i) human lung cancer cells (PC-9), with strong EpCAM expression, were efficiently captured with the CTC-chip coated with an anti-EpCAM antibody (with an average capture efficiency of 101% when tumor cells were spiked in phosphate‑buffered saline (PBS) and 88% when spiked in blood); ii) human mesothelioma cells (ACC-MESO-4), with no EpCAM expression but with podoplanin expression, were captured with the CTC-chip coated with an anti-podoplanin antibody (average capture efficiency of 78% when tumor cells were spiked in PBS and 38% when spiked in blood), whereas ACC-MESO-4 cells were not captured with the CTC-chip coated with the anti-EpCAM antibody. These results indicate that the novel 'CTC-chip' can be useful in sensitive EpCAM-independent detection of CTCs, which may provide new insights into personalized medicine.

  18. T Cell Receptor-Independent Basal Signaling via Erk and Abl Kinases Suppresses RAG Gene Expression

    PubMed Central

    Roose, Jeroen P; Diehn, Maximilian; Tomlinson, Michael G; Lin, Joseph; Alizadeh, Ash A; Botstein, David; Brown, Patrick O

    2003-01-01

    Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation. PMID:14624253

  19. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication.

    PubMed

    Piunti, Andrea; Rossi, Alessandra; Cerutti, Aurora; Albert, Mareike; Jammula, Sriganesh; Scelfo, Andrea; Cedrone, Laura; Fragola, Giulia; Olsson, Linda; Koseki, Haruhiko; Testa, Giuseppe; Casola, Stefano; Helin, Kristian; d'Adda di Fagagna, Fabrizio; Pasini, Diego

    2014-04-14

    The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints.

  20. TGF-{beta}'s delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

    SciTech Connect

    Schabort, Elske J.; Merwe, Mathilde van der; Loos, Benjamin; Moore, Frances P.; Niesler, Carola U.

    2009-02-01

    Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-{beta} (TGF-{beta}) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-{beta} on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-{beta}1, -{beta}2 and -{beta}3 on wound repair in other tissues. In the current study we compared the effect of TGF-{beta}1, -{beta}2 and -{beta}3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-{beta} increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-{beta}1, -{beta}2 and -{beta}3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-{beta} promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner.

  1. Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote.

    PubMed

    Miyagishima, Shin-ya; Fujiwara, Takayuki; Sumiya, Nobuko; Hirooka, Shunsuke; Nakano, Akihiko; Kabeya, Yukihiro; Nakamura, Mami

    2014-05-08

    Circadian rhythms of cell division have been observed in several lineages of eukaryotes, especially photosynthetic unicellular eukaryotes. However, the mechanism underlying the circadian regulation of the cell cycle and the nature of the advantage conferred remain unknown. Here, using the unicellular red alga Cyanidioschyzon merolae, we show that the G1/S regulator RBR-E2F-DP complex links the G1/S transition to circadian rhythms. Time-dependent E2F phosphorylation promotes the G1/S transition during subjective night and this phosphorylation event occurs independently of cell cycle progression, even under continuous dark or when cytosolic translation is inhibited. Constitutive expression of a phospho-mimic of E2F or depletion of RBR unlinks cell cycle progression from circadian rhythms. These transgenic lines are exposed to higher oxidative stress than the wild type. Circadian inhibition of cell cycle progression during the daytime by RBR-E2F-DP pathway likely protects cells from photosynthetic oxidative stress by temporally compartmentalizing photosynthesis and cell cycle progression.

  2. CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

    PubMed Central

    Cao, Y Z; Friedman-Kien, A E; Huang, Y X; Li, X L; Mirabile, M; Moudgil, T; Zucker-Franklin, D; Ho, D D

    1990-01-01

    Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism. Images PMID:2159530

  3. ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells.

    PubMed

    Celi, A; Cianchetti, S; Petruzzelli, S; Carnevali, S; Baliva, F; Giuntini, C

    1999-09-01

    Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 +/- 3 to 49 +/- 7% (SE). A significant increase from 17 +/- 4 to 39 +/- 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin beta-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

  4. Bile acids induce apoptosis selectively in androgen-dependent and -independent prostate cancer cells

    PubMed Central

    Goldberg, Alexander A.; Titorenko, Vladimir I.; Beach, Adam

    2013-01-01

    Prostate cancer is a prevalent age-related disease in North America, accounting for about 15% of all diagnosed cancers. We have previously identified lithocholic acid (LCA) as a potential chemotherapeutic compound that selectively kills neuroblastoma cells while sparing normal human neurons. Now, we report that LCA inhibits the proliferation of androgen-dependent (AD) LNCaP prostate cancer cells and that LCA is the most potent bile acid with respect to inducing apoptosis in LNCaP as well as androgen-independent (AI) PC-3 cells, without killing RWPE-1 immortalized normal prostate epithelial cells. In LNCaP and PC-3 cells, LCA triggered the extrinsic pathway of apoptosis and cell death induced by LCA was partially dependent on the activation of caspase-8 and -3. Moreover, LCA increased cleavage of Bid and Bax, down-regulation of Bcl-2, permeabilization of the mitochondrial outer membrane and activation of caspase-9. The cytotoxic actions of LCA occurred despite the inability of this bile acid to enter the prostate cancer cells with about 98% of the nominal test concentrations present in the extracellular culture medium. With our findings, we provide evidence to support a mechanism of action underlying the broad anticancer activity of LCA in various human tissues. PMID:23940835

  5. A Cilia Independent Role of Ift88/Polaris during Cell Migration

    PubMed Central

    Hamann, Christoph; Powelske, Christian; Mergen, Miriam; Herbst, Henriette; Kotsis, Fruzsina; Nitschke, Roland; Kuehn, E. Wolfgang

    2015-01-01

    Ift88 is a central component of the intraflagellar transport (Ift) complex B, essential for the building of cilia and flagella from single cell organisms to mammals. Loss of Ift88 results in the absence of cilia and causes left-right asymmetry defects, disordered Hedgehog signaling, and polycystic kidney disease, all of which are explained by aberrant ciliary function. In addition, a number of extraciliary functions of Ift88 have been described that affect the cell-cycle, mitosis, and targeting of the T-cell receptor to the immunological synapse. Similarly, another essential ciliary molecule, the kinesin-2 subunit Kif3a, which transports Ift-B in the cilium, affects microtubule (MT) dynamics at the leading edge of migrating cells independently of cilia. We now show that loss of Ift88 impairs cell migration irrespective of cilia. Ift88 is required for the polarization of migrating MDCK cells, and Ift88 depleted cells have fewer MTs at the leading edge. Neither MT dynamics nor MT nucleation are dependent on Ift88. Our findings dissociate the function of Ift88 from Kif3a outside the cilium and suggest a novel extraciliary function for Ift88. Future studies need to address what unifying mechanism underlies the different extraciliary functions of Ift88. PMID:26465598

  6. Yessotoxin activates cell death pathways independent of Protein Kinase C in K-562 human leukemic cell line.

    PubMed

    Fernández-Araujo, Andrea; Alfonso, Amparo; Vieytes, Mercedes R; Botana, Luis M

    2015-10-01

    Protein Kinase C (PKC) is a group of enzymes involved in pro-survival or pro-apoptotic events depending on the cellular model. Moreover, Yessotoxin (YTX) modulates its expression and activates different cell death pathways. In K-562 tumor cell line, YTX induces apoptosis and autophagy after 24 and 48 h of incubation, respectively, and the toxin carries out its action through the phosphodiesterase 4A (PDE4A). Therefore, the levels of two subtypes of PKC, conventional (cPKC) and δ isotype of novel PKC (PKCδ) were studied at these times after YTX incubation. Also their involvement in the cell death activated by the toxin and their relationship with PDE4A was checked. The expression of cPKC and PKCδ in cytosol, plasma membrane and nucleus was studied in normal and PDE4A-silenced cells. Furthermore, cell viability of normal cells, as well as cPKC-, PKCδ- and PDE4A-silenced cells was tested by Lactate Dehydrogenase (LDH) assay. As a result, PKCδ showed a key role in K-562 cell survive, since without this protein, K-562 cell decreased their viability. Furthermore, modulation of PKCs by YTX treatment was observed, however, the changes in the expression of these proteins are independent of cell death activated by the toxin. In addition, the modulation of PKCs detected is PDE4A-dependent, since the silencing of this protein change PKC expression pattern.

  7. Association of autophagy in the cell death mediated by dihydrotestosterone in autoreactive T cells independent of antigenic stimulation

    PubMed Central

    Jia, Ting; Anandhan, Annandurai; Massilamany, Chandirasegaran; Rajasekaran, Rajkumar A.; Franco, Rodrigo; Reddy, Jay

    2015-01-01

    Gender disparity is well documented in the mouse model of experimental autoimmune encephalomyelitis (EAE) induced with proteolipid protein (PLP) 139-151, in which female, but not male, SJL mice show a chronic relapsing-remitting paralysis. Furthermore, dihydrotestosterone (DHT) has been shown to ameliorate the severity of EAE, but the underlying mechanisms of its protective effects are unclear. Using major histocompatibility complex (MHC) class II dextramers for PLP 139-151, we tested the hypothesis that DHT selectively modulates the expansion and functionalities of antigen-specific T cells. Unexpectedly, we noted that DHT induced cell death in antigen-specific, autoreactive T cells, but the effects were not selective, because both proliferating and non-proliferating cells were equally affected independent of antigenic stimulation. Furthermore, DHT-exposed PLP 139-151-specific T cells did not show any shift in cytokine production; rather, frequencies of cytokine-producing PLP-specific T cells were significantly reduced, irrespective of T helper (Th) 1, Th2, and Th17 subsets of cytokines. By evaluating cell death and autophagy pathways, we provide evidence for the induction of autophagy to be associated with cell death caused by DHT. Taken together, the data provide new insights into the role of DHT and indicate that cell death and autophagy contribute to the therapeutic effects of androgens in autoreactive T cells. PMID:26416183

  8. Evolutionary convergence of cell-specific gene expression in independent lineages of C4 grasses.

    PubMed

    John, Christopher R; Smith-Unna, Richard D; Woodfield, Helen; Covshoff, Sarah; Hibberd, Julian M

    2014-05-01

    Leaves of almost all C4 lineages separate the reactions of photosynthesis into the mesophyll (M) and bundle sheath (BS). The extent to which messenger RNA profiles of M and BS cells from independent C4 lineages resemble each other is not known. To address this, we conducted deep sequencing of RNA isolated from the M and BS of Setaria viridis and compared these data with publicly available information from maize (Zea mays). This revealed a high correlation (r=0.89) between the relative abundance of transcripts encoding proteins of the core C4 pathway in M and BS cells in these species, indicating significant convergence in transcript accumulation in these evolutionarily independent C4 lineages. We also found that the vast majority of genes encoding proteins of the C4 cycle in S. viridis are syntenic to homologs used by maize. In both lineages, 122 and 212 homologous transcription factors were preferentially expressed in the M and BS, respectively. Sixteen shared regulators of chloroplast biogenesis were identified, 14 of which were syntenic homologs in maize and S. viridis. In sorghum (Sorghum bicolor), a third C4 grass, we found that 82% of these trans-factors were also differentially expressed in either M or BS cells. Taken together, these data provide, to our knowledge, the first quantification of convergence in transcript abundance in the M and BS cells from independent lineages of C4 grasses. Furthermore, the repeated recruitment of syntenic homologs from large gene families strongly implies that parallel evolution of both structural genes and trans-factors underpins the polyphyletic evolution of this highly complex trait in the monocotyledons.

  9. Genetic Dissection of γ-secretase-dependent and - independent Functions of Presenilin in Regulating Neuronal Cell Cycle and Cell Death

    PubMed Central

    Kallhoff-Munoz, Verena; Hu, Lingyun; Chen, Xiaoli; Pautler, Robia G.; Zheng, Hui

    2008-01-01

    Cell cycle markers have been shown to be upregulated and proposed to lead to apoptosis of post-mitotic neurons in Alzheimer’s disease (AD). Presenilin (PS) plays a critical role in AD pathogenesis, and loss of function studies in mice established a potent effect of PS in cell proliferation in peripheral tissues. Whether PS has a similar activity in the neuronal cell cycle has not been investigated. PS exhibits γ-secretase-dependent and -independent functions; the former requires aspartate 257 (D257) as part of the active site, and the latter involves the hydrophilic loop domain encoded by exon 10. We used two novel mouse models, one expressing the PS1 D257A mutation on a postnatal PS conditional knockout background and the other deleting exon 10 of PS1, to dissect the γ-secretase-dependent and -independent activities of PS in the adult CNS. Whereas γ-secretase plays a dominant role in neuronal survival, our studies reveal potent neuronal cell cycle regulation mediated by the PS1 hydrophilic loop. Although neurons expressing cell cycle markers do not directly succumb to apoptosis, they are more vulnerable under stress conditions. Importantly, our data identify a novel pool of cytoplasmic p53 as a downstream mediator of this cellular vulnerability. These results support a model whereby the PS γ-secretase activity is essential in maintaining neuronal viability, and the PS1 loop domain modulates neuronal homeostasis through cell cycle and cytoplasmic p53 control. PMID:18971484

  10. LIF independent JAK signalling to chromatin in embryonic stem cells uncovered from an adult stem cell disease

    PubMed Central

    Griffiths, Dean S.; Li, Juan; Dawson, Mark A.; Trotter, Matthew W.B.; Cheng, Yi-Han; Smith, Aileen M.; Mansfield, William; Liu, Pentao; Kouzarides, Tony; Nichols, Jennifer; Bannister, Andrew J.; Green, Anthony R; Göttgens, Berthold

    2010-01-01

    Activating mutations in the tyrosine kinase JAK2 cause myeloproliferative neoplasms, clonal blood stem cell disorders with a propensity for leukaemic transformation. LIF signalling through JAK-STAT enables ES cell self-renewal. Here we show that mouse ES cells carrying the human JAK2V617F mutation could self-renew in chemically defined conditions without cytokines or small molecule inhibitors independently of JAK signalling through STAT3 or PI3K pathways. Phosphorylation of histone H3Y41 by JAK2 was recently shown to interfere with HP1α binding. Chromatin bound HP1α was lower in JAK2V617F ES cells but increased following JAK2 inhibition, coincident with a global reduction in H3Y41ph. JAK2 inhibition reduced Nanog, with a reduction in H3Y41ph and concomitant increase in HP1α at the Nanog promoter. Furthermore, Nanog was required for factor-independence of JAK2V617F ES cells. Taken together, these results uncover a previously unrecognised role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal. PMID:21151131

  11. Hemolysin-producing Listeria monocytogenes affects the immune response to T-cell-dependent and T-cell-independent antigens.

    PubMed Central

    Hage-Chahine, C M; Del Giudice, G; Lambert, P H; Pechere, J C

    1992-01-01

    A murine experimental infection with a hemolysin-producing (Hly+) strain of Listeria monocytogenes and a non-hemolysin-producing (Hly-) mutant was used as an in vivo model to evaluate the role of hemolysin production in the immune response. No antilisterial antibodies were detectable following sublethal infection with Hly+ bacteria, but consistent antilisterial immunoglobulin G (IgG) and IgM antibody production was observed following sublethal infection with the Hly- mutant. Hly+ but not Hly- L. monocytogenes induced transient inhibition of antibody response to Hly- bacteria and to unrelated T-cell-dependent (tetanus toxoid) and T-cell-independent (pneumococcal polysaccharide 3) antigens. Transient inhibition of the activation of an antigen-specific T-cell clone was also observed following Hly+ infection of antigen-presenting cells but not following Hly- infection. These results suggest that hemolysin production by L. monocytogenes is an important factor in modulating the immune response to T-cell-dependent and T-cell-independent antigens in infected individuals. Images PMID:1548067

  12. Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death.

    PubMed

    Peterson, Brianna; Knotts, Taylor; Cummings, Brian S

    2007-01-01

    This study determined the roles of Ca2+-independent PLA2 (iPLA2) in phospholipid chemistry and oxidant-induced cell death in human astrocytes. A172 cells expressed both cytosolic Group VIA (iPLA2beta) and microsomal Group VIB (iPLA2gamma) PLA2 as determined by activity assays and immunoblot analysis. Inhibition of total iPLA2 activity using racemic bromoenol lactone (BEL, 2.5 microM) decreased the expression of 14:0-16:0 phosphatidylcholine (PtdCho) 15% and increased 18:0-18:1-PtdCho expression 15%. Treatment of cells with the iPLA2gamma specific inhibitor R-BEL decreased 14:0-16:0-PtdCho 35%, 16:0-16:0-PtdCho 15% and induced a 35% increase in 18:0-18:1-PtdCho. In contrast, treatment of cells with the iPLA2beta inhibitor S-BEL did not alter any phospholipid studied. To determine the roles of iPLA2 in oxidant-induced cell death, A172 cells were exposed to hydrogen peroxide (H2O2) or tert-butylhydroperoxide (TBHP); both induced time- and concentration-dependent increases in cell death as assessed by annexin V and propidium iodide staining. Treatment of cells with racemic-BEL alone did not induce cell death. However, pretreatment with BEL prior to H2O2 (500 microM) or TBHP (200 microM) significantly increased necrosis as determined by increases in propidium iodide staining. Treatment with BEL prior to exposure to oxidants accelerated the loss of ATP levels, but not the formation of reactive oxygen species. These data support the hypothesis that iPLA2 mediates oxidant-induced neural cell death and demonstrates differential roles of iPLA2 isoforms in physiological and pathological events.

  13. The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells.

    PubMed

    Guiet, Romain; Van Goethem, Emeline; Cougoule, Céline; Balor, Stéphanie; Valette, Annie; Al Saati, Talal; Lowell, Clifford A; Le Cabec, Véronique; Maridonneau-Parini, Isabelle

    2011-10-01

    Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

  14. Liver cancer cells are sensitive to Lanatoside C induced cell death independent of their PTEN status.

    PubMed

    Durmaz, Irem; Guven, Ebru Bilget; Ersahin, Tulin; Ozturk, Mehmet; Calis, Ihsan; Cetin-Atalay, Rengul

    2016-01-15

    Hepatocellular carcinoma is the second deadliest cancer with limited treatment options. Loss of PTEN causes the P13K/Akt pathway to be hyperactive which contributes to cell survival and resistance to therapeutics in various cancers, including the liver cancer. Hence molecules targeting this pathway present good therapeutic strategies for liver cancer. It was previously reported that Cardiac glycosides possessed antitumor activity by inducing apoptosis of multiple cancer cells through oxidative stress. However, whether Cardiac glycoside Lanatoside C can induce oxidative stress in liver cancer cells and induce cell death both in vitro and in vivo remains unknown. Cell viability was measured by SRB assay. Cell death analysis was investigated by propidium iodide staining with flow cytometry and PARP cleavage. DCFH-DA staining and cytometry were used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Antitumor activity was investigated on mice xenografts in vivo. In this study, we found that Cardiac glycosides, particularly Lanatoside C from Digitalis ferruginea could significantly inhibit PTEN protein adequate Huh7 and PTEN deficient Mahlavu human liver cancer cell proliferation by the induction of apoptosis and G2/M arrest in the cells. Lanatoside C was further shown to induce oxidative stress and alter ERK and Akt pathways. Consequently, JNK1 activation resulted in extrinsic apoptotic pathway stimulation in both cells while JNK2 activation involved in the inhibition of cell survival only in PTEN deficient cells. Furthermore, nude mice xenografts followed by MRI showed that Lanatoside C caused a significant decrease in the tumor size. In this study apoptosis induction by Lanatoside C was characterized through ROS altered ERK and Akt pathways in both PTEN adequate epithelial and deficient mesenchymal liver cancer cells. The results indicated that Lanatoside C could be contemplated in liver cancer therapeutics, particularly in PTEN

  15. Quantitative tumour necrosis is an independent predictor of overall survival in clear cell renal cell carcinoma.

    PubMed

    Renshaw, Andrew A; Cheville, John C

    2015-01-01

    Previous studies have reached conflicting results regarding whether tumour necrosis is a predictor of survival in clear cell renal cell carcinoma. In addition, studies quantifying the extent of necrosis are limited.The aim of this study was to determine if quantifying tumour necrosis could improve its predictive value for survival in clear cell renal cell carcinoma.We reviewed the clinical pathological information contained in The Cancer Genome Atlas for clear cell renal cell carcinoma and correlated it with overall survival using a Cox proportional hazard model. Necrosis was quantified on a single frozen section slide taken at the time of tissue harvesting for molecular studies.For all tumours, the presence of tumour necrosis was a significant predictor of overall survival (p < 0.001) on univariate analysis. When quantitated, >10% necrosis was associated with survival, but ≤10% necrosis was not. On multivariate analysis, age (p = 0.004), T3b stage (p = 0.02), M1 stage (p < 0.001), necrosis >30% (p < 0.001), and elevated serum calcium (p = 0.003) remained significant. For clinical stage 1-2 (T1-T2N0M0) tumours, necrosis >20% was significant on univariate analysis (p ≤ 0.005), and remained so on multivariate analysis (p < 0.001).We conclude that quantitating the extent of tumour necrosis adds prognostic information in clear cell renal cell carcinomas, including organ confined tumours.

  16. Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

    SciTech Connect

    Sarafian, Theodore Montes, Cindy; Harui, Airi; Beedanagari, Sudheer R.; Kiertscher, Sylvia; Stripecke, Renata; Hossepian, Derik; Kitchen, Christina; Kern, Rita; Belperio, John; Roth, Michael D.

    2008-09-15

    , did not increase cell migration. Moreover, CB2R-transduced cells displayed higher {psi}{sub m} than did control cells. Since both {psi}{sub m} and chemotaxis are regulated by intracellular signaling, we investigated the effects of THC on the activation of multiple signaling pathways. Serum exposure activated several signaling events of which phosphorylation of I{kappa}B-{alpha} and JNK was regulated in a CB2R- and THC-dependent manner. We conclude that airway epithelial cells are sensitive to both CB2R-dependent and independent effects mediated by THC.

  17. Tumor Necrosis Factor Inhibits Spread of Hepatitis C Virus Among Liver Cells, Independent From Interferons.

    PubMed

    Laidlaw, Stephen M; Marukian, Svetlana; Gilmore, Rachel H; Cashman, Siobhán B; Nechyporuk-Zloy, Volodymyr; Rice, Charles M; Dustin, Lynn B

    2017-08-01

    Tumor necrosis factor (TNF) is an inflammatory cytokine expressed by human fetal liver cells (HFLCs) after infection with cell culture-derived hepatitis C virus (HCV). TNF has been reported to increase entry of HCV pseudoparticles into hepatoma cells and inhibit signaling by interferon alpha (IFNα), but have no effect on HCV-RNA replication. We investigated the effects of TNF on HCV infection of and spread among Huh-7 hepatoma cells and primary HFLCs. Human hepatoma (Huh-7 and Huh-7.5) and primary HFLCs were incubated with TNF and/or recombinant IFNA2A, IFNB, IFNL1, and IFNL2 before or during HCV infection. We used 2 fully infectious HCV chimeric viruses of genotype 2A in these studies: J6/JFH (clone 2) and Jc1(p7-nsGluc2A) (Jc1G), which encodes a secreted luciferase reporter. We measured HCV replication, entry, spread, production, and release in hepatoma cells and HFLCs. TNF inhibited completion of the HCV infectious cycle in hepatoma cells and HFLCs in a dose-dependent and time-dependent manner. This inhibition required TNF binding to its receptor. Inhibition was independent of IFNα, IFNβ, IFNL1, IFNL2, or Janus kinase signaling via signal transducer and activator of transcription. TNF reduced production of infectious viral particles by Huh-7 and HFLC, and thereby reduced the number of infected cells and focus size. TNF had little effect on HCV replicons and increased entry of HCV pseudoparticles. When cells were incubated with TNF before infection, the subsequent antiviral effects of IFNs were increased. In a cell culture system, we found TNF to have antiviral effects independently of, as well as in combination with, IFNs. TNF inhibits HCV infection despite increased HCV envelope glycoprotein-mediated infection of liver cells. These findings contradict those from other studies, which have reported that TNF blocks signal transduction in response to IFNs. The destructive inflammatory effects of TNF must be considered along with its antiviral effects. Copyright

  18. Ambiguine I Isonitrile from Fischerella ambigua Induces Caspase-Independent Cell Death in MCF-7 Hormone Dependent Breast Cancer Cells

    PubMed Central

    Acuña, Ulyana Muñoz; Zi, Jiachen; Orjala, Jimmy; Carcache de Blanco, Esperanza J.

    2015-01-01

    Ambiguine I isonitrile (AmbI) obtained from the cultured cyanobacterium Fischerella ambigua was identified as a potent NF-κB inhibitor (IC50=30 nM). The cytotoxic effect was evaluated in both HT-29 colon cancer cell line (EC50=4.35 μM) and MCF-7 breast cancer cell line (EC50=1.7 μM) using the SRB assay. In the cells treated with AmbI, an increased population of cells was detected in sub G1-phase. The apoptotic effect was associated with block in G1-phase of the cell cycle in treated cells; however, cell death was induced independently of caspase-7. The NF-κB expression of p50 and p65 units were also examined in treated cells and compared with the positive control, rocaglamide (IC50=75 nM). Moreover, the expression of mediators of the NF-κB pathway such as kinase IKKκ was studied at increasing concentrations of AmbI. The down stream effect of NF-κB inhibition and the effect on the expression of TNF-α induced ICAM-1 was evaluated. Thus, the dose-dependent and time-dependent effect of AmbI on MCF-7 cells was examined in an attempt to investigate its potential mechanism of action on inducing apoptosis. PMID:26753095

  19. A repertoire-independent and cell-intrinsic defect in murine GVHD induction by effector memory T cells

    PubMed Central

    Juchem, Kathryn W.; Anderson, Britt E.; Zhang, Cuiling; McNiff, Jennifer M.; Demetris, Anthony J.; Farber, Donna L.; Caton, Andrew J.; Shlomchik, Warren D.

    2011-01-01

    Effector memory T cells (TEM) do not cause graft-versus-host disease (GVHD), though why this is has not been elucidated. To compare the fates of alloreactive naive (TN) or memory (TM) T cells, we developed a model of GVHD in which donor T cells express a transgene-encoded TCR specific for an antigenic peptide that is ubiquitously expressed in the recipient. Small numbers of naive TCR transgenic (Tg) T cells induced a robust syndrome of GVHD in transplanted recipients. We then used an established method to convert TCR Tg cells to TM and tested these for GVHD induction. This allowed us to control for the potentially different frequencies of alloreactive T cells among TN and TM, and to track fates of alloreactive T cells after transplantation. TEM caused minimal, transient GVHD whereas central memory T cells (TCM) caused potent GVHD. Surprisingly, TEM were not inert: they, engrafted, homed to target tissues, and proliferated extensively, but they produced less IFN-γ and their expansion in target tissues was limited at later time points, and local proliferation was reduced. Thus, cell-intrinsic properties independent of repertoire explain the impairment of TEM, which can initiate but cannot sustain expansion and tissue damage. PMID:21768295

  20. Air-Independent Solid Oxide Fuel Cells for NASA's LOX-CH4 Landers

    NASA Technical Reports Server (NTRS)

    Ryan, Abigail C.; Araghi, Koorosh R.; Farmer, Serene C.

    2013-01-01

    Gemini, Apollo, and Space Shuttle used fuel cells as main power source for vehicle and water source for life support and thermal PEM (Gemini) and Alkaline (Apollo, Shuttle) fuel cells were used Ideal for short (less than 3 weeks) missions when the required O2 and H2 can be launched with the vehicle. New missions that might require long-duration stays in orbit or at a habitat, cannot rely on the availability of pure reactants but should also aim to be sun-independent - a problem for which Solid Oxide Fuel Cells might be the answer. Recently, NASA has investigated & developed LOX/CH4-propelled landers (Altair, MORPHEUS). In order to preserve mission flexibility, fuel cells are being studied as a potential power source. Much of NASA's fuel cell development has been focused on creating a dead-headed, non-flow through PEM fuel cells which would weigh less and be more reliable than the existing Alkaline and PEM technology; however, LOX/CH4 as a propellant introduces SOFCs as a power option due to their ability to accept those reactants without much reforming.

  1. MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch.

    PubMed

    Porter, Kristine M; Jeyabalan, Nallathambi; Liton, Paloma B

    2014-06-01

    The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30mmHg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.

  2. T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells

    SciTech Connect

    Dandolo, L.; Aghion, J.; Blangy, D.

    1984-02-01

    Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, the authors investigated the fate of viral DNA upon infection of these cells; they showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, they studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). The results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

  3. Methylsulfonylmethane Induces p53 Independent Apoptosis in HCT-116 Colon Cancer Cells

    PubMed Central

    Karabay, Arzu Zeynep; Koc, Asli; Ozkan, Tulin; Hekmatshoar, Yalda; Sunguroglu, Asuman; Aktan, Fugen; Buyukbingol, Zeliha

    2016-01-01

    Methylsulfonylmethane (MSM) is an organic sulfur-containing compound which has been used as a dietary supplement for osteoarthritis. MSM has been shown to reduce oxidative stress and inflammation, as well as exhibit apoptotic or anti-apoptotic effects depending on the cell type or activating stimuli. However, there are still a lot of unknowns about the mechanisms of actions of MSM. In this study, MSM was tested on colon cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis revealed that MSM inhibited cell viability and increased apoptotic markers in both HCT-116 p53 +/+ and HCT-116 p53 −/− colon cancer cells. Increased poly (ADP-ribose) polymerase (PARP) fragmentation and caspase-3 activity by MSM also supported these findings. MSM also modulated the expression of various apoptosis-related genes and proteins. Moreover, MSM was found to increase c-Jun N-terminal kinases (JNK) phosphorylation in both cell lines, dose-dependently. In conclusion, our results show for the first time that MSM induces apoptosis in HCT-116 colon cancer cells regardless of their p53 status. Since p53 is defective in >50% of tumors, the ability of MSM to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy. Moreover, the remarkable effect of MSM on Bim, an apoptotic protein, also suggests its potential use as a novel chemotherapeutic agent for Bim-targeted anti-cancer therapies. PMID:27428957

  4. Circulating Tumour Cells as an Independent Prognostic Factor in Patients with Advanced Oesophageal Squamous Cell Carcinoma Undergoing Chemoradiotherapy

    PubMed Central

    Su, Po-Jung; Wu, Min-Hsien; Wang, Hung-Ming; Lee, Chia-Lin; Huang, Wen-Kuan; Wu, Chiao-En; Chang, Hsien-Kun; Chao, Yin-Kai; Tseng, Chen-Kan; Chiu, Tzu-Keng; Lin, Nina Ming-Jung; Ye, Siou-Ru; Lee, Jane Ying-Chieh; Hsieh, Chia-Hsun

    2016-01-01

    The role of circulating tumour cells (CTCs) in advanced oesophageal cancer (EC) patients undergoing concurrent chemoradiotherapy (CCRT) remains uncertain. A negative selection protocol plus flow cytometry was validated to efficiently identify CTCs. The CTC number was calculated and analysed for survival impact. The protocol’s efficacy in CTC identification was validated with a recovery rate of 44.6 ± 9.1% and a coefficient of variation of 20.4%. Fifty-seven patients and 20 healthy donors were enrolled. Initial staging, first response to CRT, and surgery after CRT were prognostic for overall survival, with P values of <0.0001, <0.0001, and <0.0001, respectively. The CTC number of EC patients is significantly higher (P = 0.04) than that of healthy donors. Multivariate analysis for disease-specific progression-free survival showed that surgery after response to CCRT, initial stage, and CTC number (≥21.0 cells/mL) played independent prognostic roles. For overall survival, surgery after CCRT, performance status, initial stage, and CTC number were significant independent prognostic factors. In conclusion, a negative selection plus flow cytometry protocol efficiently detected CTCs. The CTC number before CCRT was an independent prognostic factor in patients with unresectable oesophageal squamous cell carcinoma. Further large-scale prospective studies for validation are warranted. PMID:27530152

  5. Independent control of reciprocal and lateral inhibition at the axon terminal of retinal bipolar cells

    PubMed Central

    Tanaka, Masashi; Tachibana, Masao

    2013-01-01

    Bipolar cells (BCs), the second order neurons in the vertebrate retina, receive two types of GABAergic feedback inhibition at their axon terminal: reciprocal and lateral inhibition. It has been suggested that two types of inhibition may be mediated by different pathways. However, how each inhibition is controlled by excitatory BC output remains to be clarified. Here, we applied single/dual whole cell recording techniques to the axon terminal of electrically coupled BCs in slice preparation of the goldfish retina, and found that each inhibition was regulated independently. Activation voltage of each inhibition was different: strong output from a single BC activated reciprocal inhibition, but could not activate lateral inhibition. Outputs from multiple BCs were essential for activation of lateral inhibition. Pharmacological examinations revealed that composition of transmitter receptors and localization of Na+ channels were different between two inhibitory pathways, suggesting that different amacrine cells may mediate each inhibition. Depending on visual inputs, each inhibition could be driven independently. Model simulation showed that reciprocal and lateral inhibition cooperatively reduced BC outputs as well as background noise, thereby preserving high signal-to-noise ratio. Therefore, we conclude that excitatory BC output is efficiently regulated by the dual operating mechanisms of feedback inhibition without deteriorating the quality of visual signals. PMID:23690563

  6. Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin.

    PubMed

    Maniti, Ofelia; Blanchard, Elise; Trugnan, Germain; Lamazière, Antonin; Ayala-Sanmartin, Jesus

    2012-06-01

    Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. MODEL AND CELL MEMBRANE PARTITIONING OF PERFLUOROOCTANESULFONATE IS INDEPENDENT OF THE LIPID CHAIN LENGTH

    PubMed Central

    Xie, Wei; Ludewig, Gabriele; Wang, Kai; Lehmler, Hans-Joachim

    2009-01-01

    Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse health effects in humans and animals by interacting with and disturbing of the normal properties of biological lipid assemblies. To gain further insights into these interactions, we investigated the effect of PFOS potassium salt on dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) model membranes using fluorescence anisotropy measurements and differential scanning calorimetry (DSC) and on the cell membrane of HL-60 human leukemia cells and freshly isolated rat alveolar macrophages using fluorescence anisotropy measurements. PFOS caused a concentration-dependent decrease of the main phase transition temperature (Tm) and an increased peak width (ΔTw) in both the fluorescence anisotropy and the DSC experiments, with a rank order DMPC > DPPC > DSPC. PFOS caused a fluidization of the gel phase of all phosphatidylcholines investigated, but had the opposite effect on the liquid crystalline phase. The apparent partition coefficients of PFOS between the phosphatidylcholine bilayer and the bulk aqueous phase were largely independent of the phosphatidylcholine chain length and ranged from 4.4 × 104 to 8.8 × 104. PFOS also significantly increased the fluidity of membranes of cells. These findings suggest that PFOS readily partitions into lipid assemblies, independent of their composition, and may cause adverse biological effects by altering their fluidity in a manner that depends on the membrane cooperativity and state (e.g., gel versus liquid crystalline phase) of the lipid assembly. PMID:19932010

  8. A method for multiprotein assembly in cells reveals independent action of kinesins in complex

    PubMed Central

    Norris, Stephen R.; Soppina, Virupakshi; Dizaji, Aslan S.; Schimert, Kristin I.; Sept, David; Cai, Dawen; Sivaramakrishnan, Sivaraj

    2014-01-01

    Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. PMID:25365993

  9. Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

    SciTech Connect

    Ho, T.-F.; Ma, C.-J.; Lu, C.-H.; Tsai, Yo-Ting; Wei, Y.-H.; Chang, J.-S.; Lai, J.-K.; Cheuh, Pin-Ju; Yeh, C.-T.; Tang, P.-C.; Jingua, T.C.; Ko, J.-L.; Liu, F.-S.; Yen, H.E.

    2007-12-15

    Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X{sub L}, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.

  10. Riluzole mediates anti-tumor properties in breast cancer cells independent of metabotropic glutamate receptor-1.

    PubMed

    Speyer, Cecilia L; Nassar, Mahdy A; Hachem, Ali H; Bukhsh, Miriam A; Jafry, Waris S; Khansa, Rafa M; Gorski, David H

    2016-06-01

    Riluzole, the only drug approved by the FDA for treating amyotrophic lateral sclerosis, inhibits melanoma proliferation through its inhibitory effect on glutamatergic signaling. We demonstrated that riluzole also inhibits the growth of triple-negative breast cancer (TNBC) and described a role for metabotropic glutamate receptor-1 (GRM1) in regulating TNBC cell growth and progression. However, the role of GRM1 in mediating riluzole's effects in breast cancer has not been fully elucidated. In this study, we seek to determine how much of riluzole's action in breast cancer is mediated through GRM1. We investigated anti-tumor properties of riluzole in TNBC and ER+ cells using cell growth, invasion, and soft-agar assays and compared riluzole activity with GRM1 levels. Using Lentiviral vectors expressing GRM1 or shGRM1, these studies were repeated in cells expressing high or low GRM1 levels where the gene was either silenced or overexpressed. Riluzole inhibited proliferation, invasion, and colony formation in both TNBC and ER+ cells. There was a trend between GRM1 expression in TNBC cells and their response to riluzole in both cell proliferation and invasion assays. However, silencing and overexpression studies had no effect on cell sensitivity to riluzole. Our results clearly suggest a GRM1-independent mechanism through which riluzole mediates its effects on breast cancer cells. Understanding the mechanism by which riluzole mediates breast cancer progression will be useful in identifying new therapeutic targets for treating TNBC and in facilitating stratification of patients in clinical trials using riluzole in conjunction with conventional therapy.

  11. Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis

    PubMed Central

    Singh, P K; Kumar, A

    2016-01-01

    Bacterial endophthalmitis, a vision-threatening complication of ocular surgery or trauma, is characterized by increased intraocular inflammation and retinal tissue damage. Although significant vision loss in endophthalmitis has been linked to retinal cell death, the underlying mechanisms of cell death remain elusive. In this study, using a mouse model of Staphylococcus aureus endophthalmitis and cultured human retinal Müller glia (MIO-M1 cell line), we demonstrate that S. aureus caused significant apoptotic cell death in the mouse retina and Müller glia, as evidenced by increased number of terminal dUTP nick end labeling and Annexin V and propidium iodide-positive cells. Immunohistochemistry and western blot studies revealed the reduction in mitochondrial membrane potential (JC-1 staining), release of cytochrome c into the cytosol, translocation of Bax to the mitochondria and the activation of caspase-9 and -3 in S. aureus-infected retina/retinal cells. In addition, the activation of PARP-1 and the release of apoptosis inducing factor from mitochondria was also observed in S. aureus-infected retinal cells. Inhibition studies using pan-caspase (Q-VD-OPH) and PARP-1 (DPQ) inhibitors showed significant reduction in S. aureus-induced retinal cell death both in vivo and in vitro. Together, our findings demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in the both caspase-dependent and independent manners, and mitochondria have a central role in this process. Hence, targeting the identified signaling pathways may provide the rationale to design therapeutic interventions to prevent bystander retinal tissue damage in bacterial endophthalmitis. PMID:27551524

  12. MLN4924 sensitizes monocytes and maturing dendritic cells for TNF-dependent and -independent necroptosis

    PubMed Central

    El-Mesery, Mohamed; Seher, Axel; Stühmer, Thorsten; Siegmund, Daniela; Wajant, Harald

    2015-01-01

    Background and Purpose MLN4924 prevents the formation of active cullin-RING ubiquitin ligase complexes and thus inhibits NF-κB signalling. Here, we evaluated the effects of this compound on monocytes and dendritic cells (DCs). Experimental Approach Monocytes and DCs were challenged with TNF or LPS in the presence and absence of MLN4924. The effects of MLN4924 on cellular viability, pro-inflammatory gene induction and DC maturation were investigated using the MTT assay, elisa and FACS analysis. Mechanisms of cell death induction were evaluated by using inhibitors of caspases, RIPK1 and MLKL. Key Results MLN4924 inhibited NF-κB activation and sensitized monocytes and immature DCs (iDCs) for TNFR1-induced cell death. Neither the caspase inhibitor zVAD-fmk, the RIPK1 inhibitor necrostatin-1 (nec-1) nor the MLKL inhibitor necrosulfonamide (NSA) alone prevented TNF-induced cell death. A combination of zVAD-fmk and nec-1 or NSA, however, rescued monocytes and iDCs from MLN4924/TNF-induced cell death indicating that MLN4924 affects anti-apoptotic and anti-necrotic activities in TNFR1 signalling. MLN4924 also converted the response of iDCs to LPS from maturation to cell death. LPS-induced cell death in MLN4924-treated iDCs was again only effectively blocked by cotreatment with zVAD-fmk and nec-1 or NSA. Noteworthy, MLN4924/LPS-induced cell death was almost completely independent of endogenous TNF. MLN4924 also strongly inhibited maturation and activation of iDCs that were rescued from cell death by zVAD-fmk and nec-1. Conclusions and Implications Our data reveal a strong dual suppressive effect of MLN4924 on DC activity. The targeting of NAE by MLN4924 could be a new way to treat inflammatory diseases. PMID:25363690

  13. Smoking and Female Sex: Independent Predictors of Human Vascular Smooth Muscle Cells Stiffening

    PubMed Central

    Dinardo, Carla Luana; Santos, Hadassa Campos; Vaquero, André Ramos; Martelini, André Ricardo; Dallan, Luis Alberto Oliveira; Alencar, Adriano Mesquita; Krieger, José Eduardo; Pereira, Alexandre Costa

    2015-01-01

    Aims Recent evidence shows the rigidity of vascular smooth muscle cells (VSMC) contributes to vascular mechanics. Arterial rigidity is an independent cardiovascular risk factor whose associated modifications in VSMC viscoelasticity have never been investigated. This study’s objective was to evaluate if the arterial rigidity risk factors aging, African ancestry, female sex, smoking and diabetes mellitus are associated with VMSC stiffening in an experimental model using a human derived vascular smooth muscle primary cell line repository. Methods Eighty patients subjected to coronary artery bypass surgery were enrolled. VSMCs were extracted from internal thoracic artery fragments and mechanically evaluated using Optical Magnetic Twisting Cytometry assay. The obtained mechanical variables were correlated with the clinical variables: age, gender, African ancestry, smoking and diabetes mellitus. Results The mechanical variables Gr, G’r and G”r had a normal distribution, demonstrating an inter-individual variability of VSMC viscoelasticity, which has never been reported before. Female sex and smoking were independently associated with VSMC stiffening: Gr (apparent cell stiffness) p = 0.022 and p = 0.018, R2 0.164; G’r (elastic modulus) p = 0.019 and p = 0.009, R2 0.184 and G”r (dissipative modulus) p = 0.011 and p = 0.66, R2 0.141. Conclusion Female sex and smoking are independent predictors of VSMC stiffening. This pro-rigidity effect represents an important element for understanding the vascular rigidity observed in post-menopausal females and smokers, as well as a potential therapeutic target to be explored in the future. There is a significant inter-individual variation of VSMC viscoelasticity, which is slightly modulated by clinical variables and probably relies on molecular factors. PMID:26661469

  14. Autocrine stimulation of clear-cell renal carcinoma cell migration in hypoxia via HIF-independent suppression of thrombospondin-1

    PubMed Central

    Bienes-Martínez, Raquel; Ordóñez, Angel; Feijoo-Cuaresma, Mónica; Corral-Escariz, María; Mateo, Gloria; Stenina, Olga; Jiménez, Benilde; Calzada, María J.

    2012-01-01

    Thrombospondin-1 is a matricellular protein with potent antitumour activities, the levels of which determine the fate of many different tumours, including renal carcinomas. However, the factors that regulate this protein remain unclear. In renal carcinomas, hypoxic conditions enhance the expression of angiogenic factors that help adapt tumour cells to their hostile environment. Therefore, we hypothesized that anti-angiogenic factors should correspondingly be dampened. Indeed, we found that hypoxia decreased the thrombospondin-1 protein in several clear cell renal carcinoma cell lines (ccRCC), although no transcriptional regulation was observed. Furthermore, we proved that hypoxia stimulates multiple signals that independently contribute to diminish thrombospondin-1 in ccRCC, which include a decrease in the activity of oxygen-dependent prolylhydroxylases (PHDs) and activation of the PI3K/Akt signalling pathway. In addition, thrombospondin-1 regulation in hypoxia proved to be important for ccRCC cell migration and invasion. PMID:23145312

  15. Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism

    PubMed Central

    Suparji, Noor Shahirah; Chan, Gomathi; Sapili, Hani; Arshad, Norhafiza M.; In, Lionel L. A.; Awang, Khalijah; Hasima Nagoor, Noor

    2016-01-01

    Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could

  16. Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

    PubMed Central

    Wargelius, Anna; Leininger, Sven; Skaftnesmo, Kai Ove; Kleppe, Lene; Andersson, Eva; Taranger, Geir Lasse; Schulz, Rüdiger W; Edvardsen, Rolf B

    2016-01-01

    Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish. PMID:26888627

  17. Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon.

    PubMed

    Wargelius, Anna; Leininger, Sven; Skaftnesmo, Kai Ove; Kleppe, Lene; Andersson, Eva; Taranger, Geir Lasse; Schulz, Rüdiger W; Edvardsen, Rolf B

    2016-02-18

    Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.

  18. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

    PubMed Central

    Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E.; Krishnan, Aswini R.; Tsui, Tzuhan; Aguilera, Joseph A.; Advani, Sunil; Crotty Alexander, Laura E.; Brumund, Kevin T.; Wang-Rodriguez, Jessica

    2016-01-01

    Objectives Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 hours to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. Results E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. Conclusion E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. PMID:26547127

  19. Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events.

    PubMed

    Parisi, Silvia; Passaro, Fabiana; Russo, Luigi; Musto, Anna; Navarra, Angelica; Romano, Simona; Petrosino, Giuseppe; Russo, Tommaso

    2017-03-01

    Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of their expression and the mechanisms underlying their functions are still not definitively understood. Here we address the above-mentioned issues in the first steps of mouse embryonic stem cell (ESC) differentiation. We observed that the expression of Lin28 genes is transiently induced soon after the exit of ESCs from the naive ground state and that this induction is due to the Hmga2-dependent engagement of Otx2 with enhancers present at both Lin28 gene loci. These mechanisms are crucial for Lin28 regulation, as demonstrated by the abolishment of the Lin28 accumulation in Otx2- or Hmga2-knockout cells compared to the control cells. We have also found that Lin28 controls Hmga2 expression levels during ESC differentiation through a let-7-independent mechanism. Indeed, we found that Lin28 proteins bind a highly conserved element in the 3' UTR of Hmga2 mRNA, and this provokes a down-regulation of its translation. This mechanism prevents the inappropriate accumulation of Hmga2 that would modify the proliferation and physiological apoptosis of differentiating ESCs. In summary, we demonstrated that during ESC differentiation, Lin28 transient induction is dependent on Otx2 and Hmga2 and prevents an inappropriate excessive rise of Hmga2 levels.-Parisi, S., Passaro, F., Russo, L., Musto, A., Navarra, A., Romano, S., Petrosino, G., Russo, T. Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events. © FASEB.

  20. Complex gangliosides are apically sorted in polarized MDCK cells and internalized by clathrin-independent endocytosis.

    PubMed

    Crespo, Pilar M; von Muhlinen, Natalia; Iglesias-Bartolomé, Ramiro; Daniotti, Jose L

    2008-12-01

    Gangliosides are glycosphingolipids mainly present at the outer leaflet of the plasma membrane of eukaryotic cells, where they participate in recognition and signalling activities. The synthesis of gangliosides is carried out in the lumen of the Golgi apparatus by a complex system of glycosyltransferases. After synthesis, gangliosides leave the Golgi apparatus via the lumenal surface of transport vesicles destined to the plasma membrane. In this study, we analysed the synthesis and membrane distribution of GD3 and GM1 gangliosides endogenously synthesized by Madin-Darby canine kidney (MDCK) cell lines genetically modified to express appropriate ganglioside glycosyltransferases. Using biochemical techniques and confocal laser scanning microscopy analysis, we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, were transported and accumulated mainly at the plasma membrane of nonpolarized MDCK cell lines. More interestingly, both complex gangliosides were found to be enriched mainly at the apical domain when these cell lines were induced to polarize. In addition, we demonstrated that, after arrival at the plasma membrane, GD3 and GM1 gangliosides were endocytosed using a clathrin-independent pathway. Then, internalized GD3, in association with a specific monoclonal antibody, was accumulated in endosomal compartments and transported back to the plasma membrane. In contrast, endocytosed GM1, in association with cholera toxin, was transported to endosomal compartments en route to the Golgi apparatus. In conclusion, our results demonstrate that complex gangliosides are apically sorted in polarized MDCK cells, and that GD3 and GM1 gangliosides are internalized by clathrin-independent endocytosis to follow different intracellular destinations.

  1. External Na-independent Ca extrusion in cultured ventricular cells. Magnitude and functional significance

    PubMed Central

    1986-01-01

    The relative magnitudes and functional significance of Ca extrusion by Na-Ca exchange and by an Nao-independent mechanism were investigated in monolayer cultures of chick embryo ventricular cells. Abrupt exposure of cells in 0-Nao, nominally 0-Cao solution to 20 mM caffeine produced a large contracture (3.94 +/- 0.90 micron of cell shortening) that relaxed with a t1/2 of 8.60 +/- 1.22 s. An abrupt exposure to caffeine plus 140 mM Na resulted in a contracture that was smaller in amplitude (1.53 +/- 0.50 micron) and relaxed much more rapidly (t1/2 = 0.77 +/- 0.09 s). An abrupt exposure to caffeine in 0-Nao solutions produced an increase in 45Ca efflux that persisted for 20 s, and a net loss of Ca content, determined by atomic absorption spectroscopy (AAS), of approximately 4 nmol/mg protein, within 35 s. A comparable net loss of Ca was demonstrated in the presence of 100 microM [Ca]o. The abrupt exposure of cultured cells to 0 Nao in 1.8 mM Ca produced a Ca uptake, estimated with 45Ca, of 3.2 nmol/mg protein X 15 s, but produced no increase in cell Ca content (AAS). In cells in which a 30% increase in Nai was produced by 5 min exposure to 10(-6) M ouabain, the abrupt exposure to 0 Nao produced a Ca uptake of 6 nmol/mg protein X 15 s and an increase in Ca content (AAS) of 4 nmol/mg protein. We conclude that there is an Nao-independent mechanism for Ca extrusion in these cells, presumably a Ca-ATPase Ca pump, with a limited Ca transport capacity of no more than 2 nmol/mg protein X 15 s. This is five times smaller than the demonstrated maximum capacity of the Na-Ca exchanger in these cells. The relaxation of twitch tension in these cells seems to be dependent primarily on sarcoplasmic reticulum uptake of Ca, with a secondary role provided by the Na-Ca exchanger. The Ca pump appears to contribute little to beat-to-beat relaxation. PMID:3760814

  2. Natural isolates of Brome mosaic virus with the ability to move from cell to cell independently of coat protein.

    PubMed

    Takeda, Atsushi; Nakamura, Wakako; Sasaki, Nobumitsu; Goto, Kaku; Kaido, Masanori; Okuno, Tetsuro; Mise, Kazuyuki

    2005-04-01

    Brome mosaic virus (BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generated in planta that exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.

  3. Pinus radiata bark extract induces caspase-independent apoptosis-like cell death in MCF-7 human breast cancer cells.

    PubMed

    Venkatesan, Thamizhiniyan; Choi, Young-Woong; Mun, Sung-Phil; Kim, Young-Kyoon

    2016-10-01

    In the present study, we investigated the anticancer activity of Pinus radiata bark extract (PRE) against MCF-7 human breast cancer cells. First, we observed that PRE induces potent cytotoxic effects in MCF-7 cells. The cell death had features of cytoplasmic vacuolation, plasma membrane permeabilization, chromatin condensation, phosphatidylserine externalization, absence of executioner caspase activation, insensitivity to z-VAD-fmk (caspase inhibitor), increased accumulation of autophagic markers, and lysosomal membrane permeabilization (LMP). Both the inhibition of early stage autophagy flux and lysosomal cathepsins did not improve cell viability. The antioxidant, n-acetylcysteine, and the iron chelator, deferoxamine, failed to restore the lysosomal integrity indicating that PRE-induced LMP is independent of oxidative stress. This was corroborated with the absence of enhanced ROS production in PRE-treated cells. Chelation of both intracellular calcium and zinc promotes PRE-induced LMP. Geranylgeranylacetone, an inducer of Hsp70 expression, also had no significant protective effect on PRE-induced LMP. Moreover, we found that PRE induces endoplasmic reticulum (ER) stress and mitochondrial membrane depolarization in MCF-7 cells. The ER stress inhibitor, 4-PBA, did not restore the mitochondrial membrane integrity, whereas cathepsin inhibitors demonstrated significant protective effects. Collectively, our results suggest that PRE induces an autophagic block, LMP, ER stress, and mitochondrial dysfunction in MCF-7 cells. However, further studies are clearly warranted to explore the exact mechanism behind the anticancer activity of PRE in MCF-7 human breast cancer cells.

  4. Atrial natriuretic peptide degradation by CPA47 cells - Evidence for a divalent cation-independent cell-surface proteolytic activity

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Chen, Y. M.; Whitson, P. A.

    1992-01-01

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  5. Atrial natriuretic peptide degradation by CPA47 cells - Evidence for a divalent cation-independent cell-surface proteolytic activity

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Chen, Y. M.; Whitson, P. A.

    1992-01-01

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  6. Adipose Tissue Dendritic Cells Are Independent Contributors to Obesity-Induced Inflammation and Insulin Resistance.

    PubMed

    Cho, Kae Won; Zamarron, Brian F; Muir, Lindsey A; Singer, Kanakadurga; Porsche, Cara E; DelProposto, Jennifer B; Geletka, Lynn; Meyer, Kevin A; O'Rourke, Robert W; Lumeng, Carey N

    2016-11-01

    Dynamic changes of adipose tissue leukocytes, including adipose tissue macrophage (ATM) and adipose tissue dendritic cells (ATDCs), contribute to obesity-induced inflammation and metabolic disease. However, clear discrimination between ATDC and ATM in adipose tissue has limited progress in the field of immunometabolism. In this study, we use CD64 to distinguish ATM and ATDC, and investigated the temporal and functional changes in these myeloid populations during obesity. Flow cytometry and immunostaining demonstrated that the definition of ATM as F4/80(+)CD11b(+) cells overlaps with other leukocytes and that CD45(+)CD64(+) is specific for ATM. The expression of core dendritic cell genes was enriched in CD11c(+)CD64(-) cells (ATDC), whereas core macrophage genes were enriched in CD45(+)CD64(+) cells (ATM). CD11c(+)CD64(-) ATDCs expressed MHC class II and costimulatory receptors, and had similar capacity to stimulate CD4(+) T cell proliferation as ATMs. ATDCs were predominantly CD11b(+) conventional dendritic cells and made up the bulk of CD11c(+) cells in adipose tissue with moderate high-fat diet exposure. Mixed chimeric experiments with Ccr2(-/-) mice demonstrated that high-fat diet-induced ATM accumulation from monocytes was dependent on CCR2, whereas ATDC accumulation was less CCR2 dependent. ATDC accumulation during obesity was attenuated in Ccr7(-/-) mice and was associated with decreased adipose tissue inflammation and insulin resistance. CD45(+)CD64(+) ATM and CD45(+)CD64(-)CD11c(+) ATDCs were identified in human obese adipose tissue and ATDCs were increased in s.c. adipose tissue compared with omental adipose tissue. These results support a revised strategy for unambiguous delineation of ATM and ATDC, and suggest that ATDCs are independent contributors to adipose tissue inflammation during obesity. Copyright © 2016 by The American Association of Immunologists, Inc.

  7. Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

    PubMed

    Fiorentino, Ilaria; Gualtieri, Roberto; Barbato, Vincenza; Mollo, Valentina; Braun, Sabrina; Angrisani, Alberto; Turano, Mimmo; Furia, Maria; Netti, Paolo A; Guarnieri, Daniela; Fusco, Sabato; Talevi, Riccardo

    2015-01-15

    Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Kinase-independent function of RIP1, critical for mature T-cell survival and proliferation

    PubMed Central

    Dowling, John P; Cai, Yubo; Bertin, John; Gough, Peter J; Zhang, Jianke

    2016-01-01

    The death receptor, Fas, triggers apoptotic death and is essential for maintaining homeostasis in the peripheral lymphoid organs. RIP1 was originally cloned when searching for Fas-binding proteins and was later shown to associate also with the signaling complex of TNFR1. Although Fas exclusively induces apoptosis, TNFR1 primarily activates the pro-survival/pro-inflammatory NF-κB pathway. Mutations in Fas lead to lymphoproliferative (lpr) diseases, and deletion of TNFR1 results in defective innate immune responses. However, the function of RIP1 in the adult lymphoid system has not been well understood, primarily owing to perinatal lethality in mice lacking the entire RIP1 protein in germ cells. This current study investigated the requirement for RIP1 in the T lineage using viable RIP1 mutant mice containing a conditional and kinase-dead RIP1 allele. Disabling the kinase activity of RIP1 had no obvious impact on the T-cell compartment. However, T-cell-specific deletion of RIP1 led to a severe T-lymphopenic condition, owing to a dramatically reduced mature T-cell pool in the periphery. Interestingly, the immature T-cell compartment in the thymus appeared intact. Further analysis showed that mature RIP1−/− T cells were severely defective in antigen receptor-induced proliferative responses. Moreover, the RIP1−/− T cells displayed greatly increased death and contained elevated caspase activities, an indication of apoptosis. In total, these results revealed a novel, kinase-independent function of RIP1, which is essential for not only promoting TCR-induced proliferative responses but also in blocking apoptosis in mature T cells. PMID:27685623

  9. Induction of p53-independent growth inhibition in lung carcinoma cell A549 by gypenosides

    PubMed Central

    Liu, Jung-Sen; Chiang, Tzu-Hsuan; Wang, Jinn-Shyan; Lin, Li-Ju; Chao, Wei-Chih; Inbaraj, Baskaran Stephen; Lu, Jyh-Feng; Chen, Bing-Huei

    2015-01-01

    The objectives of this study are to investigate antiproliferative effect and mechanisms of bioactive compounds from Gynostemma pentaphyllum (G. pentaphyllum) on lung carcinoma cell A549. Saponins, carotenoids and chlorophylls were extracted and fractionated by column chromatography, and were subjected to high-performance liquid chromatography-mass spectrometry analyses. The saponin fraction, which consisted mainly of gypenoside (Gyp) XXII and XXIII, rather than the carotenoid and chlorophyll ones, was effective in inhibiting A549 cell growth in a concentration- and a time-dependent manner as evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The estimated half maximal inhibitory concentration (IC50) of Gyp on A549 cells was 30.6 μg/ml. Gyp was further demonstrated to induce an apparent arrest of the A549 cell cycle at both the S phase and the G2/M phase, accompanied by a concentration- and a time-dependent increase in the proportions of both the early and late apoptotic cells. Furthermore, Gyp down-regulated cellular expression of cyclin A and B as well as BCL-2, while up-regulated the expression of BAX, DNA degradation factor 35 KD, poly [ADP-ribose] polymerase 1, p53, p21 and caspase-3. Nevertheless, both the treatment of a p53 inhibitor, pifithrin-α, and the small hairpin RNA-mediated p53 knockdown in the A549 cells did not alter the growth inhibition effect induced by Gyp. As a result, the cell cycle arrest and apoptosis of A549 cells induced by Gyp would most likely proceed through p53-independent pathway(s). PMID:25781909

  10. Independent Orbiter Assessment (IOA): Analysis of the electrical power generation/fuel cell powerplant subsystem

    NASA Technical Reports Server (NTRS)

    Brown, K. L.; Bertsch, P. J.

    1986-01-01

    Results of the Independent Orbiter Assessment (IOA) of the Failure Modes and Effects Analysis (FMEA) and Critical Items List (CIL) are presented. The IOA approach features a top-down analysis of the hardware to determine failure modes, criticality, and potential critical items. To preserve independence, this analysis was accomplished without reliance upon the results contained within the NASA FMEA/CIL documentation. This report documents the independent analysis results corresponding to the Orbiter Electrical Power Generation (EPG)/Fuel Cell Powerplant (FCP) hardware. The EPG/FCP hardware is required for performing functions of electrical power generation and product water distribution in the Orbiter. Specifically, the EPG/FCP hardware consists of the following divisions: (1) Power Section Assembly (PSA); (2) Reactant Control Subsystem (RCS); (3) Thermal Control Subsystem (TCS); and (4) Water Removal Subsystem (WRS). The IOA analysis process utilized available EPG/FCP hardware drawings and schematics for defining hardware assemblies, components, and hardware items. Each level of hardware was evaluated and analyzed for possible failure modes and effects. Criticality was assigned based upon the severity of the effect for each failure mode.

  11. Polarization-independent efficiency enhancement of organic solar cells by using 3-dimensional plasmonic electrode

    NASA Astrophysics Data System (ADS)

    Li, Xuanhua; Choy, Wallace C. H.; Ren, Xingang; Xin, Jianzhuo; Lin, Peng; Leung, Dennis C. W.

    2013-04-01

    Plasmonic back reflectors have recently become a promising strategy for realizing efficient organic solar cell (OSCs). Since plasmonic effects are strongly sensitive to light polarization, it is highly desirable to simultaneously achieve polarization-independent response and enhanced power conversion efficiency (PCE) by designing the nanostructured geometry of plasmonic reflector electrode. Here, through a strategic analysis of 2-dimensional grating (2D) and 3-dimensional patterns (3D), with similar periodicity as a plasmonic back reflector, we find that the OSCs with 3D pattern achieve the best PCE enhancement by 24.6%, while the OSCs with 2D pattern can offer 17.5% PCE enhancement compared to the optimized control OSCs. Importantly, compared with the 2D pattern, the 3D pattern shows a polarization independent plasmonic response, which will greatly extend its uses in photovoltaic applications. This work shows the significances of carefully selecting and designing geometry of plasmonic nanostructures in achieving high-efficient, polarization-independent plasmonic OSCs.

  12. Exact, time-independent estimation of clone size distributions in normal and mutated cells.

    PubMed

    Roshan, A; Jones, P H; Greenman, C D

    2014-10-06

    Biological tools such as genetic lineage tracing, three-dimensional confocal microscopy and next-generation DNA sequencing are providing new ways to quantify the distribution of clones of normal and mutated cells. Understanding population-wide clone size distributions in vivo is complicated by multiple cell types within observed tissues, and overlapping birth and death processes. This has led to the increased need for mathematically informed models to understand their biological significance. Standard approaches usually require knowledge of clonal age. We show that modelling on clone size independent of time is an alternative method that offers certain analytical advantages; it can help parametrize these models, and obtain distributions for counts of mutated or proliferating cells, for example. When applied to a general birth-death process common in epithelial progenitors, this takes the form of a gambler's ruin problem, the solution of which relates to counting Motzkin lattice paths. Applying this approach to mutational processes, alternative, exact, formulations of classic Luria-Delbrück-type problems emerge. This approach can be extended beyond neutral models of mutant clonal evolution. Applications of these approaches are twofold. First, we resolve the probability of progenitor cells generating proliferating or differentiating progeny in clonal lineage tracing experiments in vivo or cell culture assays where clone age is not known. Second, we model mutation frequency distributions that deep sequencing of subclonal samples produce.

  13. Exact, time-independent estimation of clone size distributions in normal and mutated cells

    PubMed Central

    Roshan, A.; Jones, P. H.; Greenman, C. D.

    2014-01-01

    Biological tools such as genetic lineage tracing, three-dimensional confocal microscopy and next-generation DNA sequencing are providing new ways to quantify the distribution of clones of normal and mutated cells. Understanding population-wide clone size distributions in vivo is complicated by multiple cell types within observed tissues, and overlapping birth and death processes. This has led to the increased need for mathematically informed models to understand their biological significance. Standard approaches usually require knowledge of clonal age. We show that modelling on clone size independent of time is an alternative method that offers certain analytical advantages; it can help parametrize these models, and obtain distributions for counts of mutated or proliferating cells, for example. When applied to a general birth–death process common in epithelial progenitors, this takes the form of a gambler's ruin problem, the solution of which relates to counting Motzkin lattice paths. Applying this approach to mutational processes, alternative, exact, formulations of classic Luria–Delbrück-type problems emerge. This approach can be extended beyond neutral models of mutant clonal evolution. Applications of these approaches are twofold. First, we resolve the probability of progenitor cells generating proliferating or differentiating progeny in clonal lineage tracing experiments in vivo or cell culture assays where clone age is not known. Second, we model mutation frequency distributions that deep sequencing of subclonal samples produce. PMID:25079870

  14. T-cell growth transformation by herpesvirus saimiri is independent of STAT3 activation.

    PubMed

    Heck, Elke; Lengenfelder, Doris; Schmidt, Monika; Müller-Fleckenstein, Ingrid; Fleckenstein, Bernhard; Biesinger, Brigitte; Ensser, Armin

    2005-05-01

    Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 (src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.

  15. Highly polarized Th17 cells induce EAE via a T-bet independent mechanism.

    PubMed

    Grifka-Walk, Heather M; Lalor, Stephen J; Segal, Benjamin M

    2013-11-01

    In the MOG35-55 induced EAE model, autoreactive Th17 cells that accumulate in the central nervous system acquire Th1 characteristics via a T-bet dependent mechanism. It remains to be determined whether Th17 plasticity and encephalitogenicity are causally related to each other. Here, we show that IL-23 polarized T-bet(-/-) Th17 cells are unimpaired in either activation or proliferation, and induce higher quantities of the chemokines RANTES and CXCL2 than WT Th17 cells. Unlike their WT counterparts, T-bet(-/-) Th17 cells retain an IL-17(hi) IFN-γ(neg-lo) cytokine profile following adoptive transfer into syngeneic hosts. This population of highly polarized Th17 effectors is capable of mediating EAE, albeit with a milder clinical course. It has previously been reported that the signature Th1 and Th17 effector cytokines, IFN-γ and IL-17, are dispensable for the development of autoimmune demyelinating disease. The current study demonstrates that the "master regulator" transcription factor, T-bet, is also not universally required for encephalitogenicity. Our results contribute to a growing body of data showing heterogeneity of myelin-reactive T cells and the independent mechanisms they employ to inflict damage to central nervous system tissues, complicating the search for therapeutic targets relevant across the spectrum of individuals with multiple sclerosis.

  16. Terminal Uridyltransferase Enzyme Zcchc11 Promotes Cell Proliferation Independent of Its Uridyltransferase Activity*

    PubMed Central

    Blahna, Matthew T.; Jones, Matthew R.; Quinton, Lee J.; Matsuura, Kori Y.; Mizgerd, Joseph P.

    2011-01-01

    Zcchc11 is a uridyltransferase protein with enzymatic activity directed against diverse RNA species. On the basis of its known uridylation targets, we hypothesized that Zcchc11 might regulate cell proliferation. Confirming this, loss-of-function and complementary gain-of-function experiments consistently revealed that Zcchc11 promotes the transition from G1 to S phase of the cell cycle. This activity takes place through both Rb-dependent and Rb-independent mechanisms by promoting the expression of multiple G1-associated proteins, including cyclins D1 and A and CDK4. Surprisingly, a Zcchc11 construct with point mutations inactivating the uridyltransferase domain enhanced cell proliferation as effectively as wild-type Zcchc11. Furthermore, truncated mutant constructs revealed that the cell cycle effects of Zcchc11 were driven by the N-terminal region of the protein that lacks the RNA-binding domains and uridyltransferase activity of the full protein. Therefore, the N-terminal portion of Zcchc11, which lacks nucleotidyltransferase capabilities, is biologically active and mediates a previously unrecognized role for Zcchc11 in facilitating cell proliferation. PMID:22006926

  17. Agarol, an ergosterol derivative from Agaricus blazei, induces caspase-independent apoptosis in human cancer cells.

    PubMed

    Shimizu, Takamitsu; Kawai, Junya; Ouchi, Kenji; Kikuchi, Haruhisa; Osima, Yoshiteru; Hidemi, Rikiishi

    2016-04-01

    Agaricus blazei (A. blazei) is a mushroom with many biological effects and active ingredients. We purified a tumoricidal substance from A. blazei, an ergosterol derivative, and named it 'Agarol'. Cytotoxic effects of Agarol were determined by the MTT assay using A549, MKN45, HSC-3, and HSC-4 human carcinoma cell lines treated with Agarol. Apoptosis was detected by flow cytometry analysis. Reactive oxygen species (ROS) levels and mitochondria membrane potential (∆ψm) were also determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related proteins. Agarol predominantly induced apoptosis in two p53-wild cell lines (A549 and MKN45) compared to the other p53-mutant cell lines (HSC-3 and HSC-4). Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of ROS, reduced ∆ψm, release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol, upregulation of Bax, and downregulation of Bcl-2. Caspase-3 activities did not increase, and z-VAD-fmk, a caspase inhibitor, did not inhibit the Agarol-induced apoptosis. These findings indicate that Agarol induces caspase-independent apoptosis in human carcinoma cells through a mitochondrial pathway. The in vivo anticancer activity of Agarol was confirmed in a xenograft murine model. This study suggests a molecular mechanism by which Agarol induces apoptosis in human carcinoma cells and indicates the potential use of Agarol as an anticancer agent.

  18. Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin

    PubMed Central

    Mallinjoud, Pierre; Villemin, Jean-Philippe; Mortada, Hussein; Polay Espinoza, Micaela; Desmet, François-Olivier; Samaan, Samaan; Chautard, Emilie; Tranchevent, Léon-Charles; Auboeuf, Didier

    2014-01-01

    Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type–specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution. PMID:24307554

  19. p53-Independent, normal stem cell sparing epigenetic differentiation therapy for myeloid and other malignancies.

    PubMed

    Saunthararajah, Yogen; Triozzi, Pierre; Rini, Brian; Singh, Arun; Radivoyevitch, Tomas; Sekeres, Mikkael; Advani, Anjali; Tiu, Ramon; Reu, Frederic; Kalaycio, Matt; Copelan, Ed; Hsi, Eric; Lichtin, Alan; Bolwell, Brian

    2012-02-01

    Cytotoxic chemotherapy for acute myeloid leukemia (AML) usually produces only temporary remissions, at the cost of significant toxicity and risk for death. One fundamental reason for treatment failure is that it is designed to activate apoptosis genes (eg, TP53) that may be unavailable because of mutation or deletion. Unlike deletion of apoptosis genes, genes that mediate cell cycle exit by differentiation are present in myelodysplastic syndrome (MDS) and AML cells but are epigenetically repressed: MDS/AML cells express high levels of key lineage-specifying transcription factors. Mutations in these transcription factors (eg, CEBPA) or their cofactors (eg., RUNX1) affect transactivation function and produce epigenetic repression of late-differentiation genes that antagonize MYC. Importantly, this aberrant epigenetic repression can be redressed clinically by depleting DNA methyltransferase 1 (DNMT1, a central component of the epigenetic network that mediates transcription repression) using the deoxycytidine analogue decitabine at non-cytotoxic concentrations. The DNMT1 depletion is sufficient to trigger upregulation of late-differentiation genes and irreversible cell cycle exit by p53-independent differentiation mechanisms. Fortuitously, the same treatment maintains or increases self-renewal of normal hematopoietic stem cells, which do not express high levels of lineage-specifying transcription factors. The biological rationale for this approach to therapy appears to apply to cancers other than MDS/AML also. Decitabine or 5-azacytidine dose and schedule can be rationalized to emphasize this mechanism of action, as an alternative or complement to conventional apoptosis-based oncotherapy.

  20. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    SciTech Connect

    Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

    2016-03-25

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

  1. An Abscisic Acid-Activated and Calcium-Independent Protein Kinase from Guard Cells of Fava Bean.

    PubMed Central

    Li, J.; Assmann, S. M.

    1996-01-01

    Abscisic acid (ABA) regulation of stomatal aperture is known to involve both Ca2+-dependent and Ca2+-independent signal transduction pathways. Electrophysiological studies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+- independent protein kinase (AAPK) from guard cell protoplasts of fava bean. Autophosphorylation of AAPK was rapidly (~1 min) activated by ABA in a Ca2+- independent manner. ABA-activated autophosphorylation of AAPK occurred on serine but not on tyrosine residues and appeared to be guard cell specific. AAPK phosphorylated histone type III-S on serine and threonine residues, and its activity toward histone type III-S was markedly stimulated in ABA-treated guard cell protoplasts. Our results suggest that AAPK may play an important role in the Ca2+-independent ABA signaling pathways of guard cells. PMID:12239380

  2. An Abscisic Acid-Activated and Calcium-Independent Protein Kinase from Guard Cells of Fava Bean.

    PubMed

    Li, J.; Assmann, S. M.

    1996-12-01

    Abscisic acid (ABA) regulation of stomatal aperture is known to involve both Ca2+-dependent and Ca2+-independent signal transduction pathways. Electrophysiological studies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+- independent protein kinase (AAPK) from guard cell protoplasts of fava bean. Autophosphorylation of AAPK was rapidly (~1 min) activated by ABA in a Ca2+- independent manner. ABA-activated autophosphorylation of AAPK occurred on serine but not on tyrosine residues and appeared to be guard cell specific. AAPK phosphorylated histone type III-S on serine and threonine residues, and its activity toward histone type III-S was markedly stimulated in ABA-treated guard cell protoplasts. Our results suggest that AAPK may play an important role in the Ca2+-independent ABA signaling pathways of guard cells.

  3. Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells

    SciTech Connect

    Fischer, Barbara; Benzina, Sami; Jeannequin, Pierre; Dufour, Patrick; Bergerat, Jean-Pierre; Denis, Jean-Marc; Gueulette, John; Bischoff, Pierre L. . E-mail: Pierre.Bischoff@ircad.u-strasbg.fr

    2005-08-26

    We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis.

  4. Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

    PubMed Central

    Zheng, Feimeng; Yue, Caifeng; Li, Guohui; He, Bin; Cheng, Wei; Wang, Xi; Yan, Min; Long, Zijie; Qiu, Wanshou; Yuan, Zhongyu; Xu, Jie; Liu, Bing; Shi, Qian; Lam, Eric W.-F.; Hung, Mien-Chie; Liu, Quentin

    2016-01-01

    Centrosome-localized mitotic Aurora kinase A (AURKA) facilitates G2/M events. Here we show that AURKA translocates to the nucleus and causes distinct oncogenic properties in malignant cells by enhancing breast cancer stem cell (BCSC) phenotype. Unexpectedly, this function is independent of its kinase activity. Instead, AURKA preferentially interacts with heterogeneous nuclear ribonucleoprotein K (hnRNP K) in the nucleus and acts as a transcription factor in a complex that induces a shift in MYC promoter usage and activates the MYC promoter. Blocking AURKA nuclear localization inhibits this newly discovered transactivating function of AURKA, sensitizing resistant BCSC to kinase inhibition. These findings identify a previously unknown oncogenic property of the spatially deregulated AURKA in tumorigenesis and provide a potential therapeutic opportunity to overcome kinase inhibitor resistance. PMID:26782714

  5. Oxygen tension-independent protection against hypoxic cell killing in rat liver by low sodium.

    PubMed

    Ferrigno, Andrea; Di Pasqua, Laura G; Berardo, Clarissa; Siciliano, Veronica; Richelmi, Plinio; Vairetti, Mariapia

    2017-05-30

    The role of Na+ in hypoxic injury was evaluated by a time-course analysis of damage in isolated livers perfused with N2-saturated buffer containing standard (143 mM) or low (25 mM) Na+ levels. Trypan blue uptake was used to detect non-viable cells. Under hypoxia with standard-Na+, trypan blue uptake began at the border between pericentral areas and periportal regions and increased in the latter zone; using a low-Na+ buffer, no trypan blue zonation occurred but a homogenous distribution of dye was found associated with sinusoidal endothelial cell (SEC) staining. A decrease in hyaluronic acid (HA) uptake, index of SEC damage, was observed using a low-Na+ buffer. A time dependent injury was confirmed by an increase in LDH and TBARS levels with standard-Na+ buffer. Using low-Na+ buffer, SEC susceptibility appears elevated under hypoxia and hepatocytes was protected, in an oxygen independent manner.

  6. Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state

    PubMed Central

    Patani, R.; Hollins, A. J.; Wishart, T. M.; Puddifoot, C. A.; Álvarez, S.; de Lera, A. R.; Wyllie, D. J. A.; Compston, D. A. S.; Pedersen, R. A.; Gillingwater, T. H.; Hardingham, G. E.; Allen, N. D.; Chandran, S.

    2011-01-01

    A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro. PMID:21364553

  7. Methylseleninic acid potentiates apoptosis induced by chemotherapeutic drugs in androgen-independent prostate cancer cells.

    PubMed

    Hu, Hongbo; Jiang, Cheng; Ip, Clement; Rustum, Youcef M; Lü, Junxuan

    2005-03-15

    To test whether and how selenium enhances the apoptosis potency of selected chemotherapeutic drugs in prostate cancer (PCA) cells. DU145 and PC3 human androgen-independent PCA cells were exposed to minimal apoptotic doses of selenium and/or the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN38), the topoisomerase II inhibitor etoposide or the microtubule inhibitor paclitaxel/taxol. Apoptosis was measured by ELISA for histone-associated DNA fragments, by flow cytometric analysis of sub-G(1) fraction, and by immunoblot analysis of cleaved poly(ADP-ribose)polymerase. Pharmacologic inhibitors were used to manipulate caspases and c-Jun-NH(2)-terminal kinases (JNK). The methylselenol precursor methylseleninic acid (MSeA) increased the apoptosis potency of SN38, etoposide, or paclitaxel by several folds higher than the expected sum of the apoptosis induced by MSeA and each drug alone. The combination treatment did not further enhance JNK1/2 phosphorylation that was induced by each drug in DU145 cells. The JNK inhibitor SP600125 substantially decreased the activation of caspases and apoptosis induced by MSeA combination with SN38 or etoposide and completely blocked these events induced by MSeA/paclitaxel. The caspase-8 inhibitor zIETDfmk completely abolished apoptosis and caspase-9 and caspase-3 cleavage, whereas the caspase-9 inhibitor zLEHDfmk significantly decreased caspase-3 cleavage and apoptosis but had no effect on caspase-8 cleavage. None of these caspase inhibitors abolished JNK1/2 phosphorylation. A JNK-independent suppression of survivin by SN38 and etoposide, but not by paclitaxel, was also observed. In contrast to MSeA, selenite did not show any enhancing effect on the apoptosis induced by these drugs. MSeA enhanced apoptosis induced by cancer therapeutic drugs in androgen-independent PCA cells. In DU145 cells, the enhancing effect was primarily through interactions between MSeA and JNK-dependent targets to amplify the caspase-8-initiated

  8. Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non-small-cell lung cancer cell lines.

    PubMed

    Calles, Antonio; Kwiatkowski, Nicholas; Cammarata, Bernard K; Ercan, Dalia; Gray, Nathanael S; Jänne, Pasi A

    2015-01-01

    MET targeted therapies are under clinical evaluation for non-small-cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKI) against MET have varying degrees of specificity. Tivantinib (ARQ 197) is reported to be a non-ATP competitive selective MET inhibitor. We aimed to compare the activity of tivantinib to established MET TKIs in a panel of NSCLC cell lines characterized by their MET dependency and by different relevant genotypes. A549, H3122, PC9 and HCC827, their respective resistant clones PC9 GR4 and HCC827 GR6 and the MET amplified cell lines H1993 and EBC-1 were treated in vitro with tivantinib, crizotinib or PHA-665752. Crizotinib and PHA-665752 showed growth inhibition restricted to MET dependent cell lines. The pattern of activity was related to MET inhibition and downstream signaling inhibition of AKT and ERK1/2, resulting in G0/G1 cycle arrest and apoptosis. In contrast, tivantinib possessed more potent anti-proliferative activity that was not restricted to only MET dependent cell lines. Tivantinib did not inhibit cellular MET activity or phosphorylation of downstream signaling proteins AKT or ERK1/2 in either MET dependent or independent cell lines. Cell cycle analysis demonstrated that tivantinib induced a G2/M arrest and induced apoptosis. Tivantinib but not crizotinib effected microtubule dynamics, disrupting mitotic spindles by a mechanism consistent with it functioning as a microtubule depolymerizer. Tivantinib activity is independent of MET signaling in NSCLC and suggests alternative mechanisms of action that should be considered when interpreting the results from on-going clinical studies. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation.

    PubMed

    Ghosh, Paramita M; Malik, Shazli N; Bedolla, Roble G; Wang, Yu; Mikhailova, Margarita; Prihoda, Thomas J; Troyer, Dean A; Kreisberg, Jeffrey I

    2005-03-01

    In a previous report, we showed that increased activation of Akt, a downstream effector of phosphoinositide 3-kinase (PI3K) together with decreased activation of extracellular-signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, predicted poor clinical outcome in prostate cancer (Kreisberg et al. 2004 Cancer Research 64 5232-5236). We now show that Akt activation, but not ERK activation, is correlated with proliferation in human prostate tumors as estimated by the expression of the cell proliferation antigen Ki67. We verified these results in vitro, using the androgen-dependent prostate cancer cell line LNCaP and its androgen-independent clone C4-2 as models of prostate cancer of good and poor clinical outcome, respectively. C4-2 cells expressed higher Akt activation, lower ERK activation and increased proliferation compared with LNCaP cells, similar to cases of poor clinical outcome. The PI3K inhibitor LY294002, but not the MAPK/ERK kinase inhibitor PD98059, induced growth arrest in both cell lines. Transient transfection with constitutively active Akt increased proliferation while dominant negative Akt decreased it, thus showing that Akt plays an important role in prostate cancer proliferation. Akt regulates the expression and activation of the androgen receptor. Androgen receptor inhibition with Casodex induced growth arrest in LNCaP cells, but not in C4-2 cells. Another PI3K downstream effector, p70 S6 kinase, requires prior phosphorylation by mammalian target of rapamycin (mTOR) for complete activation. Activation of p70 S6 kinase was higher in C4-2 compared with LNCaP cells. Rapamycin, an mTOR inhibitor, had a growth-inhibitory effect in C4-2 cells, but not in LNCaP cells. Our data suggest a shift from a Casodex-sensitive proliferation pathway in LNCaP cells to a rapamycin-sensitive pathway in C4-2 cells.

  10. CD45/CD11b Positive Subsets of Adult Lung Anchorage-Independent Cells Harness Epithelial Stem Cells

    PubMed Central

    Peter, Yakov; Sen, Namita; Levantini, Elena; Keller, Steven; Ingenito, Edward P; Ciner, Aaron; Sackstein, Robert; Shapiro, Steven D

    2015-01-01

    Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between the stem/progenitor cells that proliferate or differentiate and the somatic cells of lung, we utilized a novel ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3, and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conserved population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. Over 50% of the AIC population was cKit+ or double-positive for CD45+ and CD11b+ antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. AICs undergo remodeling to form a cellular lining at the air/gel interface, and TGFβ1 treatment modifies protein expression, implying direct-differentiation of this population. These data confirm the active participation of clonogenic hematopietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which embrace sustained cell viability, proliferation, and differentiation, for use in studies of compensatory pulmonary growth. PMID:22585451

  11. CD45/CD11b positive subsets of adult lung anchorage-independent cells harness epithelial stem cells in culture.

    PubMed

    Peter, Yakov; Sen, Namita; Levantini, Elena; Keller, Steven; Ingenito, Edward P; Ciner, Aaron; Sackstein, Robert; Shapiro, Steven D

    2013-07-01

    Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between stem/progenitor cells that proliferate or differentiate and somatic cells of the lung, we used a novel organotypic ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free-floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3 and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conservative population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. More than 50% of the AIC population was c-KIT(+) or double-positive for CD45(+) and CD11b(+) antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. At the air/gel interface, AICs undergo remodeling to form a cellular lining, whereas TGF(β)1 treatment modifies protein expression properties to further imply a robust effect of the microenvironment on AIC phenotypic changes. These data confirm the active participation of clonogenic hematopoietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which license sustained cell viability, proliferation and differentiation, for use in studies of compensatory pulmonary growth. Copyright © 2012 John Wiley & Sons, Ltd.

  12. DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.

    PubMed

    Kalifa, Lidza; Gewandter, Jennifer S; Staversky, Rhonda J; Sia, Elaine A; Brookes, Paul S; O'Reilly, Michael A

    2014-10-01

    Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Blood Cell Palmitoleate-Palmitate Ratio Is an Independent Prognostic Factor for Amyotrophic Lateral Sclerosis

    PubMed Central

    Henriques, Alexandre; Blasco, Hélène; Fleury, Marie-Céline; Corcia, Philippe; Echaniz-Laguna, Andoni; Robelin, Laura; Rudolf, Gabrielle; Lequeu, Thiebault; Bergaentzle, Martine; Gachet, Christian; Pradat, Pierre-François; Marchioni, Eric; Andres, Christian R.; Tranchant, Christine; Gonzalez De Aguilar, Jose-Luis; Loeffler, Jean-Philippe

    2015-01-01

    Growing evidence supports a link between fatty acid metabolism and amyotrophic lateral sclerosis (ALS). Here we determined the fatty acid composition of blood lipids to identify markers of disease progression and survival. We enrolled 117 patients from two clinical centers and 48 of these were age and gender matched with healthy volunteers. We extracted total lipids from serum and blood cells, and separated fatty acid methyl esters by gas chromatography. We measured circulating biochemical parameters indicative of the metabolic status. Association between fatty acid composition and clinical readouts was studied, including ALS functional rating scale-revised (ALSFRS-R), survival, disease duration, site of onset and body mass index. Palmitoleate (16:1) and oleate (18:1) levels, and stearoyl-CoA desaturase indices (16:1/16:0 and 18:1/18:0) significantly increased in blood cells from ALS patients compared to healthy controls. Palmitoleate levels and 16:1/16:0 ratio in blood cells, but not body mass index or leptin concentrations, negatively correlated with ALSFRS-R decline over a six-month period (p<0.05). Multivariate Cox analysis, with age, body mass index, site of onset and ALSFRS-R as covariables, showed that blood cell 16:1/16:0 ratio was an independent prognostic factor for survival (hazard ratio=0.1 per unit of ratio, 95% confidence interval=0.01-0.57, p=0.009). In patients with high 16:1/16:0 ratio, survival at blood collection was extended by 10 months, as compared to patients with low ratio. The 16:1/16:0 index is an easy-to-handle parameter that predicts survival of ALS patients independently of body mass index. It therefore deserves further validation in larger cohorts for being used to assess disease outcome and effects of disease-modifying drugs. PMID:26147510

  14. DNA polymerase β-dependent cell survival independent of XRCC1 expression

    PubMed Central

    Horton, Julie K.; Gassman, Natalie R.; Dunigan, Brittany B.; Stefanick, Donna F.; Wilson, Samuel H.

    2014-01-01

    Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1+/+ and Xrcc1−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β- dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. PMID:25541391

  15. TSLP elicits IL-33–independent innate lymphoid cell responses to promote skin inflammation

    PubMed Central

    Kim, Brian S.; Siracusa, Mark C.; Saenz, Steven A.; Noti, Mario; Monticelli, Laurel A.; Sonnenberg, Gregory F.; Hepworth, Matthew R.; Van Voorhees, Abby S.; Comeau, Michael R.

    2013-01-01

    Innate lymphoid cells (ILCs) are a recently identified family of heterogeneous immune cells that can be divided into three groups based on their differential developmental requirements and expression of effector cytokines. Among these, group 2 ILCs produce the type 2 cytokines IL-5 and IL-13 and promote type 2 inflammation in the lung and intestine. However, whether group 2 ILCs reside in the skin and contribute to skin inflammation has not been characterized. Here, we identify for the first time a population of skin-resident group 2 ILCs present in healthy human skin that are enriched in lesional human skin from atopic dermatitis (AD) patients. Group 2 ILCs were also found in normal murine skin and were critical for the development of inflammation in a murine model of AD-like disease. Remarkably, in contrast to group 2 ILC responses in the intestine and lung, which are critically regulated by IL-33 and IL-25, ILC responses in the skin and skin-draining lymph nodes were independent of these canonical cytokines but were critically dependent on thymic stromal lymphopoietin (TSLP). Collectively, these results demonstrate an essential role for IL-33– and IL-25–independent group 2 ILCs in promoting skin inflammation. PMID:23363980

  16. Iron-independent phosphorylation of iron regulatory protein 2 regulates ferritin during the cell cycle.

    PubMed

    Wallander, Michelle L; Zumbrennen, Kimberly B; Rodansky, Eva S; Romney, S Joshua; Leibold, Elizabeth A

    2008-08-29

    Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

  17. Paclitaxel promotes differentiation of myeloid-derived suppressor cells into dendritic cells in vitro in a TLR4-independent manner.

    PubMed

    Michels, Tillmann; Shurin, Galina V; Naiditch, Hiam; Sevko, Alexandra; Umansky, Viktor; Shurin, Michael R

    2012-01-01

    Myeloid cells play a key role in the outcome of anti-tumor immunity and response to anti-cancer therapy, since in the tumor microenvironment they may exert both stimulatory and inhibitory pressures on the proliferative, angiogenic, metastatic, and immunomodulating potential of tumor cells. Therefore, understanding the mechanisms of myeloid regulatory cell differentiation is critical for developing strategies for the therapeutic reversal of myeloid derived suppressor cell (MDSC) accumulation in the tumor-bearing hosts. Here, using an in vitro model system, several potential mechanisms of the direct effect of paclitaxel on MDSC were tested, which might be responsible for the anti-tumor potential of low-dose paclitaxel therapy in mice. It was hypothesized that a decreased level of MDSC in vivo after paclitaxel administration might be due to (i) the blockage of MDSC generation, (ii) an induction of MDSC apoptosis, or (iii) the stimulation of MDSC differentiation. The results revealed that paclitaxel in ultra-low concentrations neither increased MDSC apoptosis nor blocked MDSC generation, but stimulated MDSC differentiation towards dendritic cells. This effect of paclitaxel was TLR4-independent since it was not diminished in cell cultures originated from TLR4-/- mice. These results support a new concept that certain chemotherapeutic agents in ultra-low non-cytotoxic doses may suppress tumor progression by targeting several cell populations in the tumor microenvironment, including MDSC.

  18. Glomerular common gamma chain confers B- and T-cell-independent protection against glomerulonephritis.

    PubMed

    Luque, Yosu; Cathelin, Dominique; Vandermeersch, Sophie; Xu, Xiaoli; Sohier, Julie; Placier, Sandrine; Xu-Dubois, Yi-Chun; Louis, Kevin; Hertig, Alexandre; Bories, Jean-Christophe; Vasseur, Florence; Campagne, Fabien; Di Santo, James P; Vosshenrich, Christian; Rondeau, Eric; Mesnard, Laurent

    2017-01-19

    Crescentic glomerulonephritis is a life-threatening renal disease that has been extensively studied by the experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN) model. Although T cells have a significant role in this model, athymic/nude mice and rats still develop severe renal disease. Here we further explored the contribution of intrinsic renal cells in the development of T-cell-independent GN lesions. Anti-GBM-GN was induced in three strains of immune-deficient mice (Rag2(-/-), Rag2(-/-)Il2rg(-/-), and Rag2(-/-)Il2rb(-/-)) that are devoid of either T/B cells or T/B/NK cells. The Rag2(-/-)Il2rg(-/-) or Rag2(-/-)Il2rb(-/-) mice harbor an additional deletion of either the common gamma chain (γC) or the interleukin-2 receptor β subunit (IL-2Rβ), respectively, impairing IL-15 signaling in particular. As expected, all these strains developed severe anti-GBM-GN. Additionally, bone marrow replenishment experiments allowed us to deduce a protective role for the glomerular-expressed γC during anti-GBM-GN. Given that IL-15 has been found highly expressed in nephritic kidneys despite the absence of lymphocytes, we then studied this cytokine in vitro on primary cultured podocytes from immune-deficient mice (Rag2(-/-)Il2rg(-/-) and Rag2(-/-)Il2rb(-/-)) compared to controls. IL-15 induced downstream activation of JAK1/3 and SYK in primary cultured podocytes. IL-15-dependent JAK/SYK induction was impaired in the absence of γC or IL-2Rβ. We found γC largely induced on podocytes during human glomerulonephritis. Thus, renal lesions are indeed modulated by intrinsic glomerular cells through the γC/IL-2Rβ receptor response, to date classically described only in immune cells.

  19. Cyclosporin A inhibits colon cancer cell growth independently of the calcineurin pathway

    PubMed Central

    Werneck, Miriam B.F.; Hottz, Eugênio; Bozza, Patrícia T.; Viola, João P.B.

    2012-01-01

    Chronic inflammation is a risk factor for the development of colon cancer, providing genotoxic insults, growth and pro-angiogenic factors that can promote tumorigenesis and tumor growth. Immunomodulatory agents can interfere with the inflammation that feeds cancer, but their impact on the transformed cell is poorly understood. The calcium/calcineurin signaling pathway, through activation of NFAT, is essential for effective immune responses, and its inhibitors cyclosporin A (CsA) and FK506 are used in the clinics to suppress immunity. Moreover, the kinases GSK3β and mTOR, modulated by PI-3K/Akt, can inhibit NFAT activity, suggesting a cross-talk between the calcium and growth factor signaling pathways. Both NFAT and mTOR activity have been associated with tumorigenesis. We therefore investigated the impact of calcineurin and PI-3K/mTOR inhibition in growth of human colon carcinoma cells. We show that despite the efficient inhibition of NFAT1 activity, FK506 promotes tumor growth, whereas CsA inhibits it due to a delay in cell cycle progression and induction of necroptosis. We found NFκB activation and mTORC1 activity not to be altered by CsA or FK506. Similarly, changes to mitochondrial homeostasis were equivalent upon treatment with these drugs. We further show that, in our model, NFAT1 activation is not modulated by PI3K/mTOR. We conclude that CsA slows cell cycle progression and induces necroptosis of human carcinoma cell lines in a TGFβ-, NFAT-, NFκB- and PI3K/mTOR-independent fashion. Nevertheless, our data suggest that CsA, in addition to its anti-inflammatory capacity, may target transformed colon and esophagus carcinoma cells without affecting non-transformed cells, promoting beneficial tumoristatic effects. PMID:22992618

  20. Spinigerin induces apoptotic like cell death in a caspase independent manner in Leishmania donovani.

    PubMed

    Sardar, A H; Das, S; Agnihorti, S; Kumar, M; Ghosh, A K; Abhishek, K; Kumar, A; Purkait, B; Ansari, M Y; Das, P

    2013-12-01

    Antimicrobial peptides (AMPs) are multifunctional components of the innate immune system. Chemotherapeutic agents used for treatment of visceral leishmaniasis (VL) are now threatened due to the emergence of acquired drug resistance and toxicity. AMPs are attractive alternative to conventional pharmaceuticals. In this study, first time we explored the antileishmanial activity of spinigerin originally derived from Pseudacanthotermes spiniger. Leishmania donovani promastigotes present apoptosis-like cell death upon exposure to spinigerin (IC50, 150 μM). The infection rate was reduced by 20% upon exposure to 150 μM spinigerin but no cytotoxicity on host macrophages was observed. Elevation of intracellular ROS level and down-regulation of two ROS detoxifying enzymes, ascorbate peroxidase (APx) and trypanothione reductase (TR) suggested essential role of ROS machinery during spinigerin mediated cell death. About 97% cell population was found to be Annexin-V positive; 44% cells being highly Annexin-V positive. Moreover, we observed morphological changes like cell rounding, nuclear condensation, oligonucleosomal DNA degradation and TUNEL positive cells without loss of membrane integrity upon spinigerin exposure, suggests apoptosis-like death. Interestingly, collapse in mitochondrial membrane potential and increased level of intracellular ROS and calcium were not associated with caspase like activity. Computational analysis suggests spiningerin interacts with trypanothione reductase and thus probably interferes its function to detoxify the toxic ROS level. Therefore, spinigerin induces apoptosis-like cell death in L. donovani in a caspase-independent manner. The study elucidates the antileishmanial property of spinigerin that may be considered for future chemotherapeutic option alone or adjunct with other drug regimens for improved treatment of visceral leishmaniasis.

  1. Molecular Mechanism of Action of Genistein and Related Phytoestrogens in Estrogen Receptor Dependent and Independent Growth of Breast Cancer Cells

    DTIC Science & Technology

    2000-07-01

    estradiol exert different effects on ER-mediated functions. In ER-negative cells, genistein and quercetin inhibited cell growth and induced apoptosis in a...dependent and -independent effects of five structurally related phytoestrogens on breast cancer cell growth and apoptosis . Our studies showed that genistein...breast cancer cells by genistein and quercetin included G2/M cell cycle arrest and alterations in cyclin B1 protein. Our results suggest that genistein and

  2. Bafilomycin A1 induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways

    PubMed Central

    Yan, Yumei; Jiang, Ke; Liu, Peng; Zhang, Xianbin; Dong, Xin; Gao, Jingchun; Liu, Quentin; Barr, Martin P.; Zhang, Quan; Hou, Xiukun; Meng, Songshu; Gong, Peng

    2016-01-01

    Hepatocellular carcinoma (HCC) is refractory to chemotherapies, necessitating novel effective agents. The lysosome inhibitor Bafilomycin A1 (BafA1) at high concentrations displays cytotoxicity in a variety of cancers. Here we show that BafA1 at nanomolar concentrations suppresses HCC cell growth in both 2 dimensional (2D) and 3D cultures. BafA1 induced cell cycle arrest in the G1 phase and triggered Cyclin D1 turnover in HCC cells in a dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) dependent manner. Notably, BafA1 induced caspase-independent cell death in HCC cells by impairing autophagy flux as demonstrated by elevated LC3 conversion and p62/SQSTM1 levels. Moreover, genetic ablation of LC3 significantly attenuated BafA1-induced cytotoxicity of HCC cells. We further demonstrate that pharmacological down-regulation or genetic depletion of p38 MAPK decreased BafA1-induced cell death via abolishment of BafA1-induced upregulation of Puma. Notably, knockdown of Puma impaired BafA1-induced HCC cell death, and overexpression of Puma enhanced BafA1-mediated HCC cell death, suggesting a role for Puma in BafA1-mediated cytotoxicity. Interestingly, pharmacological inhibition of JNK with SP600125 enhanced BafA1-mediated cytotoxicity both in vitro and in xenografts derived from HCC cells. Taken together, our data suggest that BafA1 may offer potential as an effective therapy for HCC. PMID:27845389

  3. BOK displays cell death-independent tumor suppressor activity in non-small-cell lung carcinoma.

    PubMed

    Moravcikova, Erika; Krepela, Evzen; Donnenberg, Vera S; Donnenberg, Albert D; Benkova, Kamila; Rabachini, Tatiana; Fernandez-Marrero, Yuniel; Bachmann, Daniel; Kaufmann, Thomas

    2017-11-15

    As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients. © 2017 UICC.

  4. Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis.

    PubMed

    Eyster, Craig A; Higginson, Jason D; Huebner, Robert; Porat-Shliom, Natalie; Weigert, Roberto; Wu, Wells W; Shen, Rong-Fong; Donaldson, Julie G

    2009-05-01

    Clathrin-independent endocytosis (CIE) allows internalization of plasma membrane proteins lacking clathrin-targeting sequences, such as the major histocompatibility complex class I protein (MHCI), into cells. After internalization, vesicles containing MHCI fuse with transferrin-containing endosomes generated from clathrin-dependent endocytosis. In HeLa cells, MHCI is subsequently routed to late endosomes or recycled back out to the plasma membrane (PM) in distinctive tubular carriers. Arf6 is associated with endosomal membranes carrying CIE cargo and expression of an active form of Arf6 leads to the generation of vacuolar structures that trap CIE cargo immediately after endocytosis, blocking the convergence with transferrin-containing endosomes. We isolated these trapped vacuolar structures and analyzed their protein composition by mass spectrometry. Here we identify and validate six new endogenous cargo proteins (CD44, CD55, CD98, CD147, Glut1, and ICAM1) that use CIE to enter cells. CD55 and Glut1 appear to closely parallel the trafficking of MHCI, merging with transferrin endosomes before entering the recycling tubules. In contrast, CD44, CD98, and CD147 appear to directly enter the recycling tubules and by-pass the merge with EEA1-positive, transferrin-containing endosomes. This divergent itinerary suggests that sorting may occur along this CIE pathway. Furthermore, the identification of new cargo proteins will assist others studying CIE in different cell types and tissues.

  5. Stromal fibroblasts facilitate cancer cell invasion by a novel invadopodia-independent matrix degradation process

    PubMed Central

    Krueger, Eugene W.; Chen, Jing; Qiang, Li; McNiven, Mark A.

    2015-01-01

    Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process utilizes complex, actin-based, specializations of the plasma membrane termed invadopodia that act both to sequester and release matrix metalloproteinases. Here we report that cells of mesenchymal origin, including tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. These degradative sites lack the punctate shape of conventional invadopodia to spread along the cell base and are reticular and/or fibrous in character. In marked contrast to invadopodia, this degradation does not require the action of Src kinase, Cdc42, or Dyn2. Rather, inhibition of Dyn2 causes a dramatic upregulation of stromal matrix degradation. Further, expression and activity of matrix metalloproteinases are differentially regulated between tumor cells and stromal fibroblasts. This matrix remodeling by fibroblasts increases the invasive capacity of tumor cells, thereby illustrating how the tumor microenvironment can contribute to metastasis. These findings provide evidence for a novel matrix remodeling process conducted by stromal fibroblasts that is substantially more effective than conventional invadopodia, distinct in structural organization, and regulated by disparate molecular mechanisms. PMID:25982272

  6. Verteporfin exhibits YAP-independent anti-proliferative and cytotoxic effects in endometrial cancer cells.

    PubMed

    Dasari, Venkata Ramesh; Mazack, Virginia; Feng, Wen; Nash, John; Carey, David J; Gogoi, Radhika

    2017-04-25

    Endometrial Carcinoma (EMCA) is the most common gynecologic malignancy and the fourth most common malignancy in women in the United States. Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. Verteporfin (VP), a benzoporphyrin derivative, was identified as an inhibitor of YAP-TEAD interaction. We investigated the therapeutic efficacy and mechanism of VP in EMCA. The efficacy of VP on cell viability, cytotoxicity and invasion was assayed in EMCA cell lines. An organoid model system was also developed to test the effect of VP on apoptotic markers in an in vitro model system. Treatment with VP resulted in a decrease in cell viability, invasion and an increase in cytotoxicity of EMCA cells. These effects occurred as early as 15 minutes following treatment. Similarly, VP treatment versus vehicle control increased apoptosis in human organoid model systems. Quantitative RT-PCR, cDNA based RTPCR array analysis and western blotting were performed to investigate the mechanism of VP action. The cytotoxic and anti-proliferative effects appeared to be independent of its effect on YAP. Our results suggest that VP is a promising chemotherapeutic agent for the treatment of endometrial cancer.

  7. Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

    PubMed

    Molnár, Eszter; Swamy, Mahima; Holzer, Martin; Beck-García, Katharina; Worch, Remigiusz; Thiele, Christoph; Guigas, Gernot; Boye, Kristian; Luescher, Immanuel F; Schwille, Petra; Schubert, Rolf; Schamel, Wolfgang W A

    2012-12-14

    The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

  8. Air Bubble Contact with Endothelial Cells Causes a Calcium-Independent Loss in Mitochondrial Membrane Potential

    PubMed Central

    Sobolewski, Peter; Kandel, Judith; Eckmann, David M.

    2012-01-01

    Objective Gas microembolism remains a serious risk associated with surgical procedures and decompression. Despite this, the signaling consequences of air bubbles in the vasculature are poorly understood and there is a lack of pharmacological therapies available. Here, we investigate the mitochondrial consequences of air bubble contact with endothelial cells. Methods and Results Human umbilical vein endothelial cells were loaded with an intracellular calcium indicator (Fluo-4) and either a mitochondrial calcium indicator (X-Rhod-1) or mitochondrial membrane potential indicator (TMRM). Contact with 50–150 µm air bubbles induced concurrent rises in intracellular and mitochondrial calcium, followed by a loss of mitochondrial membrane potential. Pre-treating cells with 1 µmol/L ruthenium red, a TRPV family calcium channel blocker, did not protect cells from the mitochondrial depolarization, despite blocking the intracellular calcium response. Mitigating the interactions between the air-liquid interface and the endothelial surface layer with 5% BSA or 0.1% Pluronic F-127 prevented the loss of mitochondrial membrane potential. Finally, inhibiting protein kinase C-α (PKCα), with 5 µmol/L Gö6976, protected cells from mitochondrial depolarization, but did not affect the intracellular calcium response. Conclusions Our results indicate that air bubble contact with endothelial cells activates a novel, calcium-independent, PKCα-dependent signaling pathway, which results in mitochondrial depolarization. As a result, mitochondrial dysfunction is likely to be a key contributor to the pathophysiology of gas embolism injury. Further, this connection between the endothelial surface layer and endothelial mitochondria may also play an important role in vascular homeostasis and disease. PMID:23091614

  9. ZBTB16 induces osteogenic differentiation marker genes in dental follicle cells independent from RUNX2.

    PubMed

    Felthaus, Oliver; Gosau, Martin; Morsczeck, Christian

    2014-05-01

    Dental follicle cells (DFCs) are neural crest cell-derived cells and the genuine precursor cells of cementoblast and alveolar osteoblasts. After osteogenic differentiation, expression levels of the transcription factor zinc factor and BTB domain containing 16 (ZBTB16) were significantly increased. ZBTB16 is associated with the process of osteogenic differentiation in bone marrow-derived mesenchymal stem cells and crucial for the expression of the osteogenic transcription factor runt-related transcription factor 2 (RUNX2). It is proposed that ZBTB16 plays also a crucial role for the differentiation of DFCs into osteoblasts. In this study, the differentiation of DFCs by alkaline phosphatase (ALP) activity measurement, alizarin red staining, and electron-dispersive x-ray spectrometry (EDX) analysis is investigated. The expression of ZBTB16 during osteogenic differentiation and the expression of osteogenic differentiation markers were assessed by real-time reverse transcription polymerase chain reaction. Glucocorticoid stimulation was inhibited using RU486 (11β-[p-(Dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), and ZBTB16 was overexpressed via transient transfection of an expression vector. After the initiation of osteogenic differentiation, ZBTB16 levels were increased highly in DFCs, whereas RUNX2 was expressed constitutively only. An EDX analysis verified the differentiation of DFCs into osteoblast-like cells because clusters of mineralization consisted of hydroxyapatite. ZBTB16 induced the expression of nuclear receptor subfamily 4, group A, member 3; osteocalcin; and stanniocalcin 1 (STC1) but not of RUNX2 and ALP in DFCs. STC1 was upregulated in DFCs downstream of ZBTB16 and after the osteogenic differentiation. The overexpression of STC1 in DFCs increased the expression of ZBTB16 and specific markers for biomineralization. The present study shows that ZBTB16 induced the expression of osteogenic differentiation markers independently of

  10. Adipocyte mitochondrial function is reduced in human obesity independent of fat cell size.

    PubMed

    Yin, Xiao; Lanza, Ian R; Swain, James M; Sarr, Michael G; Nair, K Sreekumaran; Jensen, Michael D

    2014-02-01

    It has been suggested that mitochondrial dysfunction in adipocytes contributes to obesity-related metabolic complications. However, obesity results in adipocyte hypertrophy, and large and small adipocytes from the same depot have different characteristics, raising the possibility that obesity-related mitochondrial defects are an inherent function of large adipocytes. Our goal was to examine whether obesity, independent of fat cell size and fat depot, is associated with mitochondria dysfunction. We compared adipocyte mitochondrial function using a cross-sectional comparison study design. The studies were performed at Mayo Clinic, an academic medical center. Omental and/or abdominal subcutaneous adipose samples were collected from 20 age-matched obese and nonobese nondiabetic men and women undergoing either elective abdominal surgery or research needle biopsy. Interventions were not conducted as part of these studies. We measured mitochondrial DNA abundance, oxygen consumption rates, and citrate synthase activity from populations of large and small adipocytes (separated with differential floatation). For both omental and subcutaneous adipocytes, at the cell and organelle level, oxygen consumption rates and citrate synthase activity were significantly reduced in cells from obese compared with nonobese volunteers, even when matched for cell size by comparing large adipocytes from nonobese and small adipocytes from obese. Adipocyte mitochondrial content was not significantly different between obese and nonobese volunteers. Mitochondrial function and content parameters were not different between small and large cells, omental, and subcutaneous adipocytes from the same person. Adipocyte mitochondrial oxidative capacity is reduced in obese compared with nonobese adults and this difference is not due to cell size differences. Adipocyte mitochondrial dysfunction in obesity is therefore related to overall adiposity rather than adipocyte hypertrophy.

  11. Galectin-3 promotes caspase-independent cell death of HIV-1-infected macrophages.

    PubMed

    Xue, Jing; Fu, Chunyan; Cong, Zhe; Peng, Lingjuan; Peng, Zhuoying; Chen, Ting; Wang, Wei; Jiang, Hong; Wei, Qiang; Qin, Chuan

    2017-01-01

    HIV-1-infected macrophages are a key contributor to the formation of a viral reservoir and new treatment strategies focus on eliminating this pool of virus. Galectin-3 is a potent apoptosis-inducing protein that regulates diverse cellular activities. In the present study, we investigated whether galectin-3 could induce cell death in HIV-1-infected macrophages using HIV-1-infected THP1 monocytes (THP1-MNs) and THP1-derived macrophages (THP1-MΦs) as in vitro cellular models. We found that THP1-MΦs were more resistant than the THP1-MNs to HIV-1 infection-induced death, and that HIV-1 infection of the THP1-MΦs increased expression of the anti-apoptotic proteins Mcl-1, Bcl-2 and Bcl-xL. Additionally, galectin-3 but not FasL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand or TNF-α, could induce cell death in HIV-1-infected THP1-MΦs. A similar result was shown for primary human monocyte-derived macrophages. Galectin-3-induced cell death was also significantly increased in macrophages obtained from SIVmac251-infected macaques compared to that of macrophages from healthy macaques. Furthermore, galectin-3-induced cell death in HIV-1-infected THP1-MΦs was caspase independent. Interestingly, endonuclease G (Endo G) was increased in the nucleus and decreased in the cytoplasm of galectin-3-treated cells; thus, galectin-3-induced cell death in HIV-1-infected THP1-MΦs is most likely related to the translocation of Endo G from the cytoplasm to the nucleus. These findings suggest that galectin-3 may potentially aid in the eradication of HIV-1/SIV-infected macrophages.

  12. A NPxY-independent {beta}5 integrin activation signal regulates phagocytosis of apoptotic cells

    SciTech Connect

    Singh, Sukhwinder; D'mello, Veera; Henegouwen, Paul van Bergen en; Birge, Raymond B.

    2007-12-21

    Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the {beta} chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 ({beta}5) integrin cDNA was expressed in {alpha}v positive, {beta}5 and {beta}3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, {beta}5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing {beta}5 mutant (Y750A) abrogated adhesion and {beta}5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of {beta}5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a {beta}5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 {mu}m diameter microspheres developed as apoptotic cell mimetics. The critical sequences in {beta}5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of {beta}5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the {beta}5 cytoplasmic tail for adhesion and phagocytosis.

  13. Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells.

    PubMed

    Sappino, André-Pascal; Buser, Raphaële; Lesne, Laurence; Gimelli, Stefania; Béna, Frédérique; Belin, Dominique; Mandriota, Stefano J

    2012-03-01

    Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics. Copyright © 2012 John Wiley & Sons, Ltd.

  14. Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination.

    PubMed

    Holtappels, Rafaela; Simon, Christian O; Munks, Michael W; Thomas, Doris; Deegen, Petra; Kühnapfel, Birgit; Däubner, Torsten; Emde, Simone F; Podlech, Jürgen; Grzimek, Natascha K A; Oehrlein-Karpi, Silke A; Hill, Ann B; Reddehase, Matthias J

    2008-06-01

    Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.

  15. Air bubble contact with endothelial cells causes a calcium-independent loss in mitochondrial membrane potential.

    PubMed

    Sobolewski, Peter; Kandel, Judith; Eckmann, David M

    2012-01-01

    Gas microembolism remains a serious risk associated with surgical procedures and decompression. Despite this, the signaling consequences of air bubbles in the vasculature are poorly understood and there is a lack of pharmacological therapies available. Here, we investigate the mitochondrial consequences of air bubble contact with endothelial cells. Human umbilical vein endothelial cells were loaded with an intracellular calcium indicator (Fluo-4) and either a mitochondrial calcium indicator (X-Rhod-1) or mitochondrial membrane potential indicator (TMRM). Contact with 50-150 µm air bubbles induced concurrent rises in intracellular and mitochondrial calcium, followed by a loss of mitochondrial membrane potential. Pre-treating cells with 1 µmol/L ruthenium red, a TRPV family calcium channel blocker, did not protect cells from the mitochondrial depolarization, despite blocking the intracellular calcium response. Mitigating the interactions between the air-liquid interface and the endothelial surface layer with 5% BSA or 0.1% Pluronic F-127 prevented the loss of mitochondrial membrane potential. Finally, inhibiting protein kinase C-α (PKCα), with 5 µmol/L Gö6976, protected cells from mitochondrial depolarization, but did not affect the intracellular calcium response. Our results indicate that air bubble contact with endothelial cells activates a novel, calcium-independent, PKCα-dependent signaling pathway, which results in mitochondrial depolarization. As a result, mitochondrial dysfunction is likely to be a key contributor to the pathophysiology of gas embolism injury. Further, this connection between the endothelial surface layer and endothelial mitochondria may also play an important role in vascular homeostasis and disease.

  16. Human adipose tissue-derived mesenchymal stem cells abrogate plasmablast formation and induce regulatory B cells independently of T helper cells.

    PubMed

    Franquesa, M; Mensah, F K; Huizinga, R; Strini, T; Boon, L; Lombardo, E; DelaRosa, O; Laman, J D; Grinyó, J M; Weimar, W; Betjes, M G H; Baan, C C; Hoogduijn, M J

    2015-03-01

    Mesenchymal or stromal stem cells (MSC) interact with cells of the immune system in multiple ways. Modulation of the immune system by MSC is believed to be a therapeutic option for autoimmune disease and transplant rejection. In recent years, B cells have moved into the focus of the attention as targets for the treatment of immune disorders. Current B-cell targeting treatment is based on the indiscriminate depletion of B cells. The aim of this study was to examine whether human adipose tissue-derived MSC (ASC) interact with B cells to affect their proliferation, differentiation, and immune function. ASC supported the survival of quiescent B cells predominantly via contact-dependent mechanisms. Coculture of B cells with activated T helper cells led to proliferation and differentiation of B cells into CD19(+) CD27(high) CD38(high) antibody-producing plasmablasts. ASC inhibited the proliferation of B cells and this effect was dependent on the presence of T cells. In contrast, ASC directly targeted B-cell differentiation, independently of T cells. In the presence of ASC, plasmablast formation was reduced and IL-10-producing CD19(+) CD24(high) CD38(high) B cells, known as regulatory B cells, were induced. These results demonstrate that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B-cell response in immune disease.

  17. Biomechanics of P-selectin PSGL-1 bonds: Shear threshold and integrin-independent cell adhesion

    SciTech Connect

    Xiao, Zhihua; Goldsmith, Harry L.; MacIntosh, Fiona A.; Shankaran, Harish; Neelamegham, Sriram

    2006-03-01

    Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP) stimulated platelets or P-selectin bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14-3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that while blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by ~60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 dissociated previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a subtle shear threshold for P-selectin PSGL-1 binding was also noted at shear rates<100/s and at force loading rates of ~300pN/sec. Results are discussed in light of biophysical computations that characterize the collision between unequal size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion that may be physiologically relevant.

  18. Bosutinib, an SRC inhibitor, induces caspase-independent cell death associated with permeabilization of lysosomal membranes in melanoma cells.

    PubMed

    Noguchi, S; Shibutani, S; Fukushima, K; Mori, T; Igase, M; Mizuno, T

    2017-03-28

    SRC kinase (SRC proto-oncogene, non-receptor tyrosine kinase) is a promising target for the treatment of solid cancers including human melanoma. Bosutinib (Bosu), a SRC inhibitor, has already been applied to the treatment of human chronic myelogenous leukemia and also has been assessed its safety in dogs. The aim of this study was to clarify a novel anti-tumour mechanism of Bosu in canine and human melanoma cells. The canine and human melanoma cells were treated with Bosu and its effects were evaluated by the cell viability, the protein expression levels such as caspase-3 and LC3, Annexin V/Propidium iodide staining, and confocal immunostaining. Bosu induced the massive caspase-independent cell death, and blocked autophagy flux, which resulted from lysosomal dysfunction. Lysosomal dysfunction caused by Bosu was due to lysosomal membrane permeabilization (LMP), which resulted in the release of lysosomal hydrolases including cathepsin B. Our data suggest that Bosu induces the cell death through induction of LMP in melanoma cells and is a promising therapeutic agent for treatment of melanoma in both dogs and humans. © 2017 John Wiley & Sons Ltd.

  19. Bisphenol A diglycidyl ether induces apoptosis in tumour cells independently of peroxisome proliferator-activated receptor-gamma, in caspase-dependent and -independent manners.

    PubMed Central

    Fehlberg, Sebastian; Trautwein, Stefan; Göke, Alexandra; Göke, Rüdiger

    2002-01-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors which are involved in many biological processes, such as regulation of cell differentiation, lipid metabolism, inflammation and cell death. PPARs consist of three families, PPAR-alpha, PPAR-delta and PPAR-gamma. Bisphenol A diglycidyl ether (BADGE) has been described as a pure antagonist of PPAR-gamma. However, recent data also revealed PPAR-gamma-agonistic activities of BADGE. Here we show that BADGE kills transformed cells by apoptosis and promotes the cytotoxic effects of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and indomethacin. The cytotoxic effect of BADGE does not require PPAR-gamma expression and is mediated in caspase-dependent and caspase-independent manners. PMID:11879183

  20. Retinoic Acid Signaling in B Cells Is Required for the Generation of an Effective T-Independent Immune Response

    PubMed Central

    Marks, Ellen; Ortiz, Carla; Pantazi, Eirini; Bailey, Charlotte S.; Lord, Graham M.; Waldschmidt, Thomas J.; Noelle, Randolph J.; Elgueta, Raul

    2016-01-01

    Retinoic acid (RA) plays an important role in the balance of inflammation and tolerance in T cells. Furthermore, it has been demonstrated that RA facilitates IgA isotype switching in B cells in vivo. However, it is unclear whether RA has a direct effect on T-independent B cell responses in vivo. To address this question, we generated a mouse model where RA signaling is specifically silenced in the B cell lineage. This was achieved through the overexpression of a dominant negative receptor α for RA (dnRARα) in the B cell lineage. In this model, we found a dramatic reduction in marginal zone (MZ) B cells and accumulation of transitional 2 B cells in the spleen. We also observed a reduction in B1 B cells in the peritoneum with a defect in the T-independent B cell response against 2,4,6-trinitrophenyl. This was not a result of inhibited development of B cells in the bone marrow, but likely the result of both defective expression of S1P1 in MZ B cells and a defect in the development of MZ and B1 B cells. This suggests that RARα expression in B cells is important for B cell frequency in the MZ and peritoneum, which is crucial for the generation of T-independent humoral responses. PMID:28066447

  1. Platelet and red blood cell utilization and transfusion independence in umbilical cord blood and allogeneic peripheral blood hematopoietic cell transplants.

    PubMed

    Solh, Melhem; Brunstein, Claudio; Morgan, Shanna; Weisdorf, Daniel

    2011-05-01

    Allogeneic hematopoietic cell transplantation (HCT) recipients have substantial transfusion requirements. Factors associated with increased transfusions and the extent of blood product use in umbilical cord blood (UCB) recipients are uncertain. We reviewed blood product use in 229 consecutive adult recipients of allogeneic HCT at the University of Minnesota: 147 with leukemia, 82 lymphoma or myeloma; 58% received unrelated UCB and 43% sibling donor peripheral blood stem cell (PBSC) grafts. Although neutrophil recovery was prompt (UCB median 17, range: 2-45 days, and PBSC 14, range: 3-34 days), only 135 of 229 (59% cumulative incidence) achieved red blood cell (RBC) independence and 157 (69%) achieved platelet independence by 6 months. Time to platelet independence was prolonged in UCB recipients (median UCB 41 versus PBSC 14 days) and in patients who had received a prior transplant (median 48 versus 32 days). Patients who received UCB grafts required more RBC through day 60 post-HCT (mean UCB 7.8 (95% confidence interval [CI] 6.7-8.9) versus PBSC 5.2 (3.7-6.7) transfusions, P = .04), and more platelet transfusions (mean 25.2 (95% CI 22.1-28.2) versus 12.9 (9.4-16.4), P < .01) compared to PBSC recipients. Patients receiving myeloablative (MA) conditioning required more RBC and platelet transfusions during the first 2 months post-HCT compared to reduced-intensity conditioning (RIC) (7.4 versus 6.2, P = .30 for RBC; 23.2 versus 17.5, P = .07 for platelets). Despite prompt neutrophil engraftment, UCB recipients had delayed platelet recovery as well as more prolonged and costly blood product requirements. Enhanced approaches to accelerate multilineage engraftment could limit the transfusion-associated morbidity and costs accompanying UCB allotransplantation.

  2. Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.

    PubMed

    Bast, Antje; Krause, Kathrin; Schmidt, Imke H E; Pudla, Matsayapan; Brakopp, Stefanie; Hopf, Verena; Breitbach, Katrin; Steinmetz, Ivo

    2014-03-01

    The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent

  3. Mip/LIN-9 can inhibit cell proliferation independent of the pocket proteins.

    PubMed

    Pilkinton, Mark; Sandoval, Raudel; Barrett, Kelly; Tian, Xinyong; Colamonici, Oscar R

    2007-01-01

    Progression through the G1-phase of the cell cycle requires that cyclin D and CDK4 phosphorylate pRB and the other pocket proteins, p107 and p130. Cyclin E and CDK2 further phosphorylate pRB to complete its inactivation and allow the cell to enter S-phase. These phosphorylation events lead to the inactivation of the antiproliferative effect of the pocket proteins. The pocket proteins are the main targets of CDK4, and its unregulated activity can contribute to carcinogenesis. Mip/LIN9 is a recently described protein with growth suppressor, as well as growth promoting effects due to its ability to stabilize B-Myb and induce genes required for S phase and mitosis. The finding that a mutation that deletes the first 84 amino acids of Mip/LIN-9 corrects the defects of the CDK4 knockout mouse suggests that it should have a growth repressor effect that is blocked by CDK4. However, overexpression of cyclin D only partially blocks the inhibitory effect of Mip/LIN-9 on cell proliferation. Here, we performed experiments to further understand the antiproliferative effect of Mip/LIN-9 within the context of the pocket proteins. Our results suggest that there is a pocket protein-independent mechanism of the Mip/LIN-9 antiproliferative effect since it can be observed in cells with ablation of the three members of the family, and in NIH3T3 cells expressing the adenovirus E1A-12S protein. Altogether, the independence from the pocket proteins and the partial blockade of the antiproliferative effect produced by expression of cyclin D suggest that the role of Mip/LIN-9 downstream of CDK4 may be more closely related to the activation of B-Myb and the induction of S/M genes. Importantly, the regulatory effect of CDK4 is not due to direct phosphorylation of Mip/LIN-9 by this kinase or even CDK2, suggesting an indirect mechanism such as phosphorylation of the pocket proteins.

  4. Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain

    PubMed Central

    Chen, Jinyu; Lee, Ho-Jeong; Wu, Xuefeng; Huo, Lei; Kim, Sun-Jin; Xu, Lei; Wang, Yan; He, Junqing; Bollu, Lakshmi Reddy; Gao, Guang; Su, Fei; Briggs, James; Liu, Xiaojing; Melman, Tamar; Asara, John M.; Fidler, Isaiah J.; Cantley, Lewis C.; Locasale, Jason W.; Weihua, Zhang

    2014-01-01

    Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain-metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the non-oxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBPs) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis, and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients. PMID:25511375

  5. Release of nitric oxide during the T cell-independent pathway of macrophage activation

    SciTech Connect

    Beckerman, K.P.; Rogers, H.W.; Corbett, J.A.; Schreiber, R.D.; McDaniel, M.L.; Unanue, E.R. )

    1993-02-01

    Immunodeficient mice are remarkably resistant to Listeria monocytogenes (LM) infection. The authors examined the role that nitric oxide (NO) plays in the CB-17/lcr SCID (SCID) response to LM. SCID spleen cells produced large quantities of NO (as measured by nitrite formation) when incubated in the presence of heat-killed LM. NO produced large quantities of nitrite in response to LM, but only in the presence of IFN-[gamma]. The production of NO induced by LM was not affected by neutralizing antibodies to TNF or IL-1. The production of NO was inhibited by addition of either of two inhibitors of NO synthase, N[sup G]-monomethyl arginine, or aminoguanidine. In a different situation, NK cells that were stimulated by TNF and Listeria products to release IFN-[gamma] did not produce NO. Macrophages cultured with IFN-[gamma] killed live LM. This increased killing of LM was significantly inhibited by amino-guanidine. In vivo, administration of aminoguanidine resulted in a marked increase in the mortality and spleen bacterial loads of LM-infected SCID or immunocompetent control mice. It is concluded that NO is a critical effector molecule of T cell-independent natural resistence of LM as studied in the SCID mouse, and that the NO-mediated response is essential for both SCID and immunocompetent host to survive after LM infection. 37 refs., 7 figs.

  6. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms

    PubMed Central

    Wörmann, Mirka E.; Horien, Corey L.; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M.

    2016-01-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host–pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus–pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms. PMID:26813911

  7. Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain.

    PubMed

    Chen, Jinyu; Lee, Ho-Jeong; Wu, Xuefeng; Huo, Lei; Kim, Sun-Jin; Xu, Lei; Wang, Yan; He, Junqing; Bollu, Lakshmi R; Gao, Guang; Su, Fei; Briggs, James; Liu, Xiaojing; Melman, Tamar; Asara, John M; Fidler, Isaiah J; Cantley, Lewis C; Locasale, Jason W; Weihua, Zhang

    2015-02-01

    Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the nonoxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBP) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients. ©2014 American Association for Cancer Research.

  8. Fenugreek induced apoptosis in breast cancer MCF-7 cells mediated independently by fas receptor change.

    PubMed

    Alshatwi, Ali Abdullah; Shafi, Gowhar; Hasan, Tarique Noorul; Syed, Naveed Ahmed; Khoja, Kholoud Khalid

    2013-01-01

    Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes, high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties, although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extract from Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in the presence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptor pathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FME induced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, as well as p53 interdependently.

  9. Indomethacin increases susceptibility to injury in human gastric cells independent of PG synthesis inhibition.

    PubMed

    Kokoska, E R; Smith, G S; Deshpande, Y; Wolff, A B; Miller, T A

    1998-10-01

    Indomethacin and other nonsteroidal anti-inflammatory drugs are commonly used to indirectly deduce the possible role of PGs in a process being studied. The objective of this study was to determine if indomethacin, at concentrations comparable to plasma and tissue levels obtained in humans taking therapeutic doses, predisposes human gastric cells to injury through inhibition of PGs or acts through an alternate mechanism. The role of intracellular Ca2+ in this damaging process was also assessed. Indomethacin pretreatment, although by itself nondamaging, was associated with elevated intracellular Ca2+ concentrations and an increased cellular permeability, an effect that was dependent on extracellular Ca2+. Furthermore, indomethacin pretreatment significantly predisposed AGS cells to injury induced by two dissimilar agents (deoxycholate and A-23187), both of which are associated with intracellular Ca2+ accumulation. The addition of exogenous PGs did not reverse the predisposition to injury induced by indomethacin. The observed effects of indomethacin were dependent on concentration and not on ability to inhibit PG synthesis. Similar effects were not observed with equipotent concentrations of ibuprofen or aspirin. Finally, the exacerbation of deoxycholate-induced injury induced by indomethacin was not observed when extracellular Ca2+ was removed. Indomethacin, by disturbing intracellular Ca2+ homeostasis, predisposes human gastric cells to injury through mechanisms independent of PG synthesis. The current study suggests that data resulting from studies employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution.

  10. p73 regulates DRAM-independent autophagy that does not contribute to programmed cell death.

    PubMed

    Crighton, D; O'Prey, J; Bell, H S; Ryan, K M

    2007-06-01

    Evading programmed cell death is a common event in tumour development. The p53 family member, p73, is a potent inducer of death and a determinant of chemotherapeutic response, but different to p53, is rarely mutated in cancer. Understanding cell death pathways downstream of p53 and p73 is therefore pivotal to understand both the development and treatment of malignant disease. Recently, p53 has been shown to modulate autophagy--a membrane trafficking process, which degrades long-lived proteins and organelles. This requires a p53 target gene, DRAM, and both DRAM and autophagy are critical for p53-mediated death. We report here that TA-p73 also regulates DRAM and autophagy, with different TA-p73 isoforms regulating DRAM and autophagy to varying extents. RNAi knockdown of DRAM, however, revealed that p73's modulation of autophagy is DRAM-independent. Also, p73's ability to induce death, again different to p53, is neither dependent on DRAM nor autophagy. In contrast to TA-p73, deltaN-p73 is a negative regulator of p53-induced and p73-induced autophagy, but does not affect autophagy induced by amino-acid starvation. These studies, therefore, represent not only the first report that p73 modulates autophagy but also highlight important differences in the mechanism by which starvation, p53 and p73 regulate autophagy and how this contributes to programmed cell death.

  11. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

    PubMed

    Wörmann, Mirka E; Horien, Corey L; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M; Exley, Rachel M

    2016-03-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

  12. Subpopulations of myeloid-derived suppressor cells impair T cell responses through independent nitric oxide-related pathways.

    PubMed

    Raber, Patrick L; Thevenot, Paul; Sierra, Rosa; Wyczechowska, Dorota; Halle, Daniel; Ramirez, Maria E; Ochoa, Augusto C; Fletcher, Matthew; Velasco, Cruz; Wilk, Anna; Reiss, Krzysztof; Rodriguez, Paulo C

    2014-06-15

    The accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing hosts is a hallmark of malignancy-associated inflammation and a major mediator for the induction of T cell suppression in cancer. MDSC can be divided phenotypically into granulocytic (G-MDSC) and monocytic (Mo-MDSC) subgroups. Several mechanisms mediate the induction of T cell anergy by MDSC; however, the specific role of these pathways in the inhibitory activity of MDSC subpopulations remains unclear. Therefore, we aimed to determine the effector mechanisms by which subsets of tumor-infiltrating MDSC block T cell function. We found that G-MDSC had a higher ability to impair proliferation and expression of effector molecules in activated T cells, as compared to Mo-MDSC. Interestingly, both MDSC subgroups inhibited T cells through nitric oxide (NO)-related pathways, but expressed different effector inhibitory mechanisms. Specifically, G-MDSC impaired T cells through the production of peroxynitrites (PNT), while Mo-MDSC suppressed by the release of NO. The production of PNT in G-MDSC depended on the expression of gp91(phox) and endothelial NO synthase (eNOS), while inducible NO synthase (iNOS) mediated the generation of NO in Mo-MDSC. Deletion of eNOS and gp91(phox) or scavenging of PNT blocked the suppressive function of G-MDSC and induced anti-tumoral effects, without altering Mo-MDSC inhibitory activity. Furthermore, NO-scavenging or iNOS knockdown prevented Mo-MDSC function, but did not affect PNT production or suppression by G-MDSC. These results suggest that MDSC subpopulations utilize independent effector mechanisms to regulate T cell function. Inhibition of these pathways is expected to specifically block MDSC subsets and overcome immune suppression in cancer.

  13. The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells.

    PubMed

    Meschini, Stefania; Condello, Maria; Calcabrini, Annarica; Marra, Manuela; Formisano, Giuseppe; Lista, Pasquale; De Milito, Angelo; Federici, Elena; Arancia, Giuseppe

    2008-11-01

    In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

  14. Whole exome sequencing of independent lung adenocarcinoma, lung squamous cell carcinoma, and malignant peritoneal mesothelioma

    PubMed Central

    Vanni, Irene; Coco, Simona; Bonfiglio, Silvia; Cittaro, Davide; Genova, Carlo; Biello, Federica; Mora, Marco; Rossella, Valeria; Dal Bello, Maria Giovanna; Truini, Anna; Banelli, Barbara; Lazarevic, Dejan; Alama, Angela; Rijavec, Erika; Barletta, Giulia; Grossi, Francesco

    2016-01-01

    Abstract The presence of multiple primary tumors (MPT) in a single patient has been identified with an increasing frequency. A critical issue is to establish if the second tumor represents an independent primary cancer or a metastasis. Therefore, the assessment of MPT clonal origin might help understand the disease behavior and improve the management/prognosis of the patient. Herein, we report a 73-year-old male smoker who developed 2 primary lung cancers (adenocarcinoma and squamous cell carcinoma) and a malignant peritoneal mesothelioma (PM). Whole exome sequencing (WES) of the 3 tumors and of germline DNA was performed to determine the clonal origin and identify genetic cancer susceptibility. Both lung cancers were characterized by a high mutational rate with distinct mutational profiles and activation of tumor-specific pathways. Conversely, the PM harbored a relative low number of genetic variants and a novel mutation in the WT1 gene that might be involved in the carcinogenesis of nonasbestos-related mesothelioma. Finally, WES of the germinal DNA displayed several single nucleotide polymorphisms in DNA repair genes likely conferring higher cancer susceptibility. Overall, WES did not disclose any somatic genetic variant shared across the 3 tumors, suggesting their clonal independency; however, the carcinogenic effect of smoke combined with a deficiency in DNA repair genes and the patient advanced age might have been responsible for the MPT development. This case highlights the WES importance to define the clonal origin of MPT and susceptibility to cancer. PMID:27902597

  15. Spreading dilatation in rat mesenteric arteries associated with calcium-independent endothelial cell hyperpolarization.

    PubMed

    Takano, Hiromichi; Dora, Kim A; Spitaler, Michaela M; Garland, Chris J

    2004-05-01

    Both ACh and levcromakalim evoke smooth muscle cell hyperpolarization and associated relaxation in rat mesenteric resistance arteries. We investigated if they could evoke conducted vasodilatation along isolated arteries, whether this reflected spreading hyperpolarization and the possible mechanism involved. Focal micropipette application of either ACh, to stimulate endothelial cell muscarinic receptors, or levcromakalim, to activate smooth muscle K(ATP) channels, each evoked a local dilatation (88 +/- 14%, n= 6 and 92 +/- 6% reversal of phenylephrine-induced tone, n= 11, respectively) that rapidly spread upstream (at 1.5 mm 46 +/- 19%, n= 6 and 57 +/- 13%, n= 9) to dilate the entire isolated artery. The local dilatation to ACh was associated with a rise in endothelial cell [Ca(2+)](i) (F/F(t = 0)= 1.22 +/- 0.33, n= 14) which did not spread beyond 0.5 mm (F/F(t = 0)= 1.01 +/- 0.01, n= 14), while the local dilatation to levcromakalim was not associated with any change in endothelial cell [Ca(2+)](i). In contrast, ACh and levcromakalim both stimulated local (12.7 +/- 1.2 mV, n= 10 and 13.5 +/- 4.7 mV, n= 10) and spreading (at 2 mm: 3.0 +/- 1.1 mV, n= 5 and 4.1 +/- 0.7 mV, n= 5) smooth muscle hyperpolarization. The spread of hyperpolarization could be prevented by cutting the artery, so was not due to a diffusible agent. Both the spreading dilatation and hyperpolarization were endothelium dependent. The injection of propidium iodide into either endothelial or smooth muscle cells revealed extensive dye coupling between the endothelial cells, but limited coupling between the smooth muscle cells. Some evidence for heterocellular spread of dye was also evident. Together, these data show that vasodilatation can spread over significant distances in mesenteric resistance arteries, and suggest this reflects an effective coupling between the endothelial cells to facilitate [Ca(2+)](i)-independent spread of hyperpolarization.

  16. Prolonged Induction Activates Cebpα Independent Adipogenesis in NIH/3T3 Cells

    PubMed Central

    Shao, Hsiao-Yun; Hsu, Hsue-Yin; Wu, Kuan-Sju; Hee, Siow-Wey; Chuang, Lee-Ming; Yeh, Jih-I

    2013-01-01

    Background 3T3-L1 cells are widely used to study adipogenesis and insulin response. Their adipogenic potential decreases with time in the culture. Expressing exogenous genes in 3T3-L1 cells can be challenging. This work tries to establish and characterize an alternative model of cultured adipocytes that is easier to work with than the 3T3-L1 cells. Methodology/Principal Findings Induced cells were identified as adipocytes based on the following three characteristics: (1) Accumulation of triglyceride droplets as demonstrated by oil red O stain. (2) Transport rate of 2-deoxyglucose increased after insulin stimulation. (3) Expression of fat specific genes such as Fabp4 (aP2), Slc2a4 (Glut4) and Pparg (PPARγ). Among the cell lines induced under different conditions in this study, only NIH/3T3 cells differentiated into adipocytes after prolonged incubation in 3T3-L1 induction medium containing 20% instead of 10% fetal bovine serum. Rosiglitazone added to the induction medium shortened the incubation period from 14 to 7 days. The PI3K/AKT pathway showed similar changes upon insulin stimulation in these two adipocytes. C/EBPα mRNA was barely detectable in NIH/3T3 adipocytes. NIH/3T3 adipocytes induced in the presence of rosiglitazone showed higher 2-deoxyglucose transport rate after insulin stimulation, expressed less Agt (angiotensinogen) and more PPARγ. Knockdown of C/EBPα using shRNA blocked 3T3-L1 but not NIH/3T3 cell differentiation. Mouse adipose tissues from various anatomical locations showed comparable levels of C/EBPα mRNA. Conclusions/Significance NIH/3T3 cells were capable of differentiating into adipocytes without genetic engineering. They were an adipocyte model that did not require the reciprocal activation between C/EBPα and PPARγ to differentiate. Future studies in the C/EBPα independent pathways leading to insulin responsiveness may reveal new targets to diabetes treatment. PMID:23326314

  17. Spreading dilatation in rat mesenteric arteries associated with calcium-independent endothelial cell hyperpolarization

    PubMed Central

    Takano, Hiromichi; Dora, Kim A; Spitaler, Michaela M; Garland, Chris J

    2004-01-01

    Both ACh and levcromakalim evoke smooth muscle cell hyperpolarization and associated relaxation in rat mesenteric resistance arteries. We investigated if they could evoke conducted vasodilatation along isolated arteries, whether this reflected spreading hyperpolarization and the possible mechanism involved. Focal micropipette application of either ACh, to stimulate endothelial cell muscarinic receptors, or levcromakalim, to activate smooth muscle KATP channels, each evoked a local dilatation (88 ± 14%, n= 6 and 92 ± 6% reversal of phenylephrine-induced tone, n= 11, respectively) that rapidly spread upstream (at 1.5 mm 46 ± 19%, n= 6 and 57 ± 13%, n= 9) to dilate the entire isolated artery. The local dilatation to ACh was associated with a rise in endothelial cell [Ca2+]i (F/Ft = 0= 1.22 ± 0.33, n= 14) which did not spread beyond 0.5 mm (F/Ft = 0= 1.01 ± 0.01, n= 14), while the local dilatation to levcromakalim was not associated with any change in endothelial cell [Ca2+]i. In contrast, ACh and levcromakalim both stimulated local (12.7 ± 1.2 mV, n= 10 and 13.5 ± 4.7 mV, n= 10) and spreading (at 2 mm: 3.0 ± 1.1 mV, n= 5 and 4.1 ± 0.7 mV, n= 5) smooth muscle hyperpolarization. The spread of hyperpolarization could be prevented by cutting the artery, so was not due to a diffusible agent. Both the spreading dilatation and hyperpolarization were endothelium dependent. The injection of propidium iodide into either endothelial or smooth muscle cells revealed extensive dye coupling between the endothelial cells, but limited coupling between the smooth muscle cells. Some evidence for heterocellular spread of dye was also evident. Together, these data show that vasodilatation can spread over significant distances in mesenteric resistance arteries, and suggest this reflects an effective coupling between the endothelial cells to facilitate [Ca2+]i-independent spread of hyperpolarization. PMID:14966304

  18. EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent.

    PubMed

    Xu, Kexin; Wu, Zhenhua Jeremy; Groner, Anna C; He, Housheng Hansen; Cai, Changmeng; Lis, Rosina T; Wu, Xiaoqiu; Stack, Edward C; Loda, Massimo; Liu, Tao; Xu, Han; Cato, Laura; Thornton, James E; Gregory, Richard I; Morrissey, Colm; Vessella, Robert L; Montironi, Rodolfo; Magi-Galluzzi, Cristina; Kantoff, Philip W; Balk, Steven P; Liu, X Shirley; Brown, Myles

    2012-12-14

    Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

  19. Tumor Cell-Independent Estrogen Signaling Drives Disease Progression through Mobilization of Myeloid-Derived Suppressor Cells.

    PubMed

    Svoronos, Nikolaos; Perales-Puchalt, Alfredo; Allegrezza, Michael J; Rutkowski, Melanie R; Payne, Kyle K; Tesone, Amelia J; Nguyen, Jenny M; Curiel, Tyler J; Cadungog, Mark G; Singhal, Sunil; Eruslanov, Evgeniy B; Zhang, Paul; Tchou, Julia; Zhang, Rugang; Conejo-Garcia, Jose R

    2017-01-01

    The role of estrogens in antitumor immunity remains poorly understood. Here, we show that estrogen signaling accelerates the progression of different estrogen-insensitive tumor models by contributing to deregulated myelopoiesis by both driving the mobilization of myeloid-derived suppressor cells (MDSC) and enhancing their intrinsic immunosuppressive activity in vivo Differences in tumor growth are dependent on blunted antitumor immunity and, correspondingly, disappear in immunodeficient hosts and upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Therefore, estrogen signaling is a crucial mechanism underlying pathologic myelopoiesis in cancer. Our work suggests that new antiestrogen drugs that have no agonistic effects may have benefits in a wide range of cancers, independently of the expression of estrogen receptors in tumor cells, and may synergize with immunotherapies to significantly extend survival.

  20. Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death1[OPEN

    PubMed Central

    2016-01-01

    Reduced cell wall invertase (CWIN) activity has been shown to be associated with poor seed and fruit set under abiotic stress. Here, we examined whether genetically increasing native CWIN activity would sustain fruit set under long-term moderate heat stress (LMHS), an important factor limiting crop production, by using transgenic tomato (Solanum lycopersicum) with its CWIN inhibitor gene silenced and focusing on ovaries and fruits at 2 d before and after pollination, respectively. We found that the increase of CWIN activity suppressed LMHS-induced programmed cell death in fruits. Surprisingly, measurement of the contents of H2O2 and malondialdehyde and the activities of a cohort of antioxidant enzymes revealed that the CWIN-mediated inhibition on programmed cell death is exerted in a reactive oxygen species-independent manner. Elevation of CWIN activity sustained Suc import into fruits and increased activities of hexokinase and fructokinase in the ovaries in response to LMHS. Compared to the wild type, the CWIN-elevated transgenic plants exhibited higher transcript levels of heat shock protein genes Hsp90 and Hsp100 in ovaries and HspII17.6 in fruits under LMHS, which corresponded to a lower transcript level of a negative auxin responsive factor IAA9 but a higher expression of the auxin biosynthesis gene ToFZY6 in fruits at 2 d after pollination. Collectively, the data indicate that CWIN enhances fruit set under LMHS through suppression of programmed cell death in a reactive oxygen species-independent manner that could involve enhanced Suc import and catabolism, HSP expression, and auxin response and biosynthesis. PMID:27462084

  1. Low plasma adiponectin level, white blood cell count and Helicobacter pylori titre independently predict abnormal pancreatic beta-cell function.

    PubMed

    So, Wing-Yee; Tong, Peter C; Ko, Gary T; Ma, Ronald C; Ozaki, Risa; Kong, Alice P; Yang, Xilin; Ho, Chung-Shun; Lam, Christopher C; Chan, Juliana C

    2009-11-01

    Adiponectin is an adipocytokine with insulin sensitizing effect while chronic inflammation damages pancreatic beta-cells leading to reduced insulin response. We aimed to prove the hypothesis that adiponectin levels and inflammatory markers (white blood cell counts [WCC], Helicobacter pylori [HP] titers, high sensitivity C-reactive protein [hs-CRP]) may interact to affect risk of diabetes. We studied 288 Chinese men (age-median: 41.0 years, IQR: 35.3-46.0 years) being recruited from the community in Hong Kong. The mean adiponectin level was 5.39+/-2.81 microg/ml and 40.9% (n=107) had low adiponectin level (<4 microg/ml). On multiple regression analysis, adiponectin was negatively associated with diabetes, HOMA insulin resistance top quartile, plasma glucose (PG) and 2h insulin; and positively associated with HOMA insulin sensitivity index. WCC was independently associated with PG and 15' insulin, and negatively associated with HOMA insulin sensitivity top quartile. HP titre was associated with 30' PG level and diabetes. hs-CRP did not enter the multivariable model. In conclusion, adiponectin, WCC and HP titer are independent predictors for hyperglycemia and reduced insulin sensitivity in Chinese men. These findings may explain the high risk for diabetes in Chinese population despite their relatively low adiposity.

  2. Combined effects of terazosin and genistein on a metastatic, hormone-independent human prostate cancer cell line.

    PubMed

    Chang, Kee-Lung; Cheng, Hsiao-Ling; Huang, Li-Wen; Hsieh, Bau-Shan; Hu, Yu-Chen; Chih, Tsai-Tung; Shyu, Huey-Wen; Su, Shu-Jem

    2009-04-08

    Metastatic prostate cancer progresses from androgen-dependent to androgen-independent. Terazosin, a long-acting selective alpha1-adrenoreceptor antagonist, induces apoptosis of prostate cancer cells in an alpha1-adrenoreceptor-independent manner, while genistein, a major soy isoflavone, inhibits the growth of several types of cancer cells. The present study was designed to test the therapeutic potential of a combination of terazosin and genistein using a metastatic, hormone-independent prostatic cancer cell line, DU-145. Terazosin or genistein treatment inhibited the growth of DU-145 cells in a dose-dependent manner, whereas had no effect on normal prostate epithelial cells. Addition of 1 microg/ml of terazosin, which was inactive alone, augmented the growth inhibitory effect of 5 microg/ml of genistein. Co-treatment with terazosin resulted in the genistein-induced arrest of DU-145 cells in G2/M phase being overridden and an increase in apoptotic cells, as evidenced by procaspase-3 activation and PARP cleavage. The combination also caused a greater decrease in the levels of the apoptosis-regulating protein, Bcl-XL, and of VEGF165 and VEGF121 than genistein alone. In conclusion, the terazosin/genistein combination was more effective in inhibiting cell growth and VEGF expression as well as inducing apoptosis of the metastatic, androgen-independent prostate cancer cell line, DU-145, than either alone. The doses used in this study are in lower and nontoxic anticancer dosage range, suggesting this combination has potential for therapeutic use.

  3. Platelet and Red Blood Cell Utilization and Transfusion Independence in Umbilical Cord Blood and Allogeneic Peripheral Blood Hematopoietic Cell Transplants

    PubMed Central

    Solh, Melhem; Brunstein, Claudio; Morgan, Shanna; Weisdorf, Daniel

    2010-01-01

    Allogeneic hematopoietic cell transplantation (HCT) recipients have substantial transfusion requirements. Factors associated with increased transfusions and the extent of blood product use in umbilical cord blood (UCB) recipients are uncertain. We reviewed blood product use in 229 consecutive adult recipients of allogeneic HCT at the University of Minnesota: 147 with leukemia, 82 lymphoma or myeloma; 58% received unrelated UCB and 43% sibling donor peripheral blood stem cell (PBSC) grafts. Although neutrophil recovery was prompt (UCB median 17, range 2–45 days, and PBSC 14, range 3–34 days), only 135 of 229 (59% cumulative incidence, CI) achieved RBC independence and 157 (69%) achieved platelet independence by 6 months. Time to platelet independence was prolonged in UCB recipients (median UCB 41 vs. PBSC 14 days) and in patients who had received a prior transplant (median 48 vs. 32 days). Patients who received UCB grafts required more RBC through day 60 post HCT (mean UCB 7.8 (95% CI 6.7–8.9) vs. PBSC 5.2 (3.7–6.7) transfusions, p=0.04), and more platelet transfusions (mean 25.2 (95% CI 22.1–28.2) vs. 12.9 (9.4–16.4), p<0.01) compared to PBSC recipients. Patient receiving myeloablative (MA) conditioning required more RBC and platelet transfusions during the first 2 months post HCT compared to reduced intensity conditioning (RIC) (7.4 vs. 6.2, p=0.3 for RBC; 23.2 vs 17.5, p=0.07 for platelets). Despite prompt neutrophil engraftment, UCB recipients had delayed platelet recovery as well as more prolonged and costly blood product requirements. Enhanced approaches to accelerate multilineage engraftment could limit the transfusion-associated morbidity and costs accompanying UCB allotransplantation. PMID:20813199

  4. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  5. MUTZ-3 Langerhans cell maturation and CXCL12 independent migration in reconstructed human gingiva.

    PubMed

    Kosten, Ilona J; Spiekstra, Sander W; de Gruijl, Tanja D; Gibbs, Susan

    2016-01-01

    Here we describe a reconstructed full thickness human oral mucosa (gingiva) equivalent with integrated Langerhans Cells (GE-LC) and use it to compare LC activation and migration from oral versus skin epithelium. The physiologically representative models consist of differentiated reconstructed epithelium (keratinocytes and Langerhans-like cells derived from the MUTZ-3 cell line) on a fibroblast-populated collagen hydrogel which serves as a lamina propria for gingiva and dermis for skin. Topical exposure of GE-LC and the skin equivalent (SE-LC) to sub-toxic concentrations of the allergens cinnamaldehyde, resorcinol and nickel sulphate, resulted in LC migration out of the epithelia. Neutralizing antibody to CXCL12 blocked allergen-induced LC migration in SE-LC but not in GE-LC. Also, gingival fibroblasts secreted very low amounts of CXCL12 compared to skin fibroblasts even when stimulated with rhTNFα or rhIL-1α. Surprisingly, cinnamaldehyde exposure of GE-LC resulted in an increase in MUTZ-3 LC and CD83 mRNA in the hydrogel but did not result in an increase in CD1a+ cells in the collagen hydrogel (as was observed for SE-LC. These results indicate that in gingiva, upon allergen exposure, MUTZ-3 LC migrate in a CXCL12 independent manner from epithelium-to-lamina propria and in doing so mature become CD1a- and increase CD83+ mRNA. These physiologically relevant in vitro models which not only are human but which also resemble specific tissues, may aid in the identification of factors regulating immune stimulation which in turn will aid the development of therapeutic interventions for allergy and inflammation, anti-cancer vaccines as well as improving diagnostics for skin and oral allergy.

  6. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    PubMed

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  7. CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway.

    PubMed

    Saiman, Yedidya; Agarwal, Ritu; Hickman, DaShawn A; Fausther, Michel; El-Shamy, Ahmed; Dranoff, Jonathan A; Friedman, Scott L; Bansal, Meena B

    2013-09-01

    Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.

  8. Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent

    PubMed Central

    Kamiyama, Haruka; Kakoki, Katsura; Yoshii, Hiroaki; Iwao, Masatomo; Igawa, Tsukasa; Sakai, Hideki; Hayashi, Hideki; Matsuyama, Toshifumi; Yamamoto, Naoki; Kubo, Yoshinao

    2011-01-01

    Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells. PMID:22022555

  9. Acute Pain and Depressive Symptoms: Independent Predictors of Insomnia Symptoms among Adults with Sickle Cell Disease

    PubMed Central

    Moscou-Jackson, Gyasi; Allen, Jerilyn; Kozachik, Sharon; Smith, Michael T.; Budhathoki, Chakra; Haywood, Carlton

    2015-01-01

    Background No studies to-date have systematically investigated insomnia symptoms among adults with sickle cell disease (SCD). The purpose of this study was to 1) describe the prevalence of insomnia symptoms and 2) identify bio-psychosocial predictors in community-dwelling adults with Sickle Cell Disease. Methods Cross-sectional analysis of baseline data from 263 African-American adults with SCD (aged 18 years or older). Measures included the Insomnia Severity Index (ISI), Center for Epidemiologic Studies in Depression scale, Urban Life Stress Scale, Brief Pain Inventory, and a chronic pain item. SCD genotype was extracted from the medical record. Results A slight majority (55%) of the sample reported clinically significant insomnia symptomatology (ISI ≥10), which suggests that insomnia symptoms are prevalent among community-dwelling African-American adults with SCD. While insomnia symptoms were associated with a number of bio-psychosocial characteristics, depressive symptoms and acute pain were the only independent predictors. Conclusion Given the high number of participants reporting clinically significant insomnia symptoms, nurses should screen for insomnia symptoms and to explore interventions to promote better sleep among adults with SCD with an emphasis on recommending treatment for pain and depression. In addition, current pain and depression interventions in this population could add insomnia measures and assess the effect of the intervention on insomnia symptomatology as a secondary outcome. PMID:26673730

  10. Squamous cell carcinomas of the skin explore angiogenesis-independent mechanisms of tumour vascularization.

    PubMed

    Pastushenko, Ievgenia; Gracia-Cazaña, Tamara; Vicente-Arregui, Sandra; Van den Eynden, Gert G; Ara, Mariano; Vermeulen, Peter B; Carapeto, Franciso José; Van Laere, Steven J

    2014-01-01

    Aims. To evaluate the vascularization in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin. Methods. We performed CD31 (i.e., panendothelial marker) and CD105 (i.e., proliferating endothelium marker) immunostaining on samples of 70 SCCs and 70 BCCs of the skin. We evaluated the relative blood vessel area using the Chalkley counting method in each histologic subtype of these tumours. We calculated the degree of proliferation of blood vessel endothelium dividing CD105-Chalkley score by CD31-Chalkley score. Results. We found significantly higher peritumoral and intratumoral blood vessel area in SCC when compared to BCC (both with CD31 and CD105). Chalkley counts differed significantly between groups with different BCC histologic subtypes and SCC with different grade of differentiation. Surprisingly, the degree of proliferation of blood vessel endothelium was higher in BCC when compared to SCC. Conclusions. While SCC exhibited significantly higher intratumoral and peritumoral blood vessel areas compared to BCC, the relatively low rate of proliferating endothelium in this tumour type suggests the existence of endothelial-sprouting-independent mechanisms of vascularization in SCC.

  11. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

    NASA Astrophysics Data System (ADS)

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  12. T Cell Immunoglobulin Mucin-3 Crystal Structure Reveals a Galectin-9-Independent Ligand-Binding Surface

    SciTech Connect

    Cao,E.; Zang, X.; Ramagopal, U.; Mukhopadhaya, A.; Fedorov, A.; Fedorov, E.; Zencheck, W.; Lary, J.; Cole, J.; et al.

    2007-01-01

    The T cell immunoglobulin mucin (Tim) family of receptors regulates effector CD4+ T cell functions and is implicated in autoimmune and allergic diseases. Tim-3 induces immunological tolerance, and engagement of the Tim-3 immunoglobulin variable (IgV) domain by galectin-9 is important for appropriate termination of T helper 1-immune responses. The 2 {angstrom} crystal structure of the Tim-3 IgV domain demonstrated that four cysteines, which are invariant within the Tim family, form two noncanonical disulfide bonds, resulting in a surface not present in other immunoglobulin superfamily members. Biochemical and biophysical studies demonstrated that this unique structural feature mediates a previously unidentified galectin-9-independent binding process and suggested that this structural feature is conserved within the entire Tim family. The current work provided a graphic example of the relationship between sequence, structure, and function and suggested that the interplay between multiple Tim-3-binding activities contributes to the regulated assembly of signaling complexes required for effective Th1-mediated immunity.

  13. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells.

    PubMed

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  14. Coordinated steroid hormone-dependent and independent expression of multiple kallikreins in breast cancer cell lines.

    PubMed

    Paliouras, Miltiadis; Diamandis, Eleftherios P

    2007-03-01

    The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well in the pathogenesis of endocrine-related cancers. Previous experiments have shown that many kallikrein genes are under steroid hormone regulation in breast cancer cell lines. We here examine the coordinated expression of multiple kallikrein genes in several breast cancer cell lines after steroid hormone stimulation. Breast cancer cell lines were treated with various steroid hormones and kallikrein (KLK/hK) expression of hK3 (prostate-specific antigen, PSA), hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 was analyzed at the RNA level via RT-PCR and at the protein level by immunofluorometric ELISA assays. We identified several distinct hK hormone-dependent and hormone-independent expression patterns. Hormone-specific modulation of expression was seen for several kallikreins in BT-474, MCF-7, and T-47D cell lines. hK6 was specifically up-regulated upon estradiol treatment in all three cell lines whereas PSA expression was induced by dihydrotestosterone (DHT) and norgestrel stimulation in BT-474 and T-47D. hK10, hK11, hK13, and hK14 were specifically up-regulated by DHT in T-47D and by estradiol in BT-474 cells. Bioinformatic analysis of upstream proximal promoter sequences for these hKs did not identify any recognizable hormone-response elements (HREs), suggesting that the coordinated activation of these four hKs represents a unique expression "cassette", utilizing a common hormone-dependent mechanism. We conclude that groups of human hKs are coordinately expressed in a steroid hormone-dependent manner. Our data supports clinical observations linking expression of multiple hKs with breast cancer prognosis.

  15. Nitric oxide regulates B cell activating factor (BAFF) expression and T-cell independent antibody responses1

    PubMed Central

    Giordano, Daniela; Draves, Kevin E.; Li, Chang; Hohl, Tobias M.; Clark, Edward A.

    2014-01-01

    While nitric oxide (NO) is known to regulate T cell responses, its role in regulating B cell responses remains unclear. Previous studies suggested that inducible NO synthase 2 (NOS2/iNOS) is required for normal IgA Ab responses but inhibits anti-viral IgG2a Ab responses. Here we used NOS2−/− mice to determine the role of NO in T cell-dependent (TD) and T cell-independent-2 (TI-2) Ab responses. While TD Ab responses were only modestly increased in NOS2−/− mice, IgM and IgG3 Ab responses as well as marginal zone (MZ) B cell plasma cell (PC) numbers and peritoneal B1b B cells were significantly elevated after immunization with the TI-2 Ag NP-Ficoll. The elevated TI-2 responses in NOS2−/− mice were accompanied by significant increases in serum levels of B cell activating factor (BAFF/BLyS) and by increases in BAFF-producing Ly6Chi inflammatory monocytes and monocyte-derived dendritic cells (Mo-DCs), suggesting that NO normally inhibits BAFF expression. Indeed, we found that NOS2−/− DCs produced more BAFF than WT DCs, and addition of a NO donor to NOS2−/− DCs reduced BAFF production. Bone marrow chimeric mice that lack NOS2 in either non-hematopoietic or hematopoietic cells, each had intermediate IgM and IgG3 Ab responses after NP-Ficoll immunization, suggesting that NOS2 from both hematopoietic and non-hematopoietic sources regulates TI-2 Ab responses. Similar to NOS2−/− mice, depletion of Ly6Chi inflammatory monocytes and Mo-DCs enhanced NP-specific IgM and IgG3 responses to NP-Ficoll. Thus, NO produced by inflammatory monocytes and their derivative DC subsets plays an important role in regulating BAFF production and TI-2 Ab responses. PMID:24951820

  16. Independent impedimetric analysis of two cell populations co-cultured on opposite sides of a porous support.

    PubMed

    Hajek, Kathrin; Wegener, Joachim

    2017-02-01

    The transepithelial or -endothelial electrical resistance (TEER) is a very common and routinely recorded parameter describing the expression of barrier-forming cell-cell contacts (tight junctions) in quantitative terms. To determine TEER, barrier-forming cell monolayers are cultured on porous filter supports that separate two fluid compartments. The frequency-dependent impedance of the cell layer is then recorded and analyzed by means of equivalent circuit modelling providing TEER and the cell layer capacitance. The latter serves as a quantitative indicator for membrane topography. When cells are co-cultured on opposite sides of such a porous support to model more complex biological barriers, TEER readings will integrate over both cell layers and the individual contributions are not assessable. This study describes the modification of commonly used porous filter inserts by coating their backside with a thin gold-film. When this gold-film is used as an additional electrode, both cell layers can be studied separately by impedance analysis. The electrical parameters of either cell layer are assessable independently by switching between different electrode combinations. The performance of this new approach is illustrated and documented by experiments that (i) follow the de novo formation of cell junctions between initially suspended cells and (ii) the manipulation of mature cell-cell junctions by cytoskeleton-active drugs. Both assays confirm that both cell layers are monitored entirely independently.

  17. Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells

    SciTech Connect

    Sakamoto, Noriho; Hayashi, Shizu; Gosselink, John; Ishii, Hiroshi; Ishimatsu, Yuji; Mukae, Hiroshi; Hogg, James C.; Eeden, Stephan F. van

    2007-12-01

    Exposure to ambient air pollution particles with a diameter of < 10 {mu}m (PM{sub 10}) has been associated with increased cardiopulmonary morbidity and mortality. We have shown that human bronchial epithelial cells (HBECs) exposed to PM{sub 10} produce pro-inflammatory mediators that contribute to a local and systemic inflammatory response. Changes in intracellular calcium concentrations ([Ca{sup 2+}]{sub i}) have been demonstrated to regulate several functions of the airway epithelium including the production of pro-inflammatory mediators. The aim of the present study was to determine the nature and mechanism of calcium responses induced by PM{sub 10} in HBECs and its relationship to cytokine synthesis. Methods: Primary HBECs were exposed to urban air pollution particles (EHC-93) and [Ca{sup 2+}]{sub i} responses were measured using the fluoroprobe (Fura-2). Cytokine levels were measured at mRNA and protein levels using real-time PCR and ELISA. Results: PM{sub 10} increased [Ca{sup 2+}]{sub i} in a dose-dependent manner. This calcium response was reduced by blocking the influx of calcium into cells (i.e. calcium-free medium, NiCl{sub 2}, LaCl{sub 3}). PM{sub 10} also decreased the activity of calcium pumps. PM{sub 10} increased the production of IL-1{beta}, IL-8, GM-CSF and LIF. Preincubation with intracellular calcium chelator (BAPTA-AM) attenuated IL-1{beta} and IL-8 production, but not GM-CSF and LIF production. Conclusion: We conclude that exposure to PM{sub 10} induces an increase in cytosolic calcium and cytokine production in bronchial epithelial cells. Our results also suggest that PM{sub 10} induces the production of pro-inflammatory mediators via either intracellular calcium-dependent (IL-1{beta}, IL-8) or -independent (GM-CSF, LIF) pathways.

  18. Mouse Oocytes Acquire Mechanisms that Permit Independent Cell Volume Regulation at the End of Oogenesis.

    PubMed

    Richard, Samantha; Tartia, Alina P; Boison, Detlev; Baltz, Jay M

    2016-09-02

    Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [(3) H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. This article is protected by copyright. All rights reserved.

  19. Human Papillomavirus as an Independent Predictor in Oral Squamous Cell Cancer

    PubMed Central

    Zhao, Dan; Xu, Qin-gan; Chen, Xin-ming; Fan, Ming-wen

    2009-01-01

    Aim There is an increasing evidence for the role of high risk human papillomavirus (HPV) in the pathogenesis of oral squamous cell carcinoma (OSCC). The purpose of this study is to evaluate the relevance of HPV infection to the survival and prognosis of OSCC. Methodology Fifty-two patients with OSCC were followed from 4 to 88 months with a median of 50.7 months. HPV DNA was identified in formalin-fixed, paraffin-embedded tumor specimens by nested PCR with MY09/MY11 and GP5+/GP6+ primer pairs and the HPV genotype was determined by direct DNA sequencing. Association between the HPV status and risk factors for cancer as well as tumor-host characteristics were analyzed. Survival curves were calculated by the Kaplan-Meier method and analyzed using the log-rank test. Results HPV was found in 40.4% of the tumors with HPV16 accounting for 63.5%, HPV18 for 30.8%, HPV6 for 3.9% and HPV11 for 1.8%. No infection with more than one HPV genotype was detected. HPV infection was significantly associated with poor histological grade, TNM stage I–II, alcohol usage and no smoking status. Multi-variate analysis showed that HPV had an independent prognostic effect on the overall survival after adjusting other confounding factors such as histological grade, TNM stage and tobacco usage. The presence of HPV was significantly correlated with a better survival in patients with OSCC. Conclusion HPV infection can act as an independent predictor for the survival and prognosis of OSCC. PMID:20695077

  20. Loss of 5-Hydroxymethylcytosine Is an Independent Unfavorable Prognostic Factor for Esophageal Squamous Cell Carcinoma

    PubMed Central

    Shi, Xuejiao; Yu, Yue; Luo, Mei; Zhang, Zhirong; Shi, Susheng; Feng, Xiaoli; Chen, Zhaoli; He, Jie

    2016-01-01

    Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxylcytosine, which result in genomic DNA demethylation. It was reported that 5-hmC levels were decreased in a variety of cancers and could be regarded as an epigenetic hallmark of cancer. In the present study, 5-hmC levels were detected by immunohistochemistry (IHC) in 173 esophageal squamous cell carcinoma (ESCC) tissues and 91 corresponding adjacent non-tumor tissues; DNA dot blot assays were used to detect the 5-hmC level in another 50 pairs of ESCC tissues and adjacent non-tumor tissues. In addition, the mRNA level of TET1, TET2 and TET3 in these 50 pairs of ESCC tissues was detected by real-time PCR. The IHC and DNA dot blot results showed that 5-hmC levels were significantly lower in ESCC tissues compared with corresponding adjacent non-tumor tissues (P = 0.029). TET2 and TET3 expression was also significantly decreased in tumor tissues compared with paired non-tumor tissues (TET2, P < 0.0001; TET3, P = 0.009), and the decrease in 5-hmC was significantly associated with the downregulation of TET2 expression (r = 0.405, P = 0.004). Moreover, the loss of 5-hmC in ESCC tissues was significantly associated with poor overall survival among patients with ESCC (P = 0.043); multivariate Cox regression analysis showed that the loss of 5-hmC in ESCC tissues was an independent unfavorable prognostic indicator for patients with ESCC (HR = 1.569, P = 0.029). In conclusion, 5-hmC levels were decreased in ESCC tissues, and the loss of 5-hmC in tumor tissues was an independent unfavorable prognostic factor for patients with ESCC. PMID:27050164

  1. Long non-coding RNA LOC283070 mediates the transition of LNCaP cells into androgen-independent cells possibly via CAMK1D

    PubMed Central

    Wang, Lina; Lin, Yani; Meng, Hui; Liu, Chunyan; Xue, Jing; Zhang, Qi; Li, Chaoyang; Zhang, Pengju; Cui, Fuai; Chen, Weiwen; Jiang, Anli

    2016-01-01

    Aims: The present study is to investigate the role of long non-coding RNAs (lncRNAs) in the development of androgen independence in prostate cancer and its underlying mechanism. Methods: We established an androgen-independent prostate carcinoma (AIPC) cell line LNCaP-AI from androgen-dependent prostate carcinoma (ADPC) cell line LNCaP. Different expression profiles of lncRNAs and mRNAs between LNCaP and LNCaP-AI cells were investigated using microarray analysis. The expression of RNAs was determined using quantitative real-time polymerase chain reaction. Protein levels were measured using Western blotting. MTT assay was used to test cell viability. Tumor formation assay was performed in nude mice to detect tumor growth in vivo. Flow cytometry was performed to detect cell cycles. Transwell assay was employed to test cell migration and invasion. Results: According to bioinformatics prediction, lncRNA LOC283070 could possibly play an important role in the transition of LNCaP cells into LNCaP-AI cells. LOC283070 was up-regulated in LNCaP-AI cells and frequently up-regulated in AIPC cell lines. Overexpression of LOC283070 in LNCaP cells accelerated cell proliferation and migration, even under androgen-independent circumstances. Knockdown of LOC283070 inhibited LNCaP-AI cell proliferation and migration. Moreover, overexpression of LOC283070 promoted tumor growth in vivo in both normal mice and castrated mice. CAMK1D overexpression had similar effect with LOC283070, and CAMK1D knockdown fully abrogated the effect of LOC283070 overexpression on the transition of LNCaP cells into androgen-independent cells. Conclusions: The present study shows that overexpression of LOC283070 mediates the transition of LNCaP cells into androgen-independent LNCaP-AI cells possibly via CAMK1D. PMID:28077997

  2. Lack of Ephrin Receptor A1 Is a Favorable Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma

    PubMed Central

    Diezel, Michael; Meinhardt, Matthias; Zastrow, Stefan; Fuessel, Susanne; Wirth, Manfred P.; Baretton, Gustavo B.

    2014-01-01

    The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma. PMID:25025847

  3. IL-15 complexes induce NK cell and T cell responses independent of type I IFN signalling during rhinovirus infection

    PubMed Central

    Jayaraman, Annabelle; Jackson, David J.; Message, Simon D.; Pearson, Rebecca M.; Aniscenko, Julia; Caramori, Gaetano; Mallia, Patrick; Papi, Alberto; Shamji, Betty; Edwards, Matt; Westwick, John; Hansel, Trevor; Stanciu, Luminita A.; Johnston, Sebastian L.; Bartlett, Nathan W.

    2014-01-01

    Rhinoviruses are the most common virus to infect man causing a range of serious respiratory diseases including exacerbations of asthma and COPD. Type I IFN and IL-15 are thought to be required for antiviral immunity however their function during rhinovirus infection in vivo is undefined. In RV infected human volunteers, IL-15 protein expression in fluid from the nasal mucosa and in bronchial biopsies was increased. In mice, RV induced type I IFN-dependent expression of IL-15 and IL-15Rα which in turn were required for NK- and CD8+ T-cell responses. Treatment with IL-15-IL-15Rα complexes (IL-15c) boosted RV-induced expression of IL-15, IL-15Rα, IFN-γ, CXCL9 and CXCL10 followed by recruitment of activated, IFN-γ expressing NK, CD8+ and CD4+ T cells. Treating infected IFNAR1−/− mice with IL-15c similarly increased IL-15, IL-15Rα, IFN-γ and CXCL9 (but not CXCL10) expression also followed by NK-, CD8+- and CD4+-T cell recruitment and activation. We have demonstrated that type I IFN induced IFN-γ and cellular immunity to RV was mediated by IL-15 and IL-15Rα. Importantly we also show that IL-15 could be induced via a type I IFN-independent mechanism by IL-15 complex treatment which in turn was sufficient to drive IFN-γ expression and lymphocyte responses. PMID:24472849

  4. The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines.

    PubMed

    Strauss, Sandra J; Higginbottom, Karen; Jüliger, Simone; Maharaj, Lenushka; Allen, Paul; Schenkein, David; Lister, T Andrew; Joel, Simon P

    2007-03-15

    Bortezomib is a proteasome inhibitor with proven efficacy in multiple myeloma and non-Hodgkin's lymphoma. This study reports the effects of bortezomib in B-cell lymphoma cell lines with differing sensitivity to bortezomib to investigate factors that influence sensitivity. Bortezomib induced a time- and concentration-dependent reduction in cell viability in five lymphoma cell lines, with EC(50) values ranging from 6 nmol/L (DHL-7 cells) to 25 nmol/L (DHL-4 cells) after 72 h. Bortezomib cytotoxicity was independent of p53 function, as all cell lines exhibited mutations by sequence analysis. The difference in sensitivity was not explained by proteasome or nuclear factor-kappaB (NF-kappaB) inhibition as these were similar in the most and least sensitive cells. NF-kappaB inhibition was less marked than that of a specific NF-kappaB inhibitor, Bay 11-7082. Cell cycle analysis showed a marked G(2)-arrested population in the least sensitive DHL-4 line only, an effect that was not present with Bay 11-7082 treatment. Conversely, in DHL-7 cells, bortezomib treatment resulted in cells moving into an aberrant mitosis, indicative of mitotic catastrophe that may contribute to increased sensitivity to bortezomib. These studies show that although bortezomib treatment had similar effects on apoptotic and NF-kappaB signaling pathways in these cell lines, different cell cycle effects were observed and induction of a further mechanism of cell death, mitotic catastrophe, was observed in the more sensitive cell line, which may provide some pointers to the difference in sensitivity between cell lines. An improved understanding of how DHL-7 cells abrogate the G(2)-M cell cycle checkpoint may help identify targets to increase the efficacy of bortezomib.

  5. Brief Report: Single-Cell Analysis Reveals Cell Division-Independent Emergence of Megakaryocytes From Phenotypic Hematopoietic Stem Cells.

    PubMed

    Roch, Aline; Trachsel, Vincent; Lutolf, Matthias P

    2015-10-01

    Despite increasingly stringent methods to isolate hematopoietic stem cells (HSCs), considerable heterogeneity remains in terms of their long-term self-renewal and differentiation potential. Recently, the existence of long-lived, self-renewing, myeloid-restricted progenitors in the phenotypically defined HSC compartment has been revealed, but these cells remain poorly characterized. Here, we used an in vitro single-cell analysis approach to track the fate of 330 long-term HSCs (LT-HSC; Lin- cKit+ Sca-1+ CD150+ CD48- CD34-) cultured for 5 days under serum-free basal conditions. Our analysis revealed a highly heterogeneous behavior with approximately 15% of all phenotypic LT-HSCs giving rise to megakaryocytes (Mk). Surprisingly, in 65% of these cases, Mk development occurred in the absence of cell division. This observation suggests that myeloid-restricted progenitors may not derive directly from LT-HSCs but instead could share an identical cell surface marker repertoire.

  6. Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng

    2014-07-01

    Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NFκB response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NFκB pathway.

  7. The alpha1-adrenoceptor antagonist terazosin induces prostate cancer cell death through a p53 and Rb independent pathway.

    PubMed

    Xu, Kexin; Wang, Xianghong; Ling, Patrick M T; Tsao, S W; Wong, Y C

    2003-01-01

    Prostate cancer is the second leading cause of cancer-related death in men. Treatment failure in prostate cancer is usually due to the development of androgen independence and resistance to chemotherapeutic drugs at an advanced stage. Recently, it was reported that the alpha1-adrenoceptor antagonist terazosin was able to inhibit prostate cancer cell growth and indicated that it may have an implication in the treatment of prostate cancer. The aim of the present study was to investigate the mechanisms involved in terazosin-induced prostate cancer cell death using two androgen-independent cell lines, PC-3 and DU145. Our results showed that terazosin inhibited not only prostate cancer cell growth but also colony forming ability, which is the main target of chemotherapy. We also found that the sensitivity of these cells to terazosin was not affected by the presence of either functional p53 or Rb, suggesting that the terazosin-induced cell death was independent of p53 and Rb. However, the terazosin-induced cell death was associated with G1 phase cell cycle arrest and up-regulation of p27KIP1. In addition, up-regulation of Bax and down-regulation of Bcl-2 was also observed indicating that these two apoptotic regulators may play important roles in terazosin-mediated cell death pathway. Our results provide evidence for the first time that terazosin may have a therapeutic potential in the treatment of advanced prostate cancer.

  8. Hop/STI1 modulates retinal proliferation and cell death independent of PrP{sup C}

    SciTech Connect

    Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.; Linden, Rafael; Chiarini, Luciana B. . E-mail: chiarini@biof.ufrj.br

    2007-09-21

    Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP{sup C}). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP{sup C} dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 ({alpha}-STI1) blocked both ganglion cell and NBL cell death independent of PrP{sup C}. cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while {alpha}-STI1 increased proliferation in the developing retina, both independent of PrP{sup C}. We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP{sup C}.

  9. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner.

    PubMed

    Takegahara, Yuki; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Disulfiram eradicates tumor-initiating hepatocellular carcinoma cells in ROS-p38 MAPK pathway-dependent and -independent manners.

    PubMed

    Chiba, Tetsuhiro; Suzuki, Eiichiro; Yuki, Kaori; Zen, Yoh; Oshima, Motohiko; Miyagi, Satoru; Saraya, Atsunori; Koide, Shuhei; Motoyama, Tenyu; Ogasawara, Sadahisa; Ooka, Yoshihiko; Tawada, Akinobu; Nakatsura, Tetsuya; Hayashi, Takehiro; Yamashita, Taro; Kaneko, Syuichi; Miyazaki, Masaru; Iwama, Atsushi; Yokosuka, Osamu

    2014-01-01

    Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)(+) HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM(+) cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM(+) cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.

  11. T cell regulation of the thymus-independent antibody response to trinitrophenylated-Brucella abortus (TNP-BA)

    SciTech Connect

    Tanay, A.; Strober, S.

    1985-06-01

    The authors have previously observed a reduction of the T cell-dependent primary antibody response to dinitrophenylated keyhole limpet hemocyanin, and an enhancement of the T cell-independent response to trinitrophenylated Brucella abortus (TNP-BA) in BALB/c mice after treatment with total lymphoid irradiation (TLI). To elucidate the relative contribution of T and B cells to the enhanced T cell-independent antibody responses after TLI, a syngeneic primary adoptive transfer system was utilized whereby irradiated hosts were reconstituted with unfractionated spleen cells or a combination of purified T and B cells from TLI-treated and untreated control mice. Antibody responses of purified splenic B cells from TLI-treated BALB/c mice (TLI/B) to TNP-BA were enhanced 10-fold as compared with those of unfractionated (UF) spleen cells or B cells from normal (NL) BALB/c mice (NL/UF and NL/B, respectively). Splenic T cells from normal animals (NL/T) suppressed the anti-TNP-BA response of TLI/B by more than 100-fold. NL/T neither suppressed nor enhanced the response of NL/B. On the other hand, T cells from TLI-treated mice (TLI/T) enhanced by 100-fold the anti-TNP-BA response of NL/B, but neither suppressed nor enhanced the response of TLI/B. Thus, T cells can regulate the T cell-independent antibody response to TNP-BA. However, experimental manipulation of the T and B cell populations is needed to demonstrate the regulatory functions.

  12. ECE-1 influences prostate cancer cell invasion via ET-1-mediated FAK phosphorylation and ET-1-independent mechanisms.

    PubMed

    Whyteside, A R; Hinsley, E E; Lambert, L A; McDermott, P J; Turner, A J

    2010-08-01

    Plasma concentrations of the mitogenic peptide endothelin-1 (ET-1) are significantly elevated in men with metastatic prostate cancer (PC). ET-1 also contributes to the transition of hormonally regulated androgen-dependent PC to androgen-independent disease. ET-1 is generated from big-ET-1 by endothelin-converting enzyme (ECE-1). ECE-1 is present in PC cell lines and primary tissue and is elevated in primary malignant stromal cells compared with benign. siRNA or shRNA-mediated knockdown of endogenous ECE-1 in either the epithelial or stromal compartment significantly reduced PC cell (PC-3) invasion and migration. The re-addition of ET-1 only partially recovered the effect, suggesting ET-1-dependent and -independent functions for ECE-1 in pPC. The ET-1-independent effect of ECE-1 on PC invasion may be due to modulation of downstream signalling events. Addition of an ECE-1 specific inhibitor to PC-3 cells reduced phosphorylation of focal adhesion kinase (FAK), a signalling molecule known to play a role in PC. siRNA-mediated knockdown of ECE-1 resulted in a significant reduction in FAK phosphorylation. Accordingly, transient ECE-1 overexpression in PNT1-a cells increased FAK phosphorylation. In conclusion, ECE-1 influences PC cell invasion via both ET-1-mediated FAK phosphorylation and ET-1 independent mechanisms.

  13. Investigation of low temperature solid oxide fuel cells for air-independent UUV applications

    NASA Astrophysics Data System (ADS)

    Moton, Jennie Mariko

    Unmanned underwater vehicles (UUVs) will benefit greatly from high energy density (> 500 Wh/L) power systems utilizing high-energy-density fuels and air-independent oxidizers. Current battery-based systems have limited energy densities (< 400 Wh/L), which motivate development of alternative power systems such as solid oxide fuel cells (SOFCs). SOFC-based power systems have the potential to achieve the required UUV energy densities, and the current study explores how SOFCs based on gadolinia-doped ceria (GDC) electrolytes with operating temperatures of 650°C and lower may operate in the unique environments of a promising UUV power plant. The plant would contain a H 2O2 decomposition reactor to supply humidified O2 to the SOFC cathode and exothermic aluminum/H2O combustor to provide heated humidified H2 fuel to the anode. To characterize low-temperature SOFC performance with these unique O2 and H2 source, SOFC button cells based on nickel/GDC (Gd0.1Ce0.9O 1.95) anodes, GDC electrolytes, and lanthanum strontium cobalt ferrite (La0.6Sr0.4Co0.2Fe0.8O3-δ or LSCF)/GDC cathodes were fabricated and tested for performance and stability with humidity on both the anode and the cathode. Cells were also tested with various reactant concentrations of H2 and O2 to simulate gas depletion down the channel of an SOFC stack. Results showed that anode performance depended primarily on fuel concentration and less on the concentration of the associated increase in product H2O. O 2 depletion with humidified cathode flows also caused significant loss in cell current density at a given voltage. With the humidified flows in either the anode or cathode, stability tests of the button cells at 650 °C showed stable voltage is maintained at low operating current (0.17 A/cm2) at up to 50 % by mole H2O, but at higher current densities (0.34 A/cm2), irreversible voltage degradation occurred at rates of 0.8-3.7 mV/hour depending on exposure time. From these button cell results, estimated average

  14. CD8+ T cells mediate antibody-independent platelet clearance in mice.

    PubMed

    Arthur, Connie M; Patel, Seema R; Sullivan, H Cliff; Winkler, Annie M; Tormey, Chris A; Hendrickson, Jeanne E; Stowell, Sean R

    2016-04-07

    Platelet transfusion provides an important therapeutic intervention in the treatment and prevention of bleeding. However, some patients rapidly clear transfused platelets, preventing the desired therapeutic outcome. Although platelet clearance can occur through a variety of mechanisms, immune-mediated platelet removal often plays a significant role. Numerous studies demonstrate that anti-platelet alloantibodies can induce significant platelet clearance following transfusion. In fact, for nearly 50 years, anti-platelet alloantibodies were considered to be the sole mediator of immune-mediated platelet clearance in platelet-refractory individuals. Although nonimmune mechanisms of platelet clearance can often explain platelet removal in the absence of anti-platelet alloantibodies, many patients experience platelet clearance following transfusion in the absence of a clear mechanism. These results suggest that other processes of antibody-independent platelet clearance may occur. Our studies demonstrate that CD8(+)T cells possess the unique ability to induce platelet clearance in the complete absence of anti-platelet alloantibodies. These results suggest a previously unrecognized form of immune-mediated platelet clearance with significant implications in the appropriate management of platelet-refractory individuals.

  15. Inhibition of immune opsonin-independent phagocytosis by antibody to a pulmonary macrophage cell surface antigen

    SciTech Connect

    Parod, R.J.; Godleski, J.J.; Brain J.D.

    1986-03-15

    Unlike other hamster phagoycytes, hamster pulmonary macrophages (PM) avidly ingest albumin-coated latex particles in the absence of serum. They also possess a highly specific cell surface antigen. To evaluate the relationship between these two characteristics, PM were incubated with mouse monoclonal antibody directed against the PM antigen. After unbound antibody was removed, the amount of bound antibody and the phagocytic capability of PM were measured by flow cytometry and fluorescence microscopy. Maximum antibody binding produced a 25% inhibition of ingestion. Particle attachment was not affected. This effect was antigen specific, since neither a nonspecific mouse myeloma protein of the same subclass nor a mouse antibody that bound to another hamster surface antigen had any effect on binding or ingestion. If antigen-specific F(ab')/sub 2/ fragments were introduced both before and during the period of phagocytosis, the inhibition of particle ingestion approached 100%. Particle binding increased at low F(ab')/sub 2/ concentrations but declined at higher concentrations. Because calcium may play a role in the ingestion process, the effect of antibody on /sup 45/Ca uptake was evaluated. It was observed that antigen-specific F(ab')/sub 2/ fragments stimulated /sup 45/Ca uptake, whereas control antibodies did not. These results suggest that the antigen reacting with the anti-hamster PM monoclonal antibody is involved in immune opsonin-independent phagocytosis and that calcium participates in this phagocytic process.

  16. Heterologous desensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells.

    PubMed Central

    Bréant, B; Keppens, S; De Wulf, H

    1981-01-01

    Vasopressin and alpha-adrenergic agonists are known to be potent cyclic AMP-independent Ca2+-dependent activators of liver glycogen phosphorylase. When hepatocytes are pre-incubated with increasing concentrations of vasopressin or of the alpha-agonist phenylephrine, they become progressively unresponsive to a second addition of the respective agonist. The relative abilities of six vasopressin analogues and of five alpha-agonists to activate glycogen phosphorylase and to cause subsequent desensitization are highly correlated, indicating that the same vasopressin and alpha-adrenergic receptors are involved in both responses. About 5-times-higher peptide concentrations are needed to desensitize the cells than to activate their glycogen phosphorylase, whereas the concentrations of alpha-agonists required for the desensitization are only twice those needed for the activation of phosphorylase. The desensitization is not mediated by a perturbation in the agonist-receptor interaction. It is clearly heterologous, i.e. it is not agonist-specific, and must therefore involve a mechanism common to both series of agonists. The evidence for a role of Ca2+ movements or phosphatidylinositol turnover is briefly discussed. PMID:6123310

  17. Testosterone alters iron metabolism and stimulates red blood cell production independently of dihydrotestosterone.

    PubMed

    Beggs, Luke A; Yarrow, Joshua F; Conover, Christine F; Meuleman, John R; Beck, Darren T; Morrow, Matthew; Zou, Baiming; Shuster, Jonathan J; Borst, Stephen E

    2014-09-01

    Testosterone (T) stimulates erythropoiesis and regulates iron homeostasis. However, it remains unknown whether the (type II) 5α-reduction of T to dihydrotestosterone (DHT) mediates these androgenic effects, as it does in some other tissues. Our purpose was to determine whether inhibition of type II 5α-reductase (via finasteride) alters red blood cell (RBC) production and serum markers of iron homeostasis subsequent to testosterone-enanthate (TE) administration in older hypogonadal men. Sixty men aged ≥60 yr with serum T <300 ng/dl or bioavailable T <70 ng/dl received treatment with TE (125 mg/wk) vs. vehicle paired with finasteride (5 mg/day) vs. placebo using a 2 × 2 factorial design. Over the course of 12 mo, TE increased RBC count 9%, hematocrit 4%, and hemoglobin 8% while suppressing serum hepcidin 57% (P < 0.001 for all measurements). Most of the aforementioned changes occurred in the first 3 mo of treatment, and finasteride coadministration did not significantly alter any of these effects. TE also reduced serum ferritin 32% (P = 0.002) within 3 mo of treatment initiation without altering iron, transferrin, or transferrin saturation. We conclude that TE stimulates erythropoiesis and alters iron homeostasis independently of the type II 5α-reductase enzyme. These results demonstrate that elevated DHT is not required for androgen-mediated erythropoiesis or for alterations in iron homeostasis that would appear to support iron incorporation into RBCs.

  18. Anchorage independency promoted tumor malignancy of melanoma cells under reattachment through elevated interleukin-8 and CXC chemokine receptor 1 expression.

    PubMed

    Uen, Wu-Ching; Hsieh, Chiao-Hui; Tseng, Ting-Ting; Jiang, Shih Sheng; Tseng, Jen-Chih; Lee, Shao-Chen

    2015-02-01

    Metastasis of melanoma cells during the recurrence or the late stage of melanoma has been characterized as the dissemination of tumor cells under anchorage independency. The secreted interleukin-8 (IL-8) and its conical receptors from melanoma cells have been associated with melanoma malignancy. However, their correlations with melanoma cells under anchorage independency were unclear. Suspension of adherent melanoma cells generated the suspended melanoma cell model of anoikis resistance. The in-vivo xenograft experiment, in-vitro cell proliferation/migration assay, microarray, and bioinformatics analysis were used to compare the malignancy and gene expression profiling in adherent and suspended melanoma cells. PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and kinase inhibition assay were adapted to validate the expression and regulation of IL-8 and CXCR1/2. Suspended melanoma cells were anoikis resistant and showed elevated malignancy in vivo and in vitro. Gene expression profiling of adherent and suspended melanoma cells showed extensive alteration associated with cell survival/death, cell signaling, and regulation of gene expression. Microarray and bioinformatics analysis on gene set enrichment analysis further showed elevated IL-8 expression in suspended melanoma cells. The upregulation of IL-8 and the effect on chemotaxis were mediated by MEK/ERK activation upon cell suspension. Change in JNK phosphorylation induced CXCR1 downregulation under cell suspension, but upregulation by cell reattachment. We suggest the possible roles of elevated IL-8 secretion and change in CXCR expression contributing toward elevated melanoma malignancy upon reattachment from cell suspension. We show that the suspension of melanoma cells is critical in promoting melanoma malignancy in vivo and in vitro.

  19. Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells

    PubMed Central

    Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

    2017-01-01

    Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death. PMID:28182713

  20. Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells.

    PubMed

    Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

    2017-01-01

    Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death.

  1. Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, gamma radiation, and cisplatin

    PubMed Central

    Vareki, Saman Maleki; Rytelewski, Mateusz; Figueredo, Rene; Chen, Di; Ferguson, Peter J.; Vincent, Mark; Min, Weiping; Zheng, Xiufen; Koropatnick, James

    2014-01-01

    Indoleamine 2,3-dioxygenase-1 (IDO) is an immunosuppressive molecule expressed by most human tumors. IDO levels correlate with poor prognosis in cancer patients and IDO inhibitors are under investigation to enhance endogenous anticancer immunosurveillance. Little is known of immune-independent functions of IDO relevant to cancer therapy. We show, for the first time, that IDO mediates human tumor cell resistance to a PARP inhibitor (olaparib), gamma radiation, cisplatin, and combined treatment with olaparib and radiation, in the absence of immune cells. Antisense-mediated reduction of IDO, alone and (in a synthetic lethal approach) in combination with antisense to the DNA repair protein BRCA2 sensitizes human lung cancer cells to olaparib and cisplatin. Antisense reduction of IDO decreased NAD+ in human tumor cells. NAD+ is essential for PARP activity and these data suggest that IDO mediates treatment resistance independent of immunity and at least partially due to a previously unrecognized role for IDO in DNA repair. Furthermore, IDO levels correlated with accumulation of tumor cells in G1 and depletion of cells in G2/M of the cell cycle, suggesting that IDO effects on cell cycle may also modulate sensitivity to radiation and chemotherapeutic agents. IDO is a potentially valuable therapeutic target in cancer treatment, independent of immune function and in combination with other therapies. PMID:24784564

  2. Influence of cations and anions on the induction of cell density-independent luminescence in Photorhabdus luminescens.

    PubMed

    Tabei, Yosuke; Ogawa, Akane; Era, Mariko; Ninomiya, Junko; Morita, Hiroshi

    2013-03-01

    Bioluminescence is emitted by various living organisms, including bacteria. While the induction mechanism in marine luminescent bacteria, such as Vibrio fischeri and V. harveyi, has been well characterized, this mechanism has not been studied in detail in the non-marine luminescent bacterium Photorhabdus luminescens. Therefore, we investigated the effect of cations and anions on the induction of luminescence by P. luminescens. Cultivation of cells in an inorganic salts solution (ISS) containing KCl, CaCl2 , MgCl2 , NaHCO3 , and MgSO4 resulted in a rapid increase in luminescence intensity. Moreover, the induction of luminescence in the ISS medium was not dependent on cell density, since cell densities remained unchanged during 48 h. Furthermore, we found that compounds containing K(+) , Mg(2+) , and HCO3(-) were necessary to induce cell density-independent luminescence. The intensity of luminescence per cell cultured in medium containing KCl, MgCl2 , and NaHCO3 was approximately 100-fold higher than that cultured in NB. In contrast, when cells actively grew in normal growth condition, the intensity of luminescence per cell was not increased even in the presence of K(+) , Mg(2+) , and HCO3(-) . Thus, these results suggest that the luminescence of P. luminescens is regulated by 2 independent cell density-dependent and -independent mechanisms. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The pure anti-androgen bicalutamide inhibits cyclin A expression both in androgen-dependent and -independent cell lines.

    PubMed

    Katayama, Hiroshi; Murashima, Teruko; Saeki, Yoshiko; Nishizawa, Yasuko

    2010-03-01

    We investigated the effects of testosterone and the pure anti-androgen, bicalutamide, on DNA synthesis and cell cycle in androgen-sensitive or -insensitive human and mouse cell lines by 3H-thymidine incorporation, flow cytometry, RT-PCR and Western blotting. In androgen-dependent mouse SC-3 cells, testosterone induced DNA synthesis, shift of cell cycle distribution from G0/G1 to S/G2/M and expression of cyclin A. The induction was preceded by that of fibroblast growth factor 8 (FGF-8), and completely blocked by monoclonal antibody to FGF-8. Dihydrotestosterone (DHT) induced cyclin A expression in androgen-sensitive human prostate cancer cells, but not in androgen-independent cell lines. Bicalutamide almost completely inhibited these androgen-dependent effects both in LNCaP and SC-3 cells, but had no or limited effect on androgen-independent or FGF-8-induced DNA synthesis, and FGF-8 induced cyclin A expression. Interestingly, bicalutamide inhibited both DNA synthesis and the cyclin A expression in androgen-independent human cell lines in serum-free condition. A MEK1/2 inhibitor U0126 blocked both androgen- and rFGF-8-induced DNA synthesis. Overall, bicalutamide inhibits the cyclin A expression possibly by inhibiting FGF-8 mRNA expression and FGF-8 protein secretion but not by inhibiting FGF receptor (FGFR) signalling in androgen-dependent cell lines, and by other mechanisms in androgen-independent cell lines. The results suggest that combination with compounds such as FGFR signalling inhibitors may provide additional benefits to anti-androgens. It is also suggested that cyclin A could be a sensitive marker for androgen-induced cancer growth and for the growth inhibitory effects of anti-androgen.

  4. Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism

    SciTech Connect

    Moreira, Liliana; Araújo, Isabel; Costa, Tito; Correia-Branco, Ana; Faria, Ana; Martel, Fátima; Keating, Elisa

    2013-07-15

    In this study we characterized {sup 3}H-2-deoxy-D-glucose ({sup 3}H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon {sup 3}H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells {sup 3}H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (V{sub max}) and affinity (K{sub m}), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that {sup 3}H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited {sup 3}H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4 h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors. -- Highlights: • Glucose uptake by MCF7 and MDA-MB-231 cells is mainly mediated by GLUT1. • QUE and EGCG inhibit cellular glucose uptake thus abolishing the Warburg effect. • This process induces cytotoxicity and proliferation arrest in MCF7 cells. • The flavonoids’ effects are independent of estrogen receptor signalling.

  5. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    SciTech Connect

    Takegahara, Yuki; Yamanouchi, Keitaro Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  6. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death.

    PubMed

    Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

    2016-03-25

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy.

  7. Reaction of germinal centers in the T-cell-independent response to the bacterial polysaccharide alpha(1-->6)dextran.

    PubMed Central

    Wang, D; Wells, S M; Stall, A M; Kabat, E A

    1994-01-01

    Primary immunization of BALB/c mice with alpha(1-->6)dextran (DEX), a native bacterial polysaccharide, induces an unexpected pattern of splenic B-cell responses. After a peak of antibody-secreting B-cell response at day 4, deposition of dextran-anti-dextran immune complexes, as revealed by staining with both dextran and antibodies to dextran, occurs and persists in splenic follicles until at least the fourth week after immunization. Antigen-specific B cells appear and proliferate in such follicles, leading by day 11 to development of DEX-specific germinal centers as characterized by the presence of distinct regions of DEX+ peanut agglutinin-positive (PNA+) cells. At this time, fluorescence-activated cell sorter analysis also reveals the appearance of a distinct population of DEX+ PNA+ splenic B cells. In contrast, DEX+ PNA- cells, characterized by intense cytoplasmic staining, are present outside of splenic follicles, peak at day 4 to day 5, and persist until at least day 28. The frequency of these cells correlates with DEX-specific antibody-secreting cells, as detected by the ELISA-spot assay. Thus, in addition to the expected plasma cellular response, the typical T-cell-independent type II antigen, DEX, surprisingly also elicits the formation of antigen-specific germinal centers. These observations raise fundamental questions about the roles of germinal centers in T-cell-independent immune responses. Images PMID:7511812

  8. Fibronectin Assembly in the Crypts of Cytokinesis-Blocked Multilobular Cells Promotes Anchorage-Independent Growth

    PubMed Central

    Gupta, Rajesh Kumar; Johansson, Staffan

    2013-01-01

    Anchorage-independent growth is a characteristic feature of cancer cells. However, it is unclear whether it represents a cause or a consequence of tumorigenesis. For normal cells, integrin-mediated adhesion is required for completion of the G1 and cytokinesis stages of the cell cycle. This study identified a mechanism that can drive anchorage-independent growth if the G1 checkpoint is suppressed. Cells with defective G1 checkpoint progressed through several rounds of the cell cycle in suspension in spite of uncompleted cytokinesis, thereby forming bi- and multilobular cells. Aurora B and CEP55 were localized to midbodies between the lobes, suggesting that the cytokinesis process reached close to abscission. Integrin-mediated re-attachment of such cells induced cytokinesis completion uncoupled from karyokinesis in most cells. However, a portion of the cells instead lost the constriction and became binucleated. Also, long-term suspension culture in soft agar produced colonies where the cytokinesis block was overcome. This process was fibronectin-dependent since fibronectin-deficient cells did not form colonies unless fibronectin was expressed or exogenously added. While fibronectin normally is not deposited on non-adherent single cells, bi/multilobular cells accumulated fibronectin in the intussusceptions. Based on our data we conclude: 1) Suppression of the G1 checkpoint allows multiple rounds of the cell cycle in detached cells and thereby enables matrix formation on their surface. 2) Uncompleted cytokinesis due to cell detachment resumes if integrin interactions are re-formed, allowing colony formation in soft agar 3) Such delayed cell division can generate binucleated cells, a feature known to cause chromosomal instability. PMID:23951336

  9. CopA3 peptide from Copris tripartitus induces apoptosis in human leukemia cells via a caspase-independent pathway.

    PubMed

    Kang, Bo Ram; Kim, Ho; Nam, Sung-Hee; Yun, Eun-Young; Kim, Seong-Ryul; Ahn, Mi-Young; Chang, Jong Soo; Hwang, Jae Sam

    2012-02-01

    Our previous study demonstrated that CopA3, a disulfide dimer of the coprisin peptide analogue (LLCIALRKK), has antibacterial activity. In this study, we assessed whether CopA3 caused cellular toxicity in various mammalian cell lines. CopA3 selectively caused a marked decrease in cell viability in Jurkat T, U937, and AML-2 cells (human leukemia cells), but was not cytotoxic to Caki or Hela cells. Fragmentation of DNA, a marker of apoptosis, was also confirmed in the leukemia cell lines, but not in the other cells. CopA3-induced apoptosis in leukemia cells was mediated by apoptosis inducing factor (AIF), indicating induction of a caspase-independent signaling pathway.

  10. Dysregulated CXCR4 expression promotes lymphoma cell survival and independently predicts disease progression in germinal center B-cell-like diffuse large B-cell lymphoma

    PubMed Central

    Deng, Lijuan; Shen, Qi; Manyam, Ganiraju C.; Martinez-Lopez, Azahara; Zhang, Li; Montes-Moreno, Santiago; Visco, Carlo; Tzankov, Alexandar; Yin, Lihui; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W.L.; van Krieken, J. Han; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J.M.; Zhao, Xiaoying; Møller, Michael B.; Farnen, John P.; Winter, Jane N.; Piris, Miguel A.; Pham, Lan; Young, Ken H.

    2015-01-01

    Abnormal expression of the chemokine receptor CXCR4 plays an essential role in tumor cell dissemination and disease progression. However, the significance of CXCR4 overexpression in de novo diffuse large B cell lymphoma (DLBCL) is unknown. In 743 patients with de novo diffuse large B cell lymphoma (DLBCL) who received standard Rituximab-CHOP immunochemotherapy, we assessed the expression of CXCR4 and dissected its prognostic significance in various DLBCL subsets. Our results showed that CXCR4+ patients was associated with male, bulky tumor, high Ki-67 index, activated B-cell-like (ABC) subtype, and Myc, Bcl-2 or p53 overexpression. Moreover, CXCR4+ was an independent factor predicting poorer progression-free survival in germinal-center B-cell-like (GCB)-DLBCL, but not in ABC-DLBCL; and in patients with an IPI of ≤2, but not in those with an IPI>2. The lack of prognostic significance of CXCR4 in ABC-DLBCL was likely due to the activation of p53 tumor suppressor attenuating CXCR4 signaling. Furthermore, concurrent CXCR4+ and BCL2 translocation showed dismal outcomes resembling but independent of MYC/BCL2 double-hit DLBCL. Gene expression profiling suggested that alterations in the tumor microenvironment and immune responses, increased tumor proliferation and survival, and the dissemination of CXCR4+ tumor cells to distant organs or tissues were underlying molecular mechanisms responsible for the CXCR4+ associated poor prognosis. PMID:25704881

  11. Differential Features of AIRE-Induced and AIRE-Independent Promiscuous Gene Expression in Thymic Epithelial Cells.

    PubMed

    St-Pierre, Charles; Trofimov, Assya; Brochu, Sylvie; Lemieux, Sébastien; Perreault, Claude

    2015-07-15

    Establishment of self-tolerance in the thymus depends on promiscuous expression of tissue-restricted Ags (TRA) by thymic epithelial cells (TEC). This promiscuous gene expression (pGE) is regulated in part by the autoimmune regulator (AIRE). To evaluate the commonalities and discrepancies between AIRE-dependent and -independent pGE, we analyzed the transcriptome of the three main TEC subsets in wild-type and Aire knockout mice. We found that the impact of AIRE-dependent pGE is not limited to generation of TRA. AIRE decreases, via non-cell autonomous mechanisms, the expression of genes coding for positive regulators of cell proliferation, and it thereby reduces the number of cortical TEC. In mature medullary TEC, AIRE-driven pGE upregulates non-TRA coding genes that enhance cell-cell interactions (e.g., claudins, integrins, and selectins) and are probably of prime relevance to tolerance induction. We also found that AIRE-dependent and -independent TRA present several distinctive features. In particular, relative to AIRE-induced TRA, AIRE-independent TRA are more numerous and show greater splicing complexity. Furthermore, we report that AIRE-dependent versus -independent TRA project nonredundant representations of peripheral tissues in the thymus. Copyright © 2015 by The American Association of Immunologists, Inc.

  12. Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

    PubMed Central

    Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

    2014-01-01

    Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. PMID:25175936

  13. Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax.

    PubMed

    Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

    2014-12-01

    Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo.

  14. Slp2-a controls renal epithelial cell size through regulation of Rap-ezrin signaling independently of Rab27.

    PubMed

    Yasuda, Takao; Fukuda, Mitsunori

    2014-02-01

    Synaptotagmin-like protein 2 (Slp2-a/Sytl2) is a Rab27 effector protein that regulates transport of Rab27-bearing vesicles and organelles through its N-terminal Rab27-binding domain and a phospholipid-binding C2A domain. Here we demonstrate a Rab27-independent function of Slp2-a in the control of renal cell size through a previously uncharacterized C2B domain. We found that by recruiting Rap1 GAPs to the plasma membrane of MDCK II cells through the C2B domain, Slp2-a inactivates Rap signaling and modulates the size of the cells. Functional ablation of Slp2-a resulted in an increase in the size of MDCK II cells. Drosophila Slp Bitesize was found to compensate for the function of Slp2-a in MDCK II cells, thereby indicating that the mechanism of the cell size control by Slp proteins has been evolutionarily conserved. Interestingly, blockade of the activity of ezrin, a downstream target of Rap, with the glucosylceramide synthase inhibitor, miglustat, effectively inhibited cell spreading of Slp2-a-knockdown cells. We also discovered aberrant expression of Slp2-a and increased activity of ezrin in pcy (Nphp3(pcy)) mice, a model of polycystic kidney disease that is characterized by renal cell spreading. Our findings indicate that Slp2-a controls renal cell size through regulation of Rap-ezrin signaling independently of Rab27.

  15. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation

    PubMed Central

    Plümpe, Tobias; Ehninger, Dan; Steiner, Barbara; Klempin, Friederike; Jessberger, Sebastian; Brandt, Moritz; Römer, Benedikt; Rodriguez, Gerardo Ramirez; Kronenberg, Golo; Kempermann, Gerd

    2006-01-01

    Background In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX) expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events. Results We found that (1) 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2) the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3) positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes. Conclusion These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis. PMID:17105671

  16. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation.

    PubMed

    Plümpe, Tobias; Ehninger, Dan; Steiner, Barbara; Klempin, Friederike; Jessberger, Sebastian; Brandt, Moritz; Römer, Benedikt; Rodriguez, Gerardo Ramirez; Kronenberg, Golo; Kempermann, Gerd

    2006-11-15

    In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX) expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events. We found that (1) 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2) the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3) positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes. These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis.

  17. Synergy of 5-aza-2'-deoxycytidine (DAC) and paclitaxel in both androgen-dependent and -independent prostate cancer cell lines.

    PubMed

    Shang, Donghao; Liu, Yuting; Liu, Qingjun; Zhang, Fengbo; Feng, Lang; Lv, Wencheng; Tian, Ye

    2009-06-08

    To determine the synergy of 5-aza-2'-deoxycytidine (DAC) and paclitaxel (PTX) against prostate carcinoma (PC) cells by isobolographic analysis. We demonstrated that DAC could significantly increase the susceptibility of PC cells to PTX, and confirmed the synergy of DAC and PTX. DAC enhanced the PTX induced up-regulation of caspase activity and antiproliferative effect, resulting in an increase of cells in subG1 and G2/M phases. In addition, the synergy was observed in both androgen-dependent and -independent PC cell lines. It suggested that combination chemotherapy with DAC and PTX might be a new strategy to improve the clinical response rate of PC.

  18. Serum HE4: An Independent Prognostic Factor in Non-Small Cell Lung Cancer.

    PubMed

    Lamy, Pierre-Jean; Plassot, Carine; Pujol, Jean-Louis

    2015-01-01

    Human epididymis secretory protein 4 (HE4) is a secreted glycosylated protein encoded by the WAP four-disulfide core domain 2 (WFDC2) gene, located on a chromosome 20 segment that is frequently amplified in many cancers. This study aimed at determining serum HE4 prognostic value in non-small cell lung cancer (NSCLC), following the REMARK guidelines. Serum samples from 346 consecutive patients with histologically proven and previously untreated NSCLC and 41 patients with benign pulmonary disease were collected at the Montpellier-Nimes Academic Hospital. Work-up investigations performed to determine the disease characteristics and treatment algorithms were congruent with international guidelines. HE4 levels in serum were measured with an ELISA test (Fujirebio Diagnostics) that uses two monoclonal antibodies, 2H5 and 3D8, against the C-WFDC domain of HE4. The area under the ROC curve (i.e., overall ability of HE4 to discriminate between controls and patients) was 0.78 (95% confidence interval [CI], 0.738-0.821; z test P <0.0001). Serum HE4 levels were significantly higher in patients with worse performance status, advanced TNM stage and positive nodal status. In the Cox model, overall survival was shorter in patients with high pretreatment serum HE4 (above 140 pmol/L) than in patients with serum H4 level ≤ 140 pmol/L [median survival: 17.7 weeks (95% CI, 11.9 to 24.9) and 46.4 weeks (95% CI, 38.6 to 56.3), respectively; hazard ratio: 1.48 (95% CI, 1.12 to 1.95) for high HE4; adjusted P = 0.0057]. High serum HE4 level at diagnosis is an independent determinant of poor prognosis in NSCLC.

  19. UV fingerprints predominate in the PTCH mutation spectra of basal cell carcinomas independent of clinical phenotype.

    PubMed

    Heitzer, Ellen; Lassacher, Anita; Quehenberger, Franz; Kerl, Helmut; Wolf, Peter

    2007-12-01

    Basal cell carcinoma (BCC) shows a wide interpatient variation in lesion accrual. To determine whether certain tumorigenic fingerprints and potentially predisposing patched (PTCH) tumor suppressor single-nucleotide polymorphisms (SNPs) are distributed differently among sporadic BCC patients, we compared the PTCH mutation spectra in early-onset BCC (first lesion at age < 35 years), regular BCC (first lesion at age > or = 35 years and < 10 lesions), and multiple BCC (> or = 10 lesions). The PTCH gene was mutated in 29 of 60 cases (48%). Most of the PTCH mutations bore the UV fingerprint (i.e., C --> T or tandem CC --> TT transitions at dipyrimidine sites). However, neither the proportion nor the spectra of exonic PTCH mutations differed significantly among the three groups. A large number of SNPs (IVS10+99C/T, IVS11-51G/C, 1665T/C, 1686C/T, IVS15+9G/C, IVS16-80G/C, IVS17+21G/A, and 3944C/T or its combinations) were also detected, but again their incidence did not differ significantly among the groups. Interestingly, expression of the IVS16-80G/C and the IVS17+21G/A genotype did not achieve the Hardy-Weinberg equilibrium in patients with regular and/or early-onset BCC. These data suggest that a (UV-) mutated PTCH gene is important for sporadic BCC formation independent of clinical phenotype and that the IVS16-80G/C and/or IVS17+21G/A SNP site might be important for tumorigenesis in certain BCC patients.

  20. (−)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells

    PubMed Central

    Ramirez-Sanchez, Israel; Maya, Lisandro; Ceballos, Guillermo

    2011-01-01

    The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (−)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca2+ depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca2+-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca2+-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca2+ depletion. Thus, under Ca2+-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca2+-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-l-arginine methyl ester, suggesting a functional relevance for this phenomenon. PMID:21209365

  1. Bax expression measured by AQUAnalysis is an independent prognostic marker in oral squamous cell carcinoma

    PubMed Central

    2012-01-01

    Background Resistance to apoptosis is a hallmark of cancer and proteins regulating apoptosis have been proposed as prognostic markers in several malignancies. However, the prognostic impact of apoptotic markers has not been consistently demonstrated in oral squamous cell carcinoma (OSCC). This inconsistency in reported associations between apoptotic proteins and prognosis can be partly attributed to the intrinsic low resolution and misclassification associated with manual, semi-quantitative methods of biomarker expression measurement. The aim of this study was to examine the association between apoptosis-regulating proteins and clinical outcomes in oral squamous cell carcinoma (OSCC) using the quantitative fluorescence immunohistochemistry (IHC) based AQUAnalysis technique. Methods Sixty-nine OSCC patients diagnosed between 1998–2005 in Calgary, Alberta, Canada were included in the study. Clinical data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded pre-treatment tumour tissue. Bax, Bcl-2 and Bcl-XL protein expression was quantified using fluorescent IHC and AQUA technology in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Survival was analyzed using Kaplan-Meier plots and the Cox proportional hazard model. Results Bax expression was predominantly nuclear in OCSE and almost exclusively cytoplasmic in OSCC. No similar differences in localization were observed for Bcl-2 or Bcl-XL. Only Bax expression associated with disease-specific survival (DSS), with 5-year survival estimates of 85.7% for high Bax versus 50.3% for low Bax (p = 0.006), in univariate analysis. High Bax expression was also significantly associated with elevated Ki67 expression, indicating that increased proliferation might lead to an improved response to radiotherapy in patients with elevated Bax expression. In multivariate analyses, Bax protein expression

  2. Discovery and optimization of new benzofuran derivatives against p53-independent malignant cancer cells through inhibition of HIF-1 pathway.

    PubMed

    Yang, Ying-Rui; Wei, Jin-Lian; Mo, Xiao-Fei; Yuan, Zhen-Wei; Wang, Jia-Lin; Zhang, Chao; Xie, Yi-Yue; You, Qi-Dong; Sun, Hao-Peng

    2016-06-01

    p53-independent malignant cancer is still severe health problem of human beings. HIF-1 pathway is believed to play an important role in the survival and developing progress of such cancers. In the present study, with the aim to inhibit the proliferation of p53-independent malignant cells, we disclose the optimization of 6a, the starting compound which is discovered in the screening of in-house compound collection. The structure-activity relationship (SAR) is summarized. The most potent derivative 8d, inhibits the proliferation of both p53-null and p53-mutated cells through inhibition of HIF-1 pathway. Our findings here provide a new chemotype in designing potent anticancer agent especially against those p53-independent malignant tumors. Copyright © 2016. Published by Elsevier Ltd.

  3. Heterogeneity of breast cancer stem cells as evidenced with Notch-dependent and Notch-independent populations.

    PubMed

    Wong, Nelson K Y; Fuller, Megan; Sung, Sandy; Wong, Fred; Karsan, Aly

    2012-10-01

    Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML-expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population.

  4. 9-cis-retinoic acid promotes cell adhesion through integrin dependent and independent mechanisms across immune lineages.

    PubMed

    Whelan, Jarrett T; Chen, Jianming; Miller, Jabin; Morrow, Rebekah L; Lingo, Joshuah D; Merrell, Kaitlin; Shaikh, Saame Raza; Bridges, Lance C

    2013-05-01

    Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) on human hematopoietic cell adhesion was investigated. 9-cis-RA treatment specifically induced cell adhesion of the human immune cell lines HuT-78, NB4, RPMI 8866 and U937. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if cell adhesion was integrin-dependent. By employing a variety of integrin antagonist including function-blocking antibodies and EDTA, we establish that 9-cis-RA prompts immune cell adhesion through established integrin receptors in addition to a novel integrin-independent process. The novel integrin-independent adhesion required the presence of retinoid and was attenuated by treatment with synthetic corticosteroids. Finally, we demonstrate that 9-cis-RA treatment of primary murine B-cells induces ex vivo adhesion that persists in the absence of integrin function. Our study is the first to demonstrate that 9-cis-RA influences immune cell adhesion through at least two functionally distinct mechanisms.

  5. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    USDA-ARS?s Scientific Manuscript database

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  6. Rabies virus interaction with various cell lines is independent of the acetylcholine receptor.

    PubMed

    Reagan, K J; Wunner, W H

    1985-01-01

    Rabies virus infects most cells in vitro. The presence of the nicotinic acetylcholine receptor on the plasma membrane of various cell lines is not an obligate factor for rabies virus susceptibility of those cells.

  7. Variation of Keratin 7 Expression and Other Phenotypic Characteristics of Independent Isolates of Cadmium Transformed Human Urothelial Cells (UROtsa)

    PubMed Central

    Somji, Seema; Zhou, Xu Dong; Mehus, Aaron; Sens, Mary Ann; Garrett, Scott H.; Lutz, Krista L.; Dunlevy, Jane R.; Zheng, Yun; Sens, Donald. A.

    2009-01-01

    This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd+2) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd+2 transformed cell lines were isolated to determine if independent exposures of the cell line to Cd+2 would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice and for the expression keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd+2 transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd+2 transformed cells have both similarities and differences in their phenotype. PMID:19921857

  8. The protein-tyrosine phosphatase CD45 reaches the cell surface via golgi-dependent and -independent pathways.

    PubMed

    Baldwin, Troy A; Ostergaard, Hanne L

    2002-12-27

    CD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis. The remaining pool of CD45 was fully glycosylated and began to arrive at the cell surface at approximately 15 min. The rapidly expressed population of CD45 possessed exclusively endoglycosidase H-sensitive N-linked carbohydrate. Additionally, this rapidly expressed pool of CD45 appeared on the cell surface in a brefeldin A (BFA)-insensitive manner, suggesting that it reached the cell surface independent of the Golgi complex. The remaining CD45 trafficked through the Golgi complex, and transport proceeded via a BFA-sensitive mechanism. These data suggest that CD45 is able to reach the cell surface via two distinct routes. The first is a conventional Golgi-dependent pathway that allows fully processed CD45 to be expressed. The second utilizes an ill defined mechanism that is independent of the Golgi, is BFA-resistant, and allows for the expression of CD45 with immature carbohydrate on the cell surface.

  9. SLI-1 Cbl Inhibits the Engulfment of Apoptotic Cells in C. elegans through a Ligase-Independent Function

    PubMed Central

    Sawin, Emma; Horvitz, H. Robert; Hurwitz, Michael E.

    2012-01-01

    The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway, which includes the small GTPase CED-10 Rac and the cytoskeletal regulator ABI-1, acts to rearrange the cytoskeleton of the engulfing cell. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The second pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Cbl, the mammalian homolog of the C. elegans E3 ubiquitin ligase and adaptor protein SLI-1, interacts with Rac and Abi2 and modulates the actin cytoskeleton, suggesting it might act in engulfment. Our genetic studies indicate that SLI-1 inhibits apoptotic cell engulfment and DTC migration independently of the CED-10 Rac and CED-1 pathways. We found that the RING finger domain of SLI-1 is not essential to rescue the effects of SLI-1 deletion on cell migration, suggesting that its role in this process is ubiquitin ligase-independent. We propose that SLI-1 opposes the engulfment of apoptotic cells via a previously unidentified pathway. PMID:23271977

  10. Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers.

    PubMed

    Losinno, Antonella D; Morales, Alfonsina; Fernández, Dario; Lopez, Luis A

    2012-05-01

    In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.

  11. Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activity.

    PubMed

    Petrs-Silva, Hilda; de Freitas, Fabíola G; Linden, Rafael; Chiarini, Luciana B

    2004-02-01

    We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system.

  12. Inositol pyrophosphates modulate cell cycle independently of alteration in telomere length.

    PubMed

    Banfic, Hrvoje; Crljen, Vladiana; Lukinovic-Skudar, Vesna; Dembitz, Vilma; Lalic, Hrvoje; Bedalov, Antonio; Visnjic, Dora

    2016-01-01

    Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the signalling response required for cell cycle progression after mating pheromone arrest. Overexpression of Kcs1 doubled the level of inositol pyrophosphates when compared to wild type cells and 30 min following the release from α-factor block further increase in inositol pyrophosphates was observed, which resulted that cells overexpressing Kcs1 reached G2/M phase earlier than wild type cells. Similar effect was observed in ipk1Δ cells, which are unable to synthesize IP6-derived inositol pyrophosphates (IP7 and IP8) but will synthesize IP5-derived inositol pyrophosphates (PP-IP4 and (PP)2-IP3). Although ipk1Δ cells have shorter telomeres than wild type cells, overexpression of Kcs1 in both strains have similar effect on cell cycle progression. As it is known that PP-IP4 regulates telomere length through Tel1, inositol polyphosphates, cell cycle and telomere length were determined in tel1Δ cells. The release of the cells from α-factor block and overexpression of Kcs1 in tel1Δ cells produced similar effects on inositol pyrophosphates level and cell cycle progression when compared to wild type cells, although tel1Δ cells possesses shorter telomeres than wild type cells. It can be concluded that telomere length does not affect cell cycle progression, since cells with short telomeres (ipk1Δ and tel1Δ) progress through cell cycle in a similar manner as wild type cells and that overexpression of Kcs1 in cells with either short or normal telomeres will increase S phase progression without affecting telomere length. Furthermore, IP5-derived inositol pyrophosphates can compensate for the loss of IP6-derived inositol pyrophosphates, in modulating S phase progression of the cell cycle.

  13. Acquired somatic mutations in PNH reveal long-term maintenance of adaptive NK cells independent of HSPCs.

    PubMed

    Corat, Marcus A F; Schlums, Heinrich; Wu, Chuanfeng; Theorell, Jakob; Espinoza, Diego A; Sellers, Stephanie E; Townsley, Danielle M; Young, Neal S; Bryceson, Yenan T; Dunbar, Cynthia E; Winkler, Thomas

    2017-04-06

    Natural killer (NK) cells have long been considered short-lived effectors of innate immunity. However, recent animal models and human studies suggest that subsets of NK cells have adaptive features. We investigate clonal relationships of various NK-cell subsets, including the adaptive population, by taking advantage of naturally occurring X-linked somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs) from patients with paroxysmal nocturnal hemoglobinuria (PNH). The affected HSPCs and their progeny lack expression of glycosylphosphatidylinositol (GPI) anchors on their cell surface, allowing quantification of PIGA-mutant (GPI-negative) HSPC-derived peripheral blood cell populations. The fraction of GPI-negative cells within the CD56(dim) NK cells was markedly lower than that of neutrophils and the CD56(bright) NK-cell compartments. This discrepancy was most prominent within the adaptive CD56(dim) NK-cell population lacking PLZF expression. The functional properties of these adaptive NK cells were similar in PNH patients and healthy individuals. Our findings support the existence of a long-lived, adaptive NK-cell population maintained independently from GPI(pos)CD56(dim).

  14. Two distinct but convergent groups of cells trigger Torso receptor tyrosine kinase activation by independently expressing torso-like.

    PubMed

    Furriols, Marc; Ventura, Gemma; Casanova, Jordi

    2007-07-10

    Cell fate determination is often the outcome of specific interactions between adjacent cells. However, cells frequently change positions during development, and thus signaling molecules might be synthesized far from their final site of action. Here, we analyze the regulation of the torso-like gene, which is required to trigger Torso receptor tyrosine kinase activation in the Drosophila embryo. Whereas torso is present in the oocyte, torso-like is expressed in the egg chamber, at the posterior follicle cells and in two separated groups of anterior cells, the border cells and the centripetal cells. We find that JAK/STAT signaling regulates torso-like expression in the posterior follicle cells and border cells but not in the centripetal cells, where torso-like is regulated by a different enhancer. The border and centripetal cells, which are originally apart, converge at the anterior end of the oocyte, and we find that both groups contribute to trigger Torso activation. Our results illustrate how independently acquired expression of a signaling molecule can constitute a mechanism by which distinct groups of cells act together in the activation of a signaling pathway.

  15. Arf6 and Rab22 mediate T cell conjugate formation by regulating clathrin-independent endosomal membrane trafficking.

    PubMed

    Johnson, Debra L; Wayt, Jessica; Wilson, Jean M; Donaldson, Julie G

    2017-07-15

    Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs. © 2017. Published by The Company of Biologists Ltd.

  16. MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

    SciTech Connect

    Liu, Lian; Zhang, Xin; Du, Chao; Zhang, Xiaoning; Hou, Nan; Zhao, Di; Sun, Jianzhi; Li, Li; Wang, Xiuwen; Ma, Chunhong

    2009-05-01

    We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even when it fails to activate MAPK as expected.

  17. Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

    PubMed

    Mingueneau, Michael; Roncagalli, Romain; Grégoire, Claude; Kissenpfennig, Adrien; Miazek, Arkadiusz; Archambaud, Cristel; Wang, Ying; Perrin, Pierre; Bertosio, Elodie; Sansoni, Amandine; Richelme, Sylvie; Locksley, Richard M; Aguado, Enrique; Malissen, Marie; Malissen, Bernard

    2009-08-21

    Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.

  18. Degree of Keratinization Is an Independent Prognostic Factor in Oral Squamous Cell Carcinoma.

    PubMed

    Wolfer, Susanne; Elstner, Stefan; Schultze-Mosgau, Stefan

    2017-06-30

    Keratinization is a routinely reported histologic feature in head and neck cancer. In contrast to numerous clinicopathologic parameters, the prognostic value of keratinization in oral squamous cell carcinoma (OSCC) is rarely reported in the literature. The purpose of this study was to review the outcome of patients with OSCC with a special focus on the degree of keratinization. In this retrospective cohort study, we evaluated the medical records at the Department of Oral and Maxillofacial Surgery, Jena University Hospital, and investigated the outcome of patients with OSCC with disease-free survival and disease-specific survival according to the degree of keratinization. This research also analyzed common clinical and histologic parameters such as age, gender, tumor site, T category, N category, resection margin, lymphovascular invasion, and extracapsular spread. Descriptive statistics were performed, and survival was calculated by the Kaplan-Meier method. Prognostic factors were analyzed by multivariate Cox analysis. In the sample of 151 OSCC patients, with a median age of 57.5 years and a male-female ratio of 4.03:1, 119 had tumors with no or low keratinization (K0 to K2) and 32 had tumors with good or high keratinization (K3 or K4). More recurrences were seen in patients with OSCC with low keratinization (P = .0008). The 5-year disease-free survival rate was significantly decreased for OSCC with low keratinization (52.9%) compared with good or high keratinization (93.2%) (P = .0008). The 5-year disease-specific survival rate was reduced to 66.1% (P = .0136) for patients with OSCC with low keratinization. Multivariate analysis showed that extracapsular spread (P = .001) and keratinization (P = .002) are independent, significant prognostic factors for recurrence in OSCC. Besides extracapsular spread, the degree of keratinization seems to be an important prognostic factor for recurrence and survival in OSCC. Our results indicate that the degree of

  19. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  20. Disulfiram Eradicates Tumor-Initiating Hepatocellular Carcinoma Cells in ROS-p38 MAPK Pathway-Dependent and -Independent Manners

    PubMed Central

    Yuki, Kaori; Zen, Yoh; Oshima, Motohiko; Miyagi, Satoru; Saraya, Atsunori; Koide, Shuhei; Motoyama, Tenyu; Ogasawara, Sadahisa; Ooka, Yoshihiko; Tawada, Akinobu; Nakatsura, Tetsuya; Hayashi, Takehiro; Yamashita, Taro; Kaneko, Syuichi; Miyazaki, Masaru; Iwama, Atsushi; Yokosuka, Osamu

    2014-01-01

    Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)+ HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM+ cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM+ cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells. PMID:24454751

  1. Independent expression of the adrenergic phenotype by neural crest cells in vitro.

    PubMed Central

    Cohen, A M

    1977-01-01

    Neural crest cells obtained from Japanese quail and grown in vitro without other embryonic tissues differentiate into adrenergic cells. These cells show intense catecholamine-specific histochemical fluorescence, and some have long, varicose neuronal processes. Ultrastructural examination shows two populations of cells, one with small (about 90 nm) dense-core vesicles resembling principal sympathetic neurons and the other with larger (about 150 nm) dense-core granules resembling chromaffin or small intensely fluorescent cells. Neuronal cells without adrenergic characteristics are also present. These results are compatible with the hypothesis that a population of cells determined along neuronal lines exists in the neural crest prior to migration. Images PMID:268641

  2. Suberoyl bishydroxamic acid-induced apoptosis in HeLa cells via ROS-independent, GSH-dependent manner.

    PubMed

    You, Bo Ra; Park, Woo Hyun

    2013-05-01

    Suberoyl bishydroxamic acid (SBHA) is a HDAC inhibitor that can regulate many biological functions including apoptosis and proliferation in various cancer cells. Here, we evaluated the effect of SBHA on the growth of HeLa cervical cancer cells in relation to apoptosis, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC50 of approximately 15 μM at 72 h. SBHA also induced apoptosis in HeLa cells, as evidenced by sub-G1 cells, annexin V-FITC staining cells, activations of caspase 3 and 8, and the loss of mitochondrial membrane potential (ΔΨm). In addition, all of the tested caspase inhibitors rescued some cells from SBHA-induced HeLa cell death. SBHA increased ROS levels including O2(•-) and induced GSH depletion in HeLa cells. Generally, caspase inhibitors did not affect ROS levels in SBHA-treated HeLa cells, but they significantly prevented GSH depletion in these cells. Furthermore, while the well-known antioxidants, N-acetyl cysteine and vitamin C, did not affect cell death, ROS level or GSH depletion in SBHA-treated HeLa cells, L-buthionine sulfoximine, a GSH synthesis inhibitor, enhanced cell death and GSH depletion in these cells. In conclusion, SBHA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis, and the inhibition is independent of ROS level changes, but dependent on GSH level changes.

  3. Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell

    PubMed Central

    Gorba, Thorsten; Reitano, Erika; Toselli, Mauro; Biella, Gerardo; Sun, Yirui; Sanzone, Sveva; Ying, Qi-Long; Cattaneo, Elena

    2005-01-01

    Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These

  4. (Pro)renin receptor mediates both angiotensin II-dependent and -independent oxidative stress in neuronal cells.

    PubMed

    Peng, Hua; Li, Wencheng; Seth, Dale M; Nair, Anand R; Francis, Joseph; Feng, Yumei

    2013-01-01

    The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.

  5. [Inhibitory effect of Genipin on uncoupling protein-2 and energy metabolism of androgen-independent prostate cancer cells].

    PubMed

    Yao, Mao-liang; Gu, Jiang; Zhang, Yong-chun; Wang, Nan; Zhu, Zhi-hui; Yang, Qing-tao; Liu, Miao; Xia, Jian-feng

    2015-11-01

    To explore whether the inhibitory effect of Genipin on uncoupling protein-2 (UCP-2) in mitochondria is involved in energy metabolism of androgen-independent PC3 prostate cancer cells. PC3 prostate cancer cells were cultured and treated with Genipin at the concentrations of 40, 80, and 160 μmol/L for 48 hours. Then the proliferation of the cells was detected by MTT assay, the expression of UCP-2 mRNA determined by RT-PCR, and the content of intracellular pyruvic acid (PA) and the activity of succinate dehydrogenase (SDH) in the mitochondria measured by visible spectrophotometry. With the increased concentration of Genipin, the proliferative activity of the PC-3 cells, the expression level of UCP-2 mRNA, the content of intracellular PA and the activity of SDH in the cells were all decreased, namely, with the enhanced inhibitory effect of Genipin on UCP-2, a trend of reduction was observed in the proliferation of the cells, intracellular PA content, and SDH activity in the mitochondria. Genipin is involved in the energy metabolism of androgen-independent PC3 prostate cancer cells by reducing the content of intracellular PA and the activity of SDH in the mitochondria, which may be associated with its inhibitory effect on UCP-2.

  6. Immunologic abnormality in NZB/W F1 mice. Thymus-independent expansion of B cells responding to interleukin-6.

    PubMed Central

    Mihara, M; Fukui, H; Koishihara, Y; Saito, M; Ohsugi, Y

    1990-01-01

    We have previously reported that B cell abnormality in NZB/W F1 mice developed independently of thymus. Here we examined further whether B cells from NZB/W F1 mice respond to interleukin-6 (IL-6), a factor for terminal differentiation of B cells. When freshly isolated splenic B cells were incubated for 5 days in the presence of human IL-6, an increased production of both IgM and IgG, including anti-DNA antibody, was evident in NZB/W F1 mice; there was no increase in BALB/c mice. A magnitude of augmentation in IgG but not IgM production by IL-6 became more apparent in older NZB/W F1 mice. The increased immunoglobulin production seen with IL-6 was neutralized by treatment with rabbit anti-recombinant human IL-6 antibody. As B cells from athymic NZB/W F1 nude mice also responded to IL-6, it was suggested that B cells in NZB/W F1 mice differentiated into the IL-6-responding state in a thymus-independent manner. This B cell abnormality may be associated with the pathogenesis of autoimmune disease in NZB/W F1 mice. PMID:2265491

  7. Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration

    PubMed Central

    Meller, Julia; Vidali, Luis; Schwartz, Martin Alexander

    2009-01-01

    Summary Rac activation by integrins is essential for cell spreading, migration, growth and survival. Based mainly on over-expression of dominant negative mutants, RhoG was proposed to mediate integrin-dependent Rac activation upstream of ELMO and Dock 180. RhoG knockout mice, however, display no significant developmental or functional abnormalities. To clarify the role of RhoG in integrin-mediated signaling, we developed a RhoG-specific antibody, which, together with shRNA mediated knockdown, allowed analysis of the endogenous protein. Despite dramatic effects of dominant negative constructs, nearly complete RhoG depletion did not substantially inhibit cell adhesion, spreading, migration or Rac activation. Additionally, RhoG was not detectably activated by adhesion to fibronectin. Using Rac1−/− cells, we found that constitutively active RhoG induced membrane ruffling via both Rac-dependent and –independent pathways. Additionally, endogenous RhoG was important for Rac-independent cell migration. However, RhoG did not significantly contribute to cell spreading even in these cells. These data therefore clarify the role of RhoG in integrin signaling and cell motility. PMID:18505794

  8. Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

    SciTech Connect

    Kakoki, Katsura; Kamiyama, Haruka; Izumida, Mai; Yashima, Yuka; Hayashi, Hideki; Yamamoto, Naoki; Matsuyama, Toshifumi; Igawa, Tsukasa; Sakai, Hideki; Kubo, Yoshinao

    2014-04-25

    Highlights: • XMRV infection induces androgen-independent growth in LNCaP cells. • XMRV infection reduces expression of androgen receptor. • XMRV promotes appearance of androgen blocker-resistant prostate cancer cells. - Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.

  9. TACI is required for efficient plasma cell differentiation in response to T-independent type 2 antigens.

    PubMed

    Mantchev, George T; Cortesão, Catarina S; Rebrovich, Michelle; Cascalho, Marilia; Bram, Richard J

    2007-08-15

    The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.

  10. The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration

    PubMed Central

    Mantuano, Elisabetta; Lam, Michael S.; Shibayama, Masataka; Campana, W. Marie; Gonias, Steven L.

    2015-01-01

    ABSTRACT NMDA receptors (NMDA-Rs) are ionotropic glutamate receptors, which associate with LDL-receptor-related protein-1 (LRP1) to trigger cell signaling in response to protein ligands in neurons. Here, we demonstrate for the first time that the NMDA-R is expressed by rat Schwann cells and functions independently and with LRP1 to regulate Schwann cell physiology. The NR1 (encoded by GRIN1) and NR2b (encoded by GRIN2B) NMDA-R subunits were expressed by cultured Schwann cells and upregulated in sciatic nerves following crush injury. The ability of LRP1 ligands to activate ERK1/2 (also known as MAPK3 and MAPK1, respectively) and promote Schwann cell migration required the NMDA-R. NR1 gene silencing compromised Schwann cell survival. Injection of the LRP1 ligands tissue-type plasminogen activator (tPA, also known as PLAT) or MMP9-PEX into crush-injured sciatic nerves activated ERK1/2 in Schwann cells in vivo, and the response was blocked by systemic treatment with the NMDA-R inhibitor MK801. tPA was unique among the LRP1 ligands examined because tPA activated cell signaling and promoted Schwann cell migration by interacting with the NMDA-R independently of LRP1, albeit with delayed kinetics. These results define the NMDA-R as a Schwann cell signaling receptor for protein ligands and a major regulator of Schwann cell physiology, which may be particularly important in peripheral nervous system (PNS) injury. PMID:26272917

  11. OSU-03012 promotes caspase-independent but PERK-, cathepsin B-, BID-, and AIF-dependent killing of transformed cells.

    PubMed

    Yacoub, Adly; Park, Margaret A; Hanna, David; Hong, Young; Mitchell, Clint; Pandya, Aditi P; Harada, Hisashi; Powis, Garth; Chen, Ching-Shih; Koumenis, Costas; Grant, Steven; Dent, Paul

    2006-08-01

    We determined one mechanism by which the putative phosphoinositide-dependent kinase (PDK)-1 inhibitor 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide (OSU-03012) killed primary human glioma and other transformed cells. OSU-03012 caused a dose-dependent induction of cell death that was not altered by p53 mutation, expression of ERBB1 vIII, or loss of phosphatase and tensin homolog deleted on chromosome 10 function. OSU-03012 promoted cell killing to a greater extent in glioma cells than in nontransformed astrocytes. OSU-03012 and ionizing radiation caused an additive, caspase-independent elevation in cell killing in 96-h viability assays and true radiosensitization in colony formation assays. In a cell type-specific manner, combined exposure to OSU-03012 with a mitogen-activated protein kinase kinase 1/2 inhibitor, phosphoinositide 3-kinase/AKT inhibitors, or parallel molecular interventions resulted in a greater than additive induction of cell killing that was independent of AKT activity and caspase function. OSU-03012 lethality as a single agent or when combined with signaling modulators was not modified in cells lacking expression of BIM or of BAX/BAK. OSU-03012 promoted the release of cathepsin B from the lysosomal compartment and release of AIF from mitochondria. Loss of BH3-interacting domain (BID) function, overexpression of BCL(XL), and inhibition of cathepsin B function suppressed cell killing and apoptosis-inducing factor (AIF) release from mitochondria. In protein kinase R-like endoplasmic reticulum kinase-/- cells, the lethality of OSU-03012 was attenuated which correlated with reduced cleavage of BID and with suppression of cathepsin B and AIF release into the cytosol. Our data demonstrate that OSU-03012 promotes glioma cell killing that is dependent on endoplasmic reticulum stress, lysosomal dysfunction, and BID-dependent release of AIF from mitochondria, and whose lethality is enhanced by irradiation or by

  12. Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

    SciTech Connect

    Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung; Kim, Hye Jin; Yang, Jin Mo; Ryu, Somi; Noh, Yoo Hun; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Yoo, Keon Hee; Koo, Hong Hoe; Sung, Ki Woong

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.

  13. Bifurcate effects of glucose on caspase-independent cell death during hypoxia

    SciTech Connect

    Aki, Toshihiko; Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi

    2010-06-04

    We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

  14. GM3 suppresses anchorage-independent growth via Rho GDP dissociation inhibitor beta in melanoma B16 cells.

    PubMed

    Wang, Pu; Xu, Su; Wang, Yinan; Wu, Peixing; Zhang, Jinghai; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2011-08-01

    Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells.

  15. PDE8 regulates rapid Teff cell adhesion and proliferation independent of ICER.

    PubMed

    Vang, Amanda G; Ben-Sasson, Shlomo Z; Dong, Hongli; Kream, Barbara; DeNinno, Michael P; Claffey, Michelle M; Housley, William; Clark, Robert B; Epstein, Paul M; Brocke, Stefan

    2010-08-09

    Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem-/- mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

  16. A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation

    PubMed Central

    2011-01-01

    Introduction Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in such breast cancers occurred through an Mdm2 mediated pathway with subsequent inactivation of p53. Methods We examined the effect of long-term 17β-estradiol (E2) treatment (five days) on the p53-Mdm2 pathway in estrogen receptor alpha (ERα) positive breast cancer cell lines that contain wild-type p53 (MCF-7 and ZR75-1). We assessed the influence of estrogen by examining cell proliferation changes, activation of transcription of p53 target genes, p53-chromatin interactions and cell cycle profile changes. To determine the effects of Mdm2 and p53 knockdown on the estrogen-mediated proliferation signals we generated MCF-7 cell lines with inducible shRNA for mdm2 or p53 and monitored their influence on estrogen-mediated outcomes. To further address the p53-independent effect of Mdm2 in ERα positive breast cancer we generated cell lines with inducible shRNA to mdm2 using the mutant p53 expressing cell line T-47D. Results Estrogen increased the Mdm2 protein level in MCF-7 cells without decreasing the p53 protein level. After estrogen treatment of MCF-7 cells, down-regulation of basal transcription of p53 target genes puma and p21 was observed. Estrogen treatment also down-regulated etoposide activated transcription of puma, but not p21. Mdm2