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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. Caspase-independent cell deaths.

    PubMed

    Lockshin, Richard A; Zakeri, Zahra

    2002-12-01

    A very common and the best understood of the mechanisms of physiological cell death is apoptosis, resulting from the activation, through either of two primary pathways, of site-specific proteases called caspases. There are, however, many other routes to cell death, prominently including autophagy and proteasomal degradation of critical constituents of cells. These routes are frequently seen in experimental situations in which initiator or effector caspases are inhibited or blocked through genetic means, but they are also encountered during normal physiological and pathological processes. Most frequently, autophagic or proteasomal degradation is used to eliminate massive cytoplasm of very large cells, especially post-mitotic cells, and these pathways are prominent even though caspase genes, messages, and pro-enzymes are found in the cells. These forms of cell death are fully physiological and not simply a default pathway for a defective cell; and they are distinct from necrosis. We do not yet understand the extent to which the pathways are linked, what mechanisms trigger the caspase-independent deaths, and how the choices are made.

  3. Gravity-Independent Orientation of Honeycomb Cells

    NASA Astrophysics Data System (ADS)

    Pratt, S. C.

    Honey bees have long been assumed to build their comb with the cells in either of two preferred orientations with respect to gravity ("vertical" or "horizontal"). I show here that these typical cell orientations in fact derive from substrate orientation and a simple building rule, rather than the influence of gravity itself. When bees were induced to build comb on substrates at four different orientations with respect to gravity, they always made cells with one vertex pointing directly toward the substrate. This produced horizontal and vertical cells on vertical and horizontal substrates, respectively, but yielded intermediate orientations on oblique substrates. The apparent preference for vertical and horizontal cells may simply reflect substrate orientation in the rectilinear hives from which cell measurements have been taken.

  4. 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.

  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. 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)

  7. 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.

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

    PubMed

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

    2005-11-01

    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. 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

  10. The price of independence: cell separation in fission yeast.

    PubMed

    Martín-García, Rebeca; Santos, Beatriz

    2016-04-01

    The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise. PMID:26931605

  11. HIF-1α Promotes A Hypoxia-Independent Cell Migration.

    PubMed

    Li, Liyuan; Madu, Chikezie O; Lu, Andrew; Lu, Yi

    2010-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.

  12. HIF-1α Promotes A Hypoxia-Independent Cell Migration

    PubMed Central

    Li, Liyuan; Madu, Chikezie O.; Lu, Andrew; Lu, Yi

    2010-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects. PMID:20882121

  13. 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

  14. 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.

  15. B cells mediate chronic allograft rejection independently of antibody production.

    PubMed

    Zeng, Qiang; Ng, Yue-Harn; Singh, Tripti; Jiang, Ke; Sheriff, Khaleefathullah A; Ippolito, Renee; Zahalka, Salwa; Li, Qi; Randhawa, Parmjeet; Hoffman, Rosemary A; Ramaswami, Balathiripurasundari; Lund, Frances E; Chalasani, Geetha

    2014-03-01

    Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

  16. 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

  17. Independent stem cell lineages regulate adipose organogenesis and adipose homeostasis

    PubMed Central

    Jiang, Yuwei; Berry, Daniel C.; Tang, Wei; Graff, Jonathan M.

    2014-01-01

    Summary Adipose tissues have striking plasticity, highlighted by childhood and adult obesity. Using adipose lineage analyses, smooth muscle actin (SMA)-mural cell fate mapping, and conditional PPARγ deletion to block adipocyte differentiation, we find two phases of adipocyte generation that emanate from two independent adipose progenitor compartments, Developmental and Adult. These two compartments are sequentially required for organ formation and maintenance. Although both Developmental and Adult progenitors are specified during the developmental period and express PPARγ, they have distinct micro-anatomical, functional, morphogenetic and molecular profiles. Further, the two compartments derive from different lineages, while adult adipose progenitors fate map from an SMA+ mural lineage, Developmental progenitors do not. Remarkably, the Adult progenitor compartment appears to be specified earlier than the Developmental cells, and then enters the already developmentally formed adipose depots. Thus, two distinct cell compartments control adipose organ development and organ homeostasis, which may provide discrete therapeutic target for childhood and adult obesity. PMID:25437556

  18. 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

  19. Osteoclast-independent bone resorption by fibroblast-like cells

    PubMed Central

    Pap, Thomas; Claus, Anja; Ohtsu, Susumu; Hummel, Klaus M; Schwartz, Peter; Drynda, Susanne; Pap, Géza; Machner, Andreas; Stein, Bernhard; George, Michael; Gay, Renate E; Neumann, Wolfram; Gay, Steffen; Aicher, Wilhelm K

    2003-01-01

    To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders. PMID:12723988

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

    DOE PAGES

    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 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

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

    PubMed

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

    2016-02-15

    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

  2. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy.

    PubMed

    Maycotte, Paola; Aryal, Suraj; Cummings, Christopher T; Thorburn, Jacqueline; Morgan, Michael J; Thorburn, Andrew

    2012-02-01

    Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research. PMID:22252008

  3. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

    PubMed Central

    Maycotte, Paola; Aryal, Suraj; Cummings, Christopher T; Thorburn, Jacqueline; Morgan, Michael J

    2012-01-01

    Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research. PMID:22252008

  4. 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.

  5. 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.

  6. 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.

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

    PubMed Central

    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. PMID:27375569

  8. 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. PMID:27375569

  9. Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

    PubMed

    Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

    2008-12-01

    Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

  10. 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. PMID:26976217

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

    PubMed

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

    2016-09-01

    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.

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

    PubMed

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

    2016-09-01

    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

  13. IL12-mediated sensitizing of T-cell receptor-dependent and -independent tumor cell killing.

    PubMed

    Braun, Matthias; Ress, Marie L; Yoo, Young-Eun; Scholz, Claus J; Eyrich, Matthias; Schlegel, Paul G; Wölfl, Matthias

    2016-07-01

    Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1. PMID:27622043

  14. 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

  15. 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

  16. 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

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

    PubMed

    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

    2015-01-09

    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.

  18. 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

  19. The TACI receptor regulates T-cell-independent marginal zone B cell responses through innate activation-induced cell death.

    PubMed

    Figgett, William A; Fairfax, Kirsten; Vincent, Fabien B; Le Page, Mélanie A; Katik, Indzi; Deliyanti, Devy; Quah, Pin Shie; Verma, Pali; Grumont, Raelene; Gerondakis, Steve; Hertzog, Paul; O'Reilly, Lorraine A; Strasser, Andreas; Mackay, Fabienne

    2013-09-19

    Activation-induced cell death (AICD) plays a critical role in immune homeostasis and tolerance. In T-cell-dependent humoral responses, AICD of B cells is initiated by Fas ligand (FasL) on T cells, stimulating the Fas receptor on B cells. In contrast, T-cell-independent B cell responses involve innate-type B lymphocytes, such as marginal zone (MZ) B cells, and little is known about the mechanisms that control AICD during innate B cell responses to Toll-like receptor (TLR) activation. Here, we show that MZ B cells undergo AICD in response to TLR4 activation in vivo. The transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) receptor and TLR4 cooperate to upregulate expression of both FasL and Fas on MZ B cells and also to repress inhibitors of Fas-induced apoptosis signaling. These findings demonstrate an unappreciated role for TACI and its ligands in the regulation of AICD during T-cell-independentcell responses.

  20. Proteasome inhibitors induce p53-independent apoptosis in human cancer cells.

    PubMed

    Pandit, Bulbul; Gartel, Andrei L

    2011-01-01

    Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.

  1. Independent Tumor Origin in Two Cases of Synchronous Bilateral Clear Cell Renal Cell Carcinoma

    PubMed Central

    Ji, Zhengguo; Zhao, Jialu; Zhao, Tian; Han, Yuying; Zhang, Yujun; Ye, Haihong

    2016-01-01

    Bilateral renal cell carcinomas (RCCs) pose a challenge for clinical treatment and management. Most bilateral RCCs are sporadic, and do not show a hereditary pattern indicative of VHL syndrome or other inherited cancers. The origin and evolution of these sporadic bilateral RCCs remains elusive. We obtained normal and tumor samples from two male patients suffering from early stage synchronous bilateral clear cell RCC (ccRCC), and analyzed genomic DNA using whole exome sequencing and bisulfite pyrosequencing. We detected distinct 3p loss of heterozygosity (LOH) in both tumors in each patient. Two tumors within the same patient harbored distinct driver mutations and different CpG hypermethylation sites in the VHL promoter. Moreover, tumors exhibit independent evolutionary trajectories. Therefore, distinct 3p LOH, combined with contingent driver gene mutations and independent VHL hypermethylation, led to independent tumor origin and parallel evolution of bilateral ccRCC in these two patients. Our results indicate that tumors in these two cases were not due to common germline oncogenic mutations. They were results of multiple de novo mutations in each kidney, rather than primary ccRCC with contralateral renal metastasis. Therefore, histopathologic and genetic profiling from single tumor specimen may underestimate the mutational burden and somatic heterogeneity of bilateral ccRCCs. PMID:27383411

  2. Independent Tumor Origin in Two Cases of Synchronous Bilateral Clear Cell Renal Cell Carcinoma.

    PubMed

    Ji, Zhengguo; Zhao, Jialu; Zhao, Tian; Han, Yuying; Zhang, Yujun; Ye, Haihong

    2016-01-01

    Bilateral renal cell carcinomas (RCCs) pose a challenge for clinical treatment and management. Most bilateral RCCs are sporadic, and do not show a hereditary pattern indicative of VHL syndrome or other inherited cancers. The origin and evolution of these sporadic bilateral RCCs remains elusive. We obtained normal and tumor samples from two male patients suffering from early stage synchronous bilateral clear cell RCC (ccRCC), and analyzed genomic DNA using whole exome sequencing and bisulfite pyrosequencing. We detected distinct 3p loss of heterozygosity (LOH) in both tumors in each patient. Two tumors within the same patient harbored distinct driver mutations and different CpG hypermethylation sites in the VHL promoter. Moreover, tumors exhibit independent evolutionary trajectories. Therefore, distinct 3p LOH, combined with contingent driver gene mutations and independent VHL hypermethylation, led to independent tumor origin and parallel evolution of bilateral ccRCC in these two patients. Our results indicate that tumors in these two cases were not due to common germline oncogenic mutations. They were results of multiple de novo mutations in each kidney, rather than primary ccRCC with contralateral renal metastasis. Therefore, histopathologic and genetic profiling from single tumor specimen may underestimate the mutational burden and somatic heterogeneity of bilateral ccRCCs. PMID:27383411

  3. 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. PMID:21333555

  4. 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. PMID:26950420

  5. 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

  6. Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes.

    PubMed

    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

    2016-02-01

    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

  7. Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes.

    PubMed

    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

    2016-02-01

    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.

  8. 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.

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

    PubMed

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

    2010-05-01

    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.

  10. Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death.

    PubMed

    Liu, Yong-Hua; Offler, Christina E; Ruan, Yong-Ling

    2016-09-01

    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

  11. 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.

  12. ANTXR-1 and -2 independent modulation of a cytotoxicity mediated by anthrax toxin in human cells.

    PubMed

    Fujikura, Daisuke; Toyomane, Kochi; Kamiya, Kozue; Mutoh, Memi; Mifune, Etsuko; Ohnuma, Miyuki; Higashi, Hideaki

    2016-09-01

    Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells. PMID:27170489

  13. Acquisition of Estrogen Independence Induces TOB1-Related Mechanisms Supporting Breast Cancer Cell Proliferation

    PubMed Central

    Zhang, Yong-Wei; Nasto, Rochelle E.; Varghese, Rency; Jablonski, Sandra A.; Serebriiskii, Ilya G; Surana, Rishi; Calvert, Valerie S.; Bebu, Ionut; Murray, Joseph; Jin, Lu; Johnson, Michael; Riggins, Rebecca; Ressom, Habtom; Petricoin, Emmanuel; Clarke, Robert; Golemis, Erica A.; Weiner, Louis M.

    2015-01-01

    Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen-receptor positive breast cancer. To address this challenge, a systems biology approach was used. A library of siRNAs targeting an estrogen receptor- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells, and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent estrogen receptor-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mTOR inhibitors in estrogen-independent cells but not estrogen-dependent cells. Phosphoproteomic profiles from reverse phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with estrogen receptor-positive breast cancers. PMID:26165839

  14. Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

    PubMed Central

    Wang, Hongkai; Wu, Junlong; Gu, Weijie; Wang, Beihe; Wan, Fangning; Dai, Bo; Zhang, Hailiang; Shi, Guohai; Shen, Yijun; Zhu, Yiping; Zhu, Yao; Ye, Dingwei

    2016-01-01

    Objectives: To examine whether serum adiponectin or leptin level has the ability to differentiate clear cell renal cell carcinoma (ccRCC) from other subtypes of renal cell carcinoma (RCC) in a Chinese population. Patients and methods: We recruited 198 consecutive patients who were treated with radical or partial nephrectomy in our department from September 2011 to June 2013. Their histological types were all malignant, including clear cell, papillary, chromophobe and unclassified RCC. We also enrolled 86 people with no cancer or cancer-related diseases as normal controls. We measured patients' preoperative blood samples for plasma adiponectin and leptin concentrations using an enzyme-linked immunosorbent assay method. Statistical methods were used to analyze ccRCC and other subtypes as they relate to serum adiponectin/leptin level and other factors such as body mass index or visceral fat area. Results: In our database, normal controls had significantly higher circulating adiponectin (p < 0.001) and leptin levels (p < 0.001) than patients with RCC. Among the 198 RCC patients, 156 patients had ccRCC while 42 patients had other histological types. Serum adiponectin levels were lower in ccRCC patients than in non-clear-cell RCC patients (p = 0.004). However, the plasma leptin level was not differently distributed between ccRCC and non-ccRCC patients (p = 0.940). In multivariate analysis, we found that serum adiponectin level may be an independent predictor for discriminating ccRCC patients from others (p = 0.004). Furthermore, in the ccRCC subgroup, we observed that men with ccRCC had lower leptin (p < 0.001) and adiponectin (p = 0.002) levels, and diabetic patients had lower plasma adiponectin levels (p = 0.001). Conclusions: Lower plasma adiponectin concentration was related to an increased incidence of ccRCC and may act as an independent predictor for ccRCC. Our study may help define the process from obesity to adipose tissue, to cytokines and finally to ccRCC. PMID

  15. DIETARY PHYTOCHEMICALS INDUCE p53- AND CASPASE-INDEPENDENT CELL DEATH IN HUMAN NEUROBLASTOMA CELLS

    PubMed Central

    Sukumari-Ramesh, Sangeetha; Bentley, J. Nicole; Laird, Melissa D.; Singh, Nagendra; Vender, John R.; Dhandapani, Krishnan M

    2013-01-01

    Neuroblastoma (NB) is the most prevalent pediatric solid tumor and a leading cause of cancer-related death in children. In the present study, a novel cytotoxic role for the dietary compounds, curcumin, andrographolide, wedelolactone, dibenzoylmethane, and tanshinone IIA was identified in human S-type NB cells, SK-N-AS and SK-N-BE(2). Mechanistically, cell death appeared apoptotic by flow cytometry; however, these effects proceeded independently from both caspase-3 and p53 activation, as assessed by both genetic (shRNA) and pharmacological approaches. Notably, cell death induced by both curcumin and andrographolide was associated with decreased NFκB activity and a reduction in Bcl-2 and Bcl-xL expression. Finally, curcumin and andrographolide increased cytotoxicity following co-treatment with either cisplatin or doxorubicin, two chemotherapeutic agents widely used in the clinical management of NB. Coupled with the documented safety in humans, dietary compounds may represent a potential adjunct therapy for NB. PMID:21704149

  16. 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

  17. Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.

    PubMed

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

    2007-08-01

    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 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, 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 strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

  18. Through-the-electrode model of a proton exchange membrane fuel cell with independently measured parameters

    SciTech Connect

    Weisbrod, K.R.; Grot, S.A.; Vanderborgh, N.E.

    1995-05-01

    A one dimensional model for a proton exchange membrane fuel cell was developed which makes use of independently measured parameters for predicting single cell performance. Optimization of catalyst layer formulation and properties are explored. Impact of temperature and cathode pressure upon system performance was investigated.

  19. Through-the-electrode model of a proton exchange membrane fuel cell with independently measured parameters

    SciTech Connect

    Weisbrod, K.R.; Grot, S.A.; Vandergborgh, N.E.

    1995-09-01

    A one dimensional model for a proton exchange membrane fuel cell was developed which makes use of independently measured parameters for predicting single cell performance. Optimization of catalyst layer formulation and properties are explored. Impact of temperature and cathode pressure upon system performance is investigated.

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

    PubMed Central

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

    2016-01-01

    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”. PMID:27177226

  1. Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

    PubMed Central

    1991-01-01

    Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'- bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D- glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)- independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits

  2. LSD1 controls metastasis of androgen-independent prostate cancer cells through PXN and LPAR6

    PubMed Central

    Ketscher, A; Jilg, C A; Willmann, D; Hummel, B; Imhof, A; Rüsseler, V; Hölz, S; Metzger, E; Müller, J M; Schüle, R

    2014-01-01

    Lysine-specific demethylase 1 (LSD1) was shown to control gene expression and cell proliferation of androgen-dependent prostate cancer (PCa) cells, whereas the role of LSD1 in androgen-independent metastatic prostate cancer remains elusive. Here, we show that depletion of LSD1 leads to increased migration and invasion of androgen-independent PCa cells. Transcriptome and cistrome analyses reveal that LSD1 regulates expression of lysophosphatidic acid receptor 6 (LPAR6) and cytoskeletal genes including the focal adhesion adaptor protein paxillin (PXN). Enhanced LPAR6 signalling upon LSD1 depletion promotes migration with concomitant phosphorylation of PXN. In mice LPAR6 overexpression enhances, whereas knockdown of LPAR6 abolishes metastasis of androgen-independent PCa cells. Taken together, we uncover a novel mechanism of how LSD1 controls metastasis and identify LPAR6 as a promising therapeutic target to treat metastatic prostate cancer. PMID:25285406

  3. 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.

  4. Chromatin-independent binding of serum amyloid P component to apoptotic cells.

    PubMed

    Familian, A; Zwart, B; Huisman, H G; Rensink, I; Roem, D; Hordijk, P L; Aarden, L A; Hack, C E

    2001-07-15

    Human serum amyloid P component (SAP) is a glycoprotein structurally belonging to the pentraxin family of proteins, which has a characteristic pentameric organization. Mice with a targeted deletion of the SAP gene develop antinuclear Abs, which was interpreted as evidence for a role of SAP in controlling the degradation of chromatin. However, in vitro SAP also can bind to phosphatidylethanolamine, a phospholipid which in normal cells is located mainly in the inner leaflet of the cell membrane, to be translocated to the outer leaflet of the cell membrane during a membrane flip-flop. We hypothesized that SAP, because of its specificity for phosphatidylethanolamine, may bind to apoptotic cells independent of its nuclear binding. Calcium-dependent binding of SAP to early, nonpermeable apoptotic Jurkat, SKW, and Raji cells was indeed observed. Experiments with flip-flopped erythrocytes confirmed that SAP bound to early apoptotic cells via exposed phosphatidylethanolamine. Binding of SAP was stronger to late, permeable apoptotic cells. Experiments with enucleated neutrophils, with DNase/RNase treatment of late apoptotic Jurkat cells, and competition experiments with histones suggested that binding of SAP to late apoptotic cells was largely independent of chromatin. Confocal laser microscopic studies indeed suggested that SAP bound to these apoptotic cells mainly via the blebs. Thus, this study shows that SAP binds to apoptotic cells already at an early stage, which raises the possibility that SAP is involved in dealing with apoptotic cells in vivo.

  5. 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.

  6. Global Ca2+ Signaling Drives Ribbon-Independent Synaptic Transmission at Rod Bipolar Cell Synapses

    PubMed Central

    Mehta, Bhupesh; Ke, Jiang-Bin; Zhang, Lei; Baden, Alexander D.; Markowitz, Alexander L.; Nayak, Subhashree; Briggman, Kevin L.

    2014-01-01

    Ribbon-type presynaptic active zones are a hallmark of excitatory retinal synapses, and the ribbon organelle is thought to serve as the organizing point of the presynaptic active zone. Imaging of exocytosis from isolated retinal neurons, however, has revealed ectopic release (i.e., release away from ribbons) in significant quantities. Here, we demonstrate in an in vitro mouse retinal slice preparation that ribbon-independent release from rod bipolar cells activates postsynaptic AMPARs on AII amacrine cells. This form of release appears to draw on a unique, ribbon-independent, vesicle pool. Experimental, anatomical, and computational analyses indicate that it is elicited by a significant, global elevation of intraterminal [Ca2+] arising following local buffer saturation. Our observations support the conclusion that ribbon-independent release provides a read-out of the average behavior of all of the active zones in a rod bipolar cell's terminal. PMID:24790194

  7. 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.

  8. Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage.

    PubMed

    Lin, H M; Lee, Y J; Li, G; Pestell, R G; Kim, H R

    2001-01-01

    Cyclin D1 expression is co-regulated by growth factor and cell adhesion signaling. Cell adhesion to the extracellular matrix activates focal adhesion kinase (FAK), which is essential for cyclin D1 expression. Upon the loss of cell adhesion, cyclin D1 expression is downregulated, followed by apoptosis in normal epithelial cells. Since bcl-2 prevents apoptosis induced by the loss of cell adhesion, we hypothesized that bcl-2 induces survival signaling complementary to cell adhesion-mediated gene regulation. In the present study, we investigated the role of bcl-2 on FAK activity and cyclin D1 expression. We found that bcl-2 overexpression induces cyclin D1 expression in human breast epithelial cell line MCF10A independent of cell anchorage. Increased cyclin D1 expression in stable bcl-2 transfectants is not related to bcl-2-increased G1 duration, but results from cyclin D1 promoter activation. Transient transfection studies confirmed anchorage-independent bcl-2 induction of cyclin D1 promoter activity in human breast epithelial cell lines (MCF10A, BT549, and MCF-7). We provide evidence that bcl-2 induction of cyclin D1 expression involves constitutive activation of focal adhesion kinase, regardless of cell adhesion. The present study suggests a potential oncogenic activity for bcl-2 through cyclin D1 induction, and provides an insight into the distinct proliferation-independent pathway leading to increased cyclin D1 expression in breast cancer.

  9. Alpha-particle emitting 213Bi-anti-EGFR immunoconjugates eradicate tumor cells independent of oxygenation.

    PubMed

    Wulbrand, Christian; Seidl, Christof; 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 (213)Bi immunoconjugates kill hypoxic and normoxic CAL33 tumor cells with identical efficiency. For that purpose CAL33 cells were incubated with (213)Bi-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 (213)Bi-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 (213)Bi-anti-EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate that α-particle emitting (213)Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, (213)Bi-radioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors.

  10. 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

  11. 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.

  12. 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

  13. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

    PubMed

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang; Wang, Jinfu

    2013-04-01

    Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the

  14. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

    PubMed

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang; Wang, Jinfu

    2013-04-01

    Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the

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

    PubMed

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

    2013-05-10

    The mitochondrial calcium uniporter (MCU) transports free ionic Ca(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(2+) levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca(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.

  16. Attenuation of natural killer cell functions by capsaicin through a direct and TRPV1-independent mechanism.

    PubMed

    Kim, Hun Sik; Kwon, Hyung-Joon; Kim, Gye Eun; Cho, Mi-Hyang; Yoon, Seung-Yong; Davies, Alexander J; Oh, Seog Bae; Lee, Heuiran; Cho, Young Keol; Joo, Chul Hyun; Kwon, Seog Woon; Kim, Sun Chang; Kim, Yoo Kyum

    2014-07-01

    The assessment of the biological activity of capsaicin, the compound responsible for the spicy flavor of chili pepper, produced controversial results, showing either carcinogenicity or cancer prevention. The innate immune system plays a pivotal role in cancer pathology and prevention; yet, the effect of capsaicin on natural killer (NK) cells, which function in cancer surveillance, is unclear. This study found that capsaicin inhibited NK cell-mediated cytotoxicity and cytokine production (interferon-γ and tumor necrosis factor-α). Capsaicin impaired the cytotoxicity of NK cells, thereby inhibiting lysis of standard target cells and gastric cancer cells by modulating calcium mobilization in NK cells. Capsaicin also induced apoptosis in gastric cancer cells, but that effect required higher concentrations and longer exposure times than those required to trigger NK cell dysfunction. Furthermore, capsaicin inhibited the cytotoxicity of isolated NK cells and of an NK cell line, suggesting a direct effect on NK cells. Antagonists of transient receptor potential vanilloid subfamily member 1 (TRPV1), a cognate capsaicin receptor, or deficiency in TRPV1 expression failed to prevent the defects induced by capsaicin in NK cells expressing functional TRPV1. Thus, the mechanism of action of capsaicin on NK cells is largely independent of TRPV1. Taken together, capsaicin may have chemotherapeutic potential but may impair NK cell function, which plays a central role in tumor surveillance. PMID:24743513

  17. Constitutive endocytosis in characean internodal cells is independent of an intact actin cytoskeleton.

    PubMed

    Foissner, Ilse; Klima, Andreas

    2008-05-01

    We have investigated constitutive endocytosis in internodal cells of the characean green algae. The endocytic tracer FM1-43 accumulated in distinct plasma membrane domains that are probably enriched in sterol-like substances. Internalization of the dye was active but independent of an intact actin or microtubule cytoskeleton.

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

    PubMed

    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

    2014-12-30

    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. 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

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

    PubMed

    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

    2014-12-30

    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. 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.

  20. 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

  1. 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

  2. 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.

  3. The copper chelator ATN-224 induces caspase-independent cell death in diffuse large B cell lymphoma

    PubMed Central

    LEE, KRISTY; HART, MATTHEW R.; BRIEHL, MARGARET M.; MAZAR, ANDREW P.; TOME, MARGARET E.

    2014-01-01

    Bcl-2 and other anti-apoptotic proteins are associated with defective caspase-dependent apoptotic pathways, resulting in chemoresistance. We have previously shown that ATN-224, a copper chelator drug, induces cell death in murine thymic lymphoma cells transfected with Bcl-2. In the current study, we tested whether ATN-224 was effective in diffuse large B cell lymphoma (DLBCL) cells, which have increased anti-apoptotic proteins through translocation or amplification. We found that nanomolar concentrations of ATN-224 induced cell death in DLBCL cells independent of Bcl-2, Bcl-xL or Mcl-1 status. ATN-224 treatment resulted in mitochondrial dysfunction, release of apoptosis-inducing factor (AIF) and induction of caspase-independent cell death. In addition, ATN-224 degraded Mcl-1 and enhanced the effect of the BH3 mimetic ABT-263. These findings indicate that ATN-224 has potential as a therapeutic for the treatment of DLBCL. Induction of caspase-independent cell death in apoptosis-resistant DLBCL would provide a therapeutic alternative for the treatment of refractory disease. PMID:24788952

  4. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    PubMed

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

    2014-10-01

    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.

  5. 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

  6. AICAR induces AMPK-independent programmed necrosis in prostate cancer cells.

    PubMed

    Guo, Feng; Liu, Shuang-Qing; Gao, Xing-Hua; Zhang, Long-Yang

    2016-05-27

    AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) is an AMP-activated protein kinase (AMPK) agonist, which induces cytotoxic effect to several cancer cells. Its potential activity in prostate cancer cells and the underlying signaling mechanisms have not been extensively studied. Here, we showed that AICAR primarily induced programmed necrosis, but not apoptosis, in prostate cancer cells (LNCaP, PC-3 and PC-82 lines). AICAR's cytotoxicity to prostate cancer cells was largely attenuated by the necrosis inhibitor necrostatin-1. Mitochondrial protein cyclophilin-D (CYPD) is required for AICAR-induced programmed necrosis. CYPD inhibitors (cyclosporin A and sanglifehrin A) as well as CYPD shRNAs dramatically attenuated AICAR-induced prostate cancer cell necrosis and cytotoxicity. Notably, AICAR-induced cell necrosis appeared independent of AMPK, yet requiring reactive oxygen species (ROS) production. ROS scavengers (N-acetylcysteine and MnTBAP), but not AMPKα shRNAs, largely inhibited prostate cancer cell necrosis and cytotoxicity by AICAR. In summary, the results of the present study demonstrate mechanistic evidences that AMPK-independent programmed necrosis contributes to AICAR's cytotoxicity in prostate cancer cells. PMID:27103440

  7. Regulation of the transferrin-independent iron transport system in cultured cells.

    PubMed

    Kaplan, J; Jordan, I; Sturrock, A

    1991-02-15

    Mammalian cells accumulate iron via the binding of transferrin to high affinity surface receptors, or through a transferrin-independent pathway which involves the uptake of iron-organic anion chelates by a membrane-based transport system. Previously we determined that the transferrin-independent transport system was present on a wide variety of cultured cells (Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). In this communication we demonstrate that the transferrin-independent iron uptake system is regulated differently than the transferrin-mediated pathway. The activity of the transferrin-independent system was unaffected by changes in cellular growth rate, induction of DNA synthesis and cell division, or depletion of cellular iron. Exposure of cells to ferric or ferrous iron, however, resulted in a time-dependent increase in transport activity, due to a change in Vmax with no change in Km. Increased transport activity was seen in a variety of cultured cell types, occurred in the presence of cycloheximide, and persisted for hours after removal of iron. The ability of other transition metals to induce changes in transport, or to compete with iron for accumulation by the transferrin-independent uptake system, was critically dependent on the composition of the media in which the cells were incubated. Metals such as Cu2+ or Zn2+, but not Cd2+ or Mn2+, when dissolved in a balanced salt solution buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, induced changes in the transferrin-independent iron transport system. The same metals which induced changes in transport were ineffective in media containing amino acids, ascorbate, or N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. The Vmax of the transferrin-independent iron transport system was also elevated by increases in intracellular Ca2+. The effect of iron on transport activity, however, did not result from an iron-induced release of

  8. Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

    PubMed Central

    Melo, Sonia A.; Sugimoto, Hikaru; O’Connell, Joyce T.; Kato, Noritoshi; Villanueva, Alberto; Vidal, August; Qiu, Le; Vitkin, Edward; Perelman, Lev T.; Melo, Carlos A.; Lucci, Anthony; Ivan, Cristina; Calin, George A.; Kalluri, Raghu

    2014-01-01

    SUMMARY Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. PMID:25446899

  9. Independent component filters of natural images compared with simple cells in primary visual cortex.

    PubMed Central

    van Hateren, J H; van der Schaaf, A

    1998-01-01

    Properties of the receptive fields of simple cells in macaque cortex were compared with properties of independent component filters generated by independent component analysis (ICA) on a large set of natural images. Histograms of spatial frequency bandwidth, orientation tuning bandwidth, aspect ratio and length of the receptive fields match well. This indicates that simple cells are well tuned to the expected statistics of natural stimuli. There is no match, however, in calculated and measured distributions for the peak of the spatial frequency response: the filters produced by ICA do not vary their spatial scale as much as simple cells do, but are fixed to scales close to the finest ones allowed by the sampling lattice. Possible ways to resolve this discrepancy are discussed. PMID:9523437

  10. Anergy and exhaustion are independent mechanisms of peripheral T cell tolerance

    PubMed Central

    1995-01-01

    We studied the interactions of male-specific T cell receptor (TCR)- alpha/beta-transgenic (TG) cells with different concentrations of male antigen in vivo. We constructed mouse chimeras expressing different amounts of male antigen by injecting thymectomized, lethally irradiated mice with various ratios of male (immunoglobulin [Ig] Ha) and female (IgHb) bone marrow. These chimeras were injected with male-specific TCR- alpha/beta-trangenic cells. These experiments allowed us to monitor antigen persistence and characterize antigen-specific T cells in terms of their frequency, reactivity, and effector functions (as tested by elimination of male B cells in vivo). In the absence of antigen, virgin TG cells persisted but did not expand. Transient exposure to antigen resulted in cell expansion, followed by the persistence of increased numbers of antigen-reactive T cells. In contrast, antigen persistence was followed by two independent mechanisms of tolerance induction: anergy (at high antigen concentrations), where T cells did not differentiate into effector functions but persisted in vivo as unresponsive T cells, and exhaustion (at lower antigen concentrations), where differentiation into effector functions (B cell elimination) occurred but was followed by the disappearance of antigen-specific T cells. PMID:7869056

  11. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    PubMed Central

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J.; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F.; Psathaki, Olympia E.; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R.; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34+ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34+ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34+ cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. PMID:25326431

  12. In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis.

    PubMed

    Vanhee, Stijn; De Mulder, Katrien; Van Caeneghem, Yasmine; Verstichel, Greet; Van Roy, Nadine; Menten, Björn; Velghe, Imke; Philippé, Jan; De Bleser, Dominique; Lambrecht, Bart N; Taghon, Tom; Leclercq, Georges; Kerre, Tessa; Vandekerckhove, Bart

    2015-02-01

    Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.

  13. Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity.

    PubMed

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J N

    2014-05-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through coculture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed 1 ligand that could activate CAR T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated that CARL aAPC propagate CAR T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Using CARL enables 1 aAPC to numerically expand all CAR T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR T cells of any specificity. PMID:24714354

  14. Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

    PubMed Central

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

    2014-01-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

  15. 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.

  16. 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

  17. Camptothecin sensitizes androgen-independent prostate cancer cells to anti-Fas-induced apoptosis

    PubMed Central

    Costa-Pereira, A P; Cotter, T G

    1999-01-01

    Despite expressing both Fas and Fas ligand, DU145 and LNCaP prostate cancer cells were resistant to anti-Fas-induced cell death. Resistance to Fas-mediated cytotoxicity could be overcome in DU145, but not in LNCaP, cells by pretreating cells with sublethal doses of cytotoxic drugs, such as camptothecin. Activated caspases were shown to be required for this cytotoxicity. Indeed, poly(ADP-Ribose) polymerase was shown to be proteolytically cleaved in cells treated with camptothecin plus anti-Fas, but not in cells treated with anti-Fas only. Moreover, pretreatment of cells with ZVAD completely blocked camptothecin-mediated Fas-induced apoptosis. Sensitization of cells to Fas-induced cell death did not involve up-regulation of Fas or FasL, and it was independent of alterations in the cell cycle. Reactive oxygen intermediates (ROI) have been shown to be important mediators of drug-induced apoptosis. Here, we demonstrate that treatment of DU145 cells with camptothecin, anti-Fas, or both, did not alter the intracellular levels of peroxide or superoxide anion. © 1999 Cancer Research Campaign PMID:10408840

  18. Bistratified ganglion cells of rabbit retina: neural architecture for contrast-independent visual responses.

    PubMed

    Famiglietti, Edward V

    2009-01-01

    Bistratified (BS) ganglion cells have long been recognized in vertebrate retina. Thirty years ago, it became clear that bistratification allows the integration of ON and OFF retinal pathways to produce contrast-independent responses in ganglion cells. Best studied is the type 1 bistratified (BS1) ganglion cell of rabbit retina, the physiologically well-characterized ON-OFF directionally selective (DS) ganglion cell, which is co-stratified with the two types of starburst amacrine (SA) cells in sublaminae a and b of the inner plexiform layer (IPL). DS responses have recently been documented in the latter. In this report, BS1 cells are further studied and are used as "fiducials" to characterize a second type of BS ganglion cell. An example of a possible third type is shown to be distinct from examples of BS1 and BS2 cells. All three have two distinct, narrowly stratified arborizations, one in sublamina a and one in sublamina b. All have similar dimensions, except for their dendritic trees, differing also in branching pattern. BS1 cells have compact, regular, highly branched trees; BS2 cells have significantly larger, more sparsely branched, irregular, radiate trees; the proposed BS3 type is intermediate in field size, and its branching pattern is different from the first two. BS2 and BS3 cells are co-stratified, branching nearer to the margins of the IPL, out of range of SA cells. In a previous report by others, illustrating the morphology of intracellularly stained ganglion cells, one example each of both "orientation-selective" ganglion cells and "uniformity detectors" resembles the BS2 cell. A rationale is presented for correlating BS2 cells with uniformity detectors.

  19. Studies on Culture and Osteogenic Induction of Human Mesenchymal Stem Cells under CO2-Independent Conditions

    PubMed Central

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang

    2013-01-01

    Abstract Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce

  20. 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

  1. Stem cell senescence. Effects of REAC technology on telomerase-independent and telomerase-dependent pathways

    PubMed Central

    Rinaldi, S.; Maioli, M.; Pigliaru, G.; Castagna, A.; Santaniello, S.; Basoli, V.; Fontani, V.; Ventura, C.

    2014-01-01

    Decline in the gene expression of senescence repressor Bmi1, and telomerase, together with telomere shortening, underlay senescence of stem cells cultured for multiple passages. Here, we investigated whether the impairment of senescence preventing mechanisms can be efficiently counteracted by exposure of human adipose-derived stem cells to radio electric asymmetrically conveyed fields by an innovative technology, named Radio Electric Asymmetric Conveyer (REAC). Due to REAC exposure, the number of stem cells positively stained for senescence associated β-galactosidase was significantly reduced along multiple culturing passages. After a 90-day culture, REAC-treated cells exhibited significantly higher transcription of Bmi1 and enhanced expression of other stem cell pluripotency genes and related proteins, compared to unexposed cells. Transcription of the catalytic telomerase subunit (TERT) was also increased in REAC-treated cells at all passages. Moreover, while telomere shortening occurred at early passages in both REAC-treated and untreated cells, a significant rescue of telomere length could be observed at late passages only in REAC-exposed cells. Thus, REAC-asymmetrically conveyed radio electric fields acted on a gene and protein expression program of both telomerase-independent and telomerase-dependent patterning to optimize stem cell ability to cope with senescence progression. PMID:25224681

  2. Monocarboxylate transporter 1 contributes to growth factor-induced tumor cell migration independent of transporter activity

    PubMed Central

    Gray, Alana L.; Coleman, David T.; Shi, Runhua; Cardelli, James A.

    2016-01-01

    Tumor progression to metastatic disease contributes to the vast majority of incurable cancer. Understanding the processes leading to advanced stage cancer is important for the development of future therapeutic strategies. Here, we establish a connection between tumor cell migration, a prerequisite to metastasis, and monocarboxylate transporter 1 (MCT1). MCT1 transporter activity is known to regulate aspects of tumor progression and, as such, is a clinically relevant target for treating cancer. Knockdown of MCT1 expression caused decreased hepatocyte growth factor (HGF)-induced as well as epidermal growth factor (EGF)-induced tumor cell scattering and wound healing. Western blot analysis suggested that MCT1 knockdown (KD) hinders signaling through the HGF receptor (c-Met) but not the EGF receptor. Exogenous, membrane-permeable MCT1 substrates were not able to rescue motility in MCT1 KD cells, nor was pharmacologic inhibition of MCT1 able to recapitulate decreased cell motility as seen with MCT1 KD cells, indicating transporter activity of MCT1 was dispensable for EGF- and HGF-induced motility. These results indicate MCT1 expression, independent of transporter activity, is required for growth factor-induced tumor cell motility. The findings presented herein suggest a novel function for MCT1 in tumor progression independent of its role as a monocarboxylate transporter. PMID:27127175

  3. 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

  4. Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells.

    PubMed

    Moutinho-Pereira, Sara; Stuurman, Nico; Afonso, Olga; Hornsveld, Marten; Aguiar, Paulo; Goshima, Gohta; Vale, Ronald D; Maiato, Helder

    2013-12-01

    Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.

  5. 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.

  6. Erythropoietin-independent regeneration of erythroid progenitor cells following multiple injections of hydroxyurea.

    PubMed

    Wagemaker, G; Visser, T P

    1980-09-01

    It wa shown previously that colony formation in vitro by early erythroid progenitor cells (BFUe) requires sequential stimulation with a specific glycoprotein termed BFA and erythropoietin (EP). The action exerted by BFA was characterized as induction of proliferation in BFUe resulting after several cell divisions in EP-responsive progeny. The present study is directed at detection of EP-independent regulation of erythroid progenitor cells in vivo. Haemopoietic regeneration was induced by multiple administrations of hydroxyurea (HU). The femoral regeneration patterns of haemopoietic stem cells (CFUs), granulocyte/macrophage progenitor cells (CFUgm) and erythroid progenitor cells (BFUe, day 3 BFUe and CFUe) were studied in hypertransfused mice in comparison to nontransfused controls. The results show that (1) the phase of exponential regeneration of none of the cell populations studied is affected by hypertransfusion; (2) each of these cell populations exhibit a distinct regeneration pattern, indicating that they behave as separate functional entities; and (3) the three erythroid cell populations are suppressed by hypertransfusion in the post-exponential phase of regeneration in contrast to CFUs and CFUgm. The results support a two-regulator model of erythropoiesis. PMID:7459981

  7. 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.

  8. Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila

    PubMed Central

    Qi, Suozhi; Calvi, Brian R.

    2016-01-01

    Apoptotic cell death is important for the normal development of a variety of organisms. Apoptosis is also a response to DNA damage and an important barrier to oncogenesis. The apoptotic response to DNA damage is dampened in specific cell types during development. Developmental signaling pathways can repress apoptosis, and reduced cell proliferation also correlates with a lower apoptotic response. However, because developmental signaling regulates both cell proliferation and apoptosis, the relative contribution of cell division to the apoptotic response has been hard to discern in vivo. Here we use Drosophila oogenesis as an in vivo model system to determine the extent to which cell proliferation influences the apoptotic response to DNA damage. We find that different types of cell cycle modifications are sufficient to repress the apoptotic response to ionizing radiation independent of developmental signaling. The step(s) at which the apoptosis pathway was repressed depended on the type of cell cycle modification—either upstream or downstream of expression of the p53-regulated proapoptotic genes. Our findings have important implications for understanding the coordination of cell proliferation with the apoptotic response in development and disease, including cancer and the tissue-specific responses to radiation therapy. PMID:27075174

  9. Derivation of LIF-independent mouse iPS cells with modified Oct4

    PubMed Central

    Hirai, Hiroyuki; Firpo, Meri; Kikyo, Nobuaki

    2015-01-01

    It has been very difficult, if not impossible, to establish mouse induced pluripotent stem cells (iPSCs) from differentiated cells, such as fibroblasts, without leukemia inhibitory factor (LIF). We have established and maintained LIF-independent iPSCs for longer than 120 days with modified Oct4 along with Sox2, Klf4, and c-Myc. The iPSCs will provide a novel tool to investigate the roles of the LIF-Stat3 signaling pathway in mouse pluripotent stem cells. Resource TableBiological reagent: induced pluripotent stem (iPS) cellName of Stem Cell constructM3O-lenti-iPSC-LIF(−)InstitutionUniversity of MinnesotaPerson who created resourceHiroyuki HiraiContact person and emailNobuaki Kikyo, kikyo001@umn.eduDate archived/stock date2014–2015OriginMouse embryonic fibroblastsType of resourceBiological reagent: induced pluripotent stem (iPS) cellSub-typeCell lineKey transcription factorsM3O, Sox2, Klf4, c-MycAuthenticationIdentity and purity of cell line confirmed (Figure 1)Link to related literature (direct URL links and full references)Hirai H., et al. In press, Stem Cell ResearchInformation in public databases PMID:26318720

  10. 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. PMID:24957291

  11. 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

  12. PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion

    PubMed Central

    Dart, Anna E.; Box, Gary M.; Court, William; Gale, Madeline E.; Brown, John P.; Pinder, Sarah E.; Eccles, Suzanne A.

    2015-01-01

    P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A–Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration. PMID:26598620

  13. Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells.

    PubMed

    Talhouk, Rabih S; Khalil, Antoine A; Bajjani, Rachid; Rahme, Gilbert J; El-Sabban, Marwan E

    2011-10-01

    Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.

  14. 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.

  15. Oral Mucosal Progenitor Cells Are Potently Immunosuppressive in a Dose-Independent Manner

    PubMed Central

    Davies, Lindsay C.; Lönnies, Helena; Locke, Matthew; Sundberg, Berit; Rosendahl, Kerstin; Götherström, Cecilia; Le Blanc, Katarina

    2012-01-01

    Oral mucosal lamina propria progenitor cells (OMLP-PCs) are a novel, clonally derived PC population of neural crest origin with the potential to differentiate down both mesenchymal and neuronal cell lineages. In this study we aimed to determine the immunological properties of OMLP-PCs and to establish whether they would be suitable candidates for allogeneic tissue engineering and in the treatment of immune-related diseases. OMLP-PCs demonstrated no inherent immunogenicity with insignificant expression of costimulatory molecules (CD40, CD80, CD86, CD154, and CD178) or human leukocyte antigen (HLA) class II. OMLP-PCs required 7 days of stimulation with interferon-γ (IFN-γ) to induce cell surface expression of HLA II. Mixed lymphocyte cultures and mitogen stimulation demonstrated the potent immunosuppressive capability of OMLP-PCs in a contact-independent manner. Complete inhibition of lymphocyte proliferation was seen at doses as low as 0.001% OMLP-PCs to responder lymphocytes, while annexin V staining confirmed that this immunosuppressive effect was not due to the induction of lymphocyte apoptosis. These data demonstrate, for the first time, that OMLP-PC immunomodulation, unlike that for mesenchymal stem cells, occurs via a dose- and HLA II–independent mechanism by the release of immunosuppressive soluble factors and suggests these cells may have wide ranging potential in future immune-related therapies. PMID:21988324

  16. TIMP-1 inhibits microvascular endothelial cell migration by MMP-dependent and MMP-independent mechanisms.

    PubMed

    Akahane, Takemi; Akahane, Manabu; Shah, Amy; Connor, Christine M; Thorgeirsson, Unnur P

    2004-12-10

    It was reported over a decade ago that tissue inhibitor of metalloproteinases-1 (TIMP-1) suppresses angiogenesis in experimental models but the mechanism is still incompletely understood. This in vitro study focused on the molecular basis of TIMP-1-mediated inhibition of endothelial cell (EC) migration, a key step in the angiogenic process. Both recombinant human TIMP-1 and the synthetic MMP inhibitors, GM6001 and MMP-2-MMP-9 Inhibitor III, suppressed migration of human dermal microvascular endothelial cells (HDMVEC) in a dose-dependent fashion. The MMP-dependent inhibition of migration was associated with increased expression of the junctional adhesion proteins, VE-cadherin and PECAM-1, and VE-cadherin accumulation at cell-cell junctions. TIMP-1 also caused MMP-independent dephosphorylation of focal adhesion kinase (FAK) (pY397) and paxillin, which was associated with reduced number of F-actin stress fibers and focal adhesions. Moreover, TIMP-1 stimulated expression of PTEN that has been shown to reduce phosphorylation of FAK and inhibit cell migration. Our data suggest that TIMP-1 inhibits HDMVEC migration through MMP-dependent stimulation of VE-cadherin and MMP-independent stimulation of PTEN with subsequent dephosphorylation of FAK and cytoskeletal remodeling. PMID:15530852

  17. 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.

  18. VE-Cadherin-Independent Cancer Cell Incorporation into the Vascular Endothelium Precedes Transmigration

    PubMed Central

    Aranda-Espinoza, Helim

    2014-01-01

    Metastasis is accountable for 90% of cancer deaths. During metastasis, tumor cells break away from the primary tumor, enter the blood and the lymph vessels, and use them as highways to travel to distant sites in the body to form secondary tumors. Cancer cell migration through the endothelium and into the basement membrane represents a critical step in the metastatic cascade, yet it is not well understood. This process is well characterized for immune cells that routinely transmigrate through the endothelium to sites of infection, inflammation, or injury. Previous studies with leukocytes have demonstrated that this step depends heavily on the activation status of the endothelium and subendothelial substrate stiffness. Here, we used a previously established in vitro model of the endothelium and live cell imaging, in order to observe cancer cell transmigration and compare this process to leukocytes. Interestingly, cancer cell transmigration includes an additional step, which we term ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs, leading to the dislocation of EC VE-cadherin away from EC junctions bordering cancer cells, and spread into the monolayer. In some cases, ECs completely detach from the matrix. Furthermore, cancer cell incorporation occurs independently of the activation status and the subendothelial substrate stiffness for breast cancer and melanoma cells, a notable difference from the process by which leukocytes transmigrate. Meanwhile, pancreatic cancer cell incorporation was dependent on the activation status of the endothelium and changed on very stiff subendothelial substrates. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation is one of the earliest steps. PMID:25275457

  19. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

    PubMed

    Cannon, J L; Labno, C M; Bosco, G; Seth, A; McGavin, M H; Siminovitch, K A; Rosen, M K; Burkhardt, J K

    2001-08-01

    Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site. PMID:11520460

  20. Progesterone regulates chicken embryonic germ cell meiotic initiation independent of retinoic acid signaling.

    PubMed

    Mi, Yuling; He, Bin; Li, Jian; Zhang, Caiqiao

    2014-07-15

    The signaling molecule retinoic acid (RA) is known to trigger germ cells to enter meiosis. However, RA may not be the only secreted inducer of meiosis. Our previous data indicate that luteinizing hormone also promotes germ cell meiotic initiation by upregulating 3βHSDII transcription. Here, using chicken embryos, we investigate the role of progesterone (P4) in regulating germ cell meiotic initiation. Progesterone treatment at embryonic Day 9.5 accelerated germ cell meiosis entry in the female chicken embryos. However, P4 treatment in vivo did no influence on testicular germ cells but triggered their meiotic initiation in the cultured testes. As treatment with an RA receptor (RAR) inhibitor did not block the stimulatory effect of P4 on germ cell meiotic initiation, this P4 stimulatory effect seems to be independent of RAR-mediated signaling. The abundance of RA metabolism-related enzymes and RAR (RARβ) mRNAs did not differ significantly between P4-treated and control individuals. The RA concentration in the ovaries remained unchanged by P4 treatment in vivo. Because no inhibition by the P4 receptor (PR) nuclear receptor antagonist mifepristone on P4 effect was observed in either in vitro or in vivo experiments, the effect of P4 on germ cell meiotic initiation is probably mediated by membrane PRs (mPR). The mPRα, mPRβ, and mPRγ mRNAs were all expressed in the embryonic ovaries. The expression of mPRα and mPRβ was higher than that of mPRγ. Immunohistochemical results showed that mPRα-positive cells were mainly scattered in the ovarian cortex area where most germ cells were distributed. The mPRβ-positive cells were widely distributed in the ovaries, and positive cells were clustered with a similar morphology to that of germ cell clusters. In conclusion, P4 may regulate embryonic germ cell meiotic initiation independent of RA signaling through the membrane PRs. This study provides a new insight into the mechanisms of germ cell meiotic initiation in the chicken

  1. TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

    PubMed

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

    2011-01-27

    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 Ca(2+) 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.

  2. Proliferation-independent growth factor modulation of the radiation sensitivity of human prostate cells

    SciTech Connect

    Howard, S.P.; Groch, K.M.; Lindstrom, M.J.

    1995-08-01

    The survival of human prostatic epithelial cells irradiated in different physiological states is reported. Exponentially growing cells and contact-inhibited cells grown and irradiated in the presence of the growth factors epidermal growth factor (EGF) and bovine pituitary extract (bPE) had overlapping radiation dose-cell survival curves. However, when EGF and bPE were removed from exponentially growing cells before irradiation, an increase in radiosensitivity was observed if the cells were replated into medium containing growth factors (EGF and bPE) immediately after irradiation. Treating cells with the nonspecific growth factor receptor antagonist suramin had similar effects as did growth factor deprivation. In contrast, when growth factor-deprived cells were maintained in this same medium for 12 h postirradiation, an increase in radiation survival was observed. This increase in survival is attributed to the repair of potentially lethal damage (PLD). Both the increase in radiosensitivity induced by deprivation of growth factor before irradiation and the repair of PLD caused by deprivation of growth factor after irradiation were independent of changes in cellular proliferation. 22 refs., 1 fig., 2 tab.

  3. 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.

  4. A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants.

    PubMed

    Drake, Andrew W; Klakamp, Scott L

    2007-01-10

    A new 4-parameter nonlinear equation based on the standard multiple independent binding site model (MIBS) is presented for fitting cell-based ligand titration data in order to calculate the ligand/cell receptor equilibrium dissociation constant and the number of receptors/cell. The most commonly used linear (Scatchard Plot) or nonlinear 2-parameter model (a single binding site model found in commercial programs like Prism(R)) used for analysis of ligand/receptor binding data assumes only the K(D) influences the shape of the titration curve. We demonstrate using simulated data sets that, depending upon the cell surface receptor expression level, the number of cells titrated, and the magnitude of the K(D) being measured, this assumption of always being under K(D)-controlled conditions can be erroneous and can lead to unreliable estimates for the binding parameters. We also compare and contrast the fitting of simulated data sets to the commonly used cell-based binding equation versus our more rigorous 4-parameter nonlinear MIBS model. It is shown through these simulations that the new 4-parameter MIBS model, when used for cell-based titrations under optimal conditions, yields highly accurate estimates of all binding parameters and hence should be the preferred model to fit cell-based experimental nonlinear titration data. PMID:17141800

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

    PubMed Central

    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. 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.

  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. 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.

  9. Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    PubMed Central

    Lee, Yoonjin; Dominy, John E.; Choi, Yoon Jong; Jurczak, Michael; Tolliday, Nicola; Camporez, Joao Paulo; Chim, Helen; Lim, Ji-Hong; Ruan, Hai-Bin; Yang, Xiaoyong; Vazquez, Francisca; Sicinski, Piotr; Shulman, Gerald I.; Puigserver, Pere

    2014-01-01

    Insulin constitutes a major evolutionarily conserved hormonal axis for maintaining glucose homeostasis1-3; dysregulation of this axis causes diabetes2,4. PGC-1α links insulin signaling to the expression of glucose and lipid metabolic genes5-7. GCN5 acetylates PGC-1α and suppresses its transcriptional activity, whereas SIRT1 deacetylates and activates PGC-1α8,9. Although insulin is a mitogenic signal in proliferative cells10,11, whether components of the cell cycle machinery contribute to insulin’s metabolic action is poorly understood. Herein, we report that insulin activates cyclin D1-CDK4, which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high throughput chemical screen, we identified a CDK4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK3β signaling induces cyclin D1 protein stability via sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 mRNA transcripts. Activated cyclin D1-CDK4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycemia. In diabetic models, cyclin D1-CDK4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division. PMID:24870244

  10. In vivo CD40 ligation can induce T-cell-independent antitumor effects that involve macrophages.

    PubMed

    Lum, Hillary D; Buhtoiarov, Ilia N; Schmidt, Brian E; Berke, Gideon; Paulnock, Donna M; Sondel, Paul M; Rakhmilevich, Alexander L

    2006-06-01

    We have previously demonstrated T cell-independent antitumor and antimetastatic effects of CD40 ligation that involved natural killer (NK) cells. As CD40 molecules are expressed on the surface of macrophages (Mphi), we hypothesized that Mphi may also serve as antitumor effector cells when activated by CD40 ligation. Progression of subcutaneous NXS2 murine neuroblastomas was delayed significantly by agonistic CD40 monoclonal antibody (anti-CD40 mAb) therapy in immunocompetent A/J mice, as well as in T and B cell-deficient severe combined immunodeficiency (SCID) mice. Although NK cells can be activated by anti-CD40 mAb, anti-CD40 mAb treatment also induced a significant antitumor effect in SCID/beige mice in the absence of T and NK effector cells, even when noncytolytic NK cells and polymorphonuclear cells (PMN) were depleted. Furthermore, in vivo treatment with anti-CD40 mAb resulted in enhanced expression of cytokines and cell surface activation markers, as well as Mphi-mediated tumor inhibition in A/J mice, C57BL/6 mice, and SCID/beige mice, as measured in vitro. A role for Mphi was shown by reduction in the antitumor effect of anti-CD40 mAb when Mphi functions were inhibited in vivo by silica. In addition, activation of peritoneal Mphi by anti-CD40 mAb resulted in survival benefits in mice bearing intraperitoneal tumors. Taken together, our results show that anti-CD40 mAb immunotherapy of mice can inhibit tumor growth in the absence of T cells, NK cells, and PMN through the involvement of activated Mphi.

  11. Association of Autophagy in the Cell Death Mediated by Dihydrotestosterone in Autoreactive T Cells Independent of Antigenic Stimulation.

    PubMed

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

    2015-12-01

    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.

  12. 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

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

    PubMed

    Boehlke, Christopher; Janusch, Heike; 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

  14. 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

  15. 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.

  16. Cell surface localised Hsp70 is a cancer specific regulator of clathrin-independent endocytosis.

    PubMed

    Nimmervoll, Benedikt; Chtcheglova, Lilia A; Juhasz, Kata; Cremades, Nunilo; Aprile, Francesco A; Sonnleitner, Alois; Hinterdorfer, Peter; Vigh, Laszlo; Preiner, Johannes; Balogi, Zsolt

    2015-09-14

    The stress inducible heat shock protein 70 (Hsp70) is present specifically on the tumour cell surface yet without a pro-tumour function revealed. We show here that cell surface localised Hsp70 (sHsp70) supports clathrin-independent endocytosis (CIE) in melanoma models. Remarkably, ability of Hsp70 to cluster on lipid rafts in vitro correlated with larger nano-domain sizes of sHsp70 in high sHsp70 expressing cell membranes. Interfering with Hsp70 oligomerisation impaired sHsp70-mediated facilitation of endocytosis. Altogether our findings suggest that a sub-fraction of sHsp70 co-localising with lipid rafts enhances CIE through oligomerisation and clustering. Targeting or utilising this tumour specific mechanism may represent an additional benefit for anti-cancer therapy.

  17. 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.

  18. RNase III-independent microRNA biogenesis in mammalian cells

    PubMed Central

    Maurin, Thomas; Cazalla, Demián; Yang, Jr-Shiuan; Bortolamiol-Becet, Diane; Lai, Eric C.

    2012-01-01

    RNase III enzymes are fundamental to the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in all species studied. Although alternative miRNA pathways independent of Drosha or Dicer exist, each still requires one RNase III-type enzyme. Here, we describe two strategies that marry either RNase Z or the Integrator complex with the slicing activity of Argonaute2 to generate highly functional mature miRNAs. We provide stringent validation of their RNase III independence by demonstrating efficient miRNA biogenesis and activity in Drosha and Dicer knockout cells. These data provide proof-of-principle evidence for additional mechanistic possibilities for efficient generation of small regulatory RNAs, and represent novel silencing triggers that may be exploited for technical purposes. PMID:23097423

  19. Both Canonical and Non-Canonical Wnt Signaling Independently Promote Stem Cell Growth in Mammospheres

    PubMed Central

    Many, Alexander M.; Brown, Anthony M. C.

    2014-01-01

    The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms. PMID:25019931

  20. Berberine hydrochloride IL-8 dependently inhibits invasion and IL-8-independently promotes cell apoptosis in MDA-MB-231 cells.

    PubMed

    Li, Xiang; Zhao, Shu-Juan; Shi, Hai-Lian; Qiu, Shui-Ping; Xie, Jian-Qun; Wu, Hui; Zhang, Bei-Bei; Wang, Zheng-Tao; Yuan, Jian-Ye; Wu, Xiao-Jun

    2014-12-01

    Breast cancer, the leading cause of cancer-related mortality worldwide in females, has high metastastic and recurrence rates. The aim of the present study was to evaluate the anti-metastatic and anticancer in situ effect of berberine hydrochloride (BER) in MDA-MB-231 cells. BER dose-dependently inhibited proliferation and the IL-8 secretion of MDA-MB-231 cells. Additional experiments revealed that the inactivation of PI3K, JAK2, NF-κB and AP-1 by BER contributed to the decreased IL-8 secretion. BER abrogated cell invasion induced by IL-8 accompanied with the downregulation of the gene expression of MMP-2, EGF, E-cadherin, bFGF and fibronectin. In addition, BER reduced cell motility but induced G2/M arrest and cell apoptosis in an IL-8‑independent manner. BER modulated multiple signaling pathway molecules involved in the regulation of cell apoptosis, including activation of p38 MAPK and JNK and deactivation of JAK2, p85 PI3K, Akt and NF-κB. The enhanced cell apoptosis induced by BER was eliminated by inhibitors of p38 MAPK and JNK but was strengthened by activator of p38 MAPK. Thus, BER inhibited cell metastasis partly through the IL-8 mediated pathway while it induced G2/M arrest and promoted cell apoptosis through the IL-8 independent pathway. Apoptosis induced by BER was mediated by crosstalks of various pathways including activation of p38 MAPK and JNK pathways and inactivation of Jak2/PI3K/NF-κB/AP-1 pathways. The results suggested that BER may be an efficient and safe drug candidate for treating highly metastatic breast cancer.

  1. T cell and non-T cell compartments can independently determine resistance to Leishmania major

    PubMed Central

    1995-01-01

    In experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm), the cellular determinants governing development of protective or exacerbative T cells are not well understood. We, therefore, attempted to determine the influence of T cell and non-T cell compartments on disease outcome. To this end, T cell chimeric mice were constructed using adult thymectomized lethally irradiated, bone marrow-reconstituted (ATXBM) animals of genetically resistant, C57BL/6, or susceptible, BALB/c, backgrounds. These hosts were engrafted with naive T cell populations from H-2-congenic susceptible, BALB.B6-H-2b, or resistant, C57BL/6.C-H-2d, animals, respectively. Chimeric mice were then infected with Lm, and disease outcome was monitored. BALB/c T cell chimeric mice, BALB/c ATXBM hosts given naive C57BL/6.C-H-2d T cells, resolved their infections as indicated by reductions in both lesion size and parasite numbers. Furthermore, the mice developed typical Th1 (interferon[IFN]-gamma hiinterleukin[IL]-4lo) cytokine patterns. In contrast, both sham chimeric, BALB/c ATXBM hosts given naive BALB/c T cells, and control irradiated euthymic mice succumbed to infection, producing Th2 profiles (IFN-gamma loIL-4hiIL-10hi). C57BL/6 T cell chimeras, C57BL/6 ATXBM hosts given naive BALB.B6-H-2b T cells, resolved their infections as did C57BL/6 sham chimeras and euthymic controls. Interestingly, whereas C57BL/6 control animals produced Th1 cytokines, chimeric animals progressed from Th0 (IFN-gamma hiIL-4hiIL- 10hi) to Th2 (IFN-gamma loIL-4hiIL-10hi) cytokine profiles as cure ensued. Both reconstitution and chimeric status of all mice were confirmed by flow cytometry. In addition, T cell receptor V beta usage of Lm-specific blasts was determined. In all cases, V beta use was multiclonal, involving primarily V beta 2, 4, 6, 8.1, 8.2, 8.3, 10, and 14, with relative V beta frequencies differing between H-2b and H-2d animals. Most importantly, however, these differences did not segregate

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

    PubMed

    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

  3. 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

  4. 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.

  5. 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.

  6. 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

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

    PubMed

    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

  8. Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements.

    PubMed

    Bartoszewski, Grzegorz; Malepszy, Stefan; Havey, Michael J

    2004-02-01

    Passage of the highly inbred cucumber ( Cucumis sativus L.) line B through cell culture produces progenies with paternally transmitted, mosaic (MSC) phenotypes. Because the mitochondrial genome of cucumber shows paternal transmission, we evaluated for structural polymorphisms by hybridizing cosmids spanning the entire mitochondrial genome of Arabidopsis thaliana L. to DNA-gel blots of four independently generated MSC and four wild-type cucumbers. Polymorphisms were identified by cosmids carrying rrn18, nad5-exon2, rpl5, and the previously described JLV5 deletion. Polymorphisms revealed by rrn18 and nad5-exon2 were due to one rearrangement bringing together these two coding regions. The polymorphism revealed by rpl5 was unique to MSC16 and was due to rearrangement(s) placing the rpl5 region next to the forward junction of the JLV5 deletion. The rearrangement near rpl5 existed as a sublimon in wild-type inbred B, but was not detected in the cultivar Calypso. Although RNA-gel blots revealed reduced transcription of rpl5 in MSC16 relative to wild-type cucumber, Western analyses revealed no differences for the RPL5 protein and the genetic basis of the MSC16 phenotype remains enigmatic. We evaluated 17 MSC and wild-type lines regenerated from independent cell-culture experiments for these structural polymorphisms and identified eight different patterns, indicating that the passage of cucumber through cell culture may be a unique mechanism to induce or select for novel rearrangements affecting mitochondrial gene expression.

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

    PubMed

    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

  10. Inflammasome-independent NLRP3 is required for epithelial-mesenchymal transition in colon cancer cells.

    PubMed

    Wang, Hong; Wang, Yajing; Du, Qianming; Lu, Ping; Fan, Huimin; Lu, Jinrong; Hu, Rong

    2016-03-15

    Inflammasome NLRP3 plays a crucial role in the process of colitis and colitis--associated colon cancer. Even though much is known regarding the NLRP3 inflammasome that regulates pro-inflammatory cytokine release in innate immune cells, the role of NLRP3 in non-immune cells is still unclear. In this study, we showed that NLRP3 was highly expressed in mesenchymal-like colon cancer cells (SW620), and was upregulated by tumor necrosis factors-α (TNF-α) and transforming growth factor-β1 (TGF-β1) respectively, during EMT in colon cancer epithelial cells HCT116 and HT29. Knockdown of NLRP3 retained epithelial spindle-like morphology of HCT116 and HT29 cells and reversed the mesenchymal characteristic of SW620 cells, indicated by the decreased expression of vimentin and MMP9 and increased expression of E-cadherin. In addition, knockdown of NLRP3 in colorectal carcinoma cells displayed diminished cell migration and invasion. Interestingly, during the EMT process induced by TNF-α or TGF-β1, the cleaved caspase-1 and ASC speck were not detected, indicating that NLRP3 functions in an inflammasome-independent way. Further studies demonstrated that NLRP3 protein expression was regulated by NF-κB signaling in TNF-α or TGF-β1-induced EMT, as verified by the NF-κB inhibitor Bay 11-7082. Moreover, NLRP3 knockdown reduced the expression of Snail1, indicating that NLRP3 may promote EMT through regulating Snail1. In summary, our results showed that the NLRP3 expression, not the inflammasome activation, was required for EMT in colorectal cancer cells. PMID:26968633

  11. 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.

  12. 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. PMID:27146584

  13. 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

  14. 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

  15. Stool-fermented Plantago ovata husk induces apoptosis in colorectal cancer cells independently of molecular phenotype.

    PubMed

    Sohn, Vanessa R; Giros, Anna; Xicola, Rosa M; Fluvià, Lourdes; Grzybowski, Mike; Anguera, Anna; Llor, Xavier

    2012-06-01

    Several studies have suggested that the partially fermentable fibre Plantago ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We studied the potentially pro-apoptotic effect of PO and the implicated mechanisms in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29, SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the human colon. The fermentation products of PO induced apoptosis in all primary tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli, β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both intrinsic and extrinsic pathways were implicated. The intrinsic pathway was activated through a shift in the balance towards a pro-apoptotic environment with an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and LoVo cells. This resulted in mitochondrial membrane depolarisation, increased expression of caspase activators second mitochondria-derived activator of caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome member apoptotic protease activating factor 1 and down-regulation of inhibitors of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The extrinsic pathway was activated presumably through the up-regulation of death receptor (DR5). Some important differences were seen between primary tumour and metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable up-regulation of TNF-α ligand along with death-inducing signalling complex components receptor interacting protein and TNF-α receptor 1-associated death domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), an inhibitor of both spontaneous death ligand-independent and death receptor-mediated apoptosis, was significantly down-regulated after PO treatment in all primary tumour cells, but not

  16. 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.

  17. Cell Density Sensing Alters TGF-β Signaling in a Cell-Type-Specific Manner, Independent from Hippo Pathway Activation

    PubMed Central

    Nallet-Staub, Flore; Yin, Xueqian; Gilbert, Cristèle; Marsaud, Véronique; Ben Mimoun, Saber; Javelaud, Delphine; Leof, Edward B.; Mauviel, Alain

    2015-01-01

    SUMMARY Cell-cell contacts inhibit cell growth and proliferation in part by activating the Hippo pathway that drives the phosphorylation and nuclear exclusion of the transcriptional coactivators YAP and TAZ. Cell density and Hippo signaling have also been reported to block transforming growth factor β (TGF-β) responses, based on the ability of phospho-YAP/TAZ to sequester TGF-β-activated SMAD complexes in the cytoplasm. Herein, we provide evidence that epithelial cell polarization interferes with TGF-β signaling well upstream and independent of cytoplasmic YAP/TAZ. Rather, polarized basolateral presentation of TGF-β receptors I and II deprives apically delivered TGF-β of access to its receptors. Basolateral ligand delivery nonetheless remains entirely effective to induce TGF-β responses. These data demonstrate that cell-type-specific inhibition of TGF-β signaling by cell density is restricted to polarized epithelial cells and reflects the polarized distribution of TGF-β receptors, which thus affects SMAD activation irrespective of Hippo pathway activation. PMID:25758862

  18. 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.

  19. 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

  20. 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.

  1. 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. PMID:12239380

  2. 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

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

    PubMed

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

    2015-07-01

    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

  4. eIF3 controls cell size independently of S6K1-activity

    PubMed Central

    Schipany, Katharina; Rosner, Margit; Ionce, Loredana; Hengstschläger, Markus; Kovacic, Boris

    2015-01-01

    All multicellular organisms require a life-long regulation of the number and the size of cells, which build up their organs. mTOR acts as a signaling nodule for the regulation of protein synthesis and growth. To activate the translational cascade, mTOR phosphorylates S6 kinase (S6K1), which is liberated from the eIF3-complex and mobilized for activation of its downstream targets. How S6K1 regulates cell size remains unclear. Here, we challenged cell size control through S6K1 by specifically depleting its binding partner eIF3 in normal and transformed cell lines. We show that loss of eIF3 leads to a massive reduction of cell size and cell number accompanied with an unexpected increase in S6K1-activity. The hyperactive S6K1-signaling was rapamycin-sensitive, suggesting an upstream mTOR-regulation. A selective S6K1 inhibitor (PF-4708671) was unable to interfere with the reduced size, despite efficiently inhibiting S6K1-activity. Restoration of eIF3 expression recovered size defects, without affecting the p-S6 levels. We further show that two, yet uncharacterized, cancer-associated mutations in the eIF3-complex, have the capacity to recover from reduced size phenotype, suggesting a possible role for eIF3 in regulating cancer cell size. Collectively, our results uncover a role for eIF3-complex in maintenance of normal and neoplastic cell size - independent of S6K1-signaling. PMID:26172298

  5. eIF3 controls cell size independently of S6K1-activity.

    PubMed

    Schipany, Katharina; Rosner, Margit; Ionce, Loredana; Hengstschläger, Markus; Kovacic, Boris

    2015-09-15

    All multicellular organisms require a life-long regulation of the number and the size of cells, which build up their organs. mTOR acts as a signaling nodule for the regulation of protein synthesis and growth. To activate the translational cascade, mTOR phosphorylates S6 kinase (S6K1), which is liberated from the eIF3-complex and mobilized for activation of its downstream targets. How S6K1 regulates cell size remains unclear. Here, we challenged cell size control through S6K1 by specifically depleting its binding partner eIF3 in normal and transformed cell lines. We show that loss of eIF3 leads to a massive reduction of cell size and cell number accompanied with an unexpected increase in S6K1-activity. The hyperactive S6K1-signaling was rapamycin-sensitive, suggesting an upstream mTOR-regulation. A selective S6K1 inhibitor (PF-4708671) was unable to interfere with the reduced size, despite efficiently inhibiting S6K1-activity. Restoration of eIF3 expression recovered size defects, without affecting the p-S6 levels. We further show that two, yet uncharacterized, cancer-associated mutations in the eIF3-complex, have the capacity to recover from reduced size phenotype, suggesting a possible role for eIF3 in regulating cancer cell size. Collectively, our results uncover a role for eIF3-complex in maintenance of normal and neoplastic cell size - independent of S6K1-signaling.

  6. 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

  7. 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.

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

    PubMed

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

    2014-10-01

    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.

  9. Hydrogen peroxide-induced necrotic cell death in cardiomyocytes is independent of matrix metalloproteinase-2.

    PubMed

    Ali, Mohammad A M; Kandasamy, Arulmozhi D; Fan, Xiaohu; Schulz, Richard

    2013-09-01

    Matrix metalloproteinase-2 (MMP-2) is well known to proteolyse both extracellular and intracellular proteins. Reactive oxygen species activate MMP-2 at both transcriptional and post-translational levels, thus MMP-2 activation is considered an early event in oxidative stress injury. Although hydrogen peroxide is widely used to trigger oxidative stress-induced cell death, the type of cell death (apoptosis vs. necrosis) in cardiomyocytes is still controversial depending on the concentration used and the exposure time. We carefully investigated the mode of cell death in neonatal rat cardiomyocytes induced by different concentrations (50-500 μM) of hydrogen peroxide at various time intervals after exposure and determined whether MMP-2 is implicated in hydrogen peroxide-induced cardiomyocyte death. Treating cardiomyocytes with hydrogen peroxide led to elevated MMP-2 level/activity with maximal effects seen at 200 μM. Hydrogen peroxide caused necrotic cell death by disrupting the plasmalemma as evidenced by the release of lactate dehydrogenase in a concentration- and time-dependent manner as well as the necrotic cleavage of PARP-1. The absence of both caspase-3 cleavage/activation and apoptotic cleavage of PARP-1 illustrated the weak contribution of apoptosis. Pre-treatment with selective MMP inhibitors did not protect against hydrogen peroxide-induced necrosis. In conclusion hydrogen peroxide increases MMP-2 level/activity in cardiomyocytes and induces necrotic cell death, however, the later effect is MMP-2 independent.

  10. Phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by carbon nanotube agglomerates.

    PubMed

    Shigemoto-Mogami, Yukari; Hoshikawa, Kazue; Hirose, Akihiko; Sato, Kaoru

    2016-01-01

    Although carbon nanotubes (CNTs) are used in many fields, including energy, healthcare, environmental technology, materials, and electronics, the adverse effects of CNTs in the brain are poorly understood. In this study, we investigated the effects of CNTs on cultured microglia, as microglia are the first responders to foreign materials. We compared the effects of sonicated suspensions of 5 kinds of CNTs and their flow-through filtered with a 0.22 µm membrane filter on microglial viability. We found that sonicated suspensions caused microglial cell damage, but their flow-through did not. The number of microglial aggregates was well correlated with the extent of the damage. We also determined that the CNT agglomerates consisted of two groups: one was phagocytosed by microglia and caused microglial cell damage, and the other caused cell damage without phagocytosis. These results suggest that phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by CNT agglomerates and it is important to conduct studies about the relationships between physical properties of nanomaterial-agglomerates and cell damage. PMID:27432236

  11. Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells.

    PubMed

    Huang, Jong-Khing; Huang, Chun-Jen; Chen, Wei-Chuan; Liu, Shiuh-Inn; Hsu, Shu-Shong; Chang, Hong-Tai; Tseng, Li-Ling; Chou, Chiang-Ting; Chang, Chih-Hung; Jan, Chung-Ren

    2005-07-01

    The effect of the carcinogen safrole on intracellular Ca2+ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca2+ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at a concentration of 325 microM started to increase [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 40% by removing extracellular Ca2+, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca2+-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) barely induced a [Ca2+]i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca2+]i rise. Neither inhibition of phospholipase C with 2 microM U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 1 microM safrole did not alter cell proliferation, but incubation with 10-1000 microM safrole increased cell proliferation by 60+/-10%. This increase was not reversed by pre-chelating Ca2+ with 10 microM of the Ca2+ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca2+]i rise by causing release of stored Ca2+ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca2+ influx via nifedipine-sensitive Ca2+ entry. Furthermore, safrole can enhance cell growth in a Ca2+-independent manner.

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

    PubMed

    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

  13. 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

  14. A caspase-independent cell clearance program. The LEI/L-DNase II pathway.

    PubMed

    Torriglia, A; Perani, P; Brossas, J Y; Altairac, S; Zeggai, S; Martin, E; Tréton, J; Courtois, Y; Counis, M F

    2000-01-01

    The discovery of caspase-mitochondrial pathway counts as one of the most important discovery in apoptosis biochemistry. Today, however, we begin to recognize its limits. Inhibition of caspase does not prevent cell death in many mammalian models. Targeted disruption of caspases does not impair every type of apoptosis. Other pathways, caspase independent, are now described. Here we present one of these pathways. It is a serine-protease dependent pathway and its key event is the transformation of LEI (a serine protease inhibitor) into L-DNase II (an endonuclease). When using this apoptotic pathway the cell activates, at the same time, its endonuclease activity (L-DNase II appears) and its protease activity (there is a release of inhibition of proteases).

  15. 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.

  16. The Helicobacter pylori Vacuolating Toxin Inhibits T Cell Activation by Two Independent Mechanisms

    PubMed Central

    Boncristiano, Marianna; Paccani, Silvia Rossi; Barone, Silvia; Ulivieri, Cristina; Patrussi, Laura; Ilver, Dag; Amedei, Amedeo; D'Elios, Mario Milco; Telford, John L.; Baldari, Cosima T.

    2003-01-01

    Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche. PMID:14676300

  17. Understanding the angle-independent photon harvesting in organic homo-tandem solar cells.

    PubMed

    Mertens, Adrian; Mescher, Jan; Bahro, Daniel; Koppitz, Manuel; Colsmann, Alexander

    2016-05-16

    The effective device photo current of organic tandem solar cells is independent of the angle of light incidence up to 65°. This feature renders these devices particularly suitable for stationary applications where they receive mainly indirect light. In a combined experimental and simulative study, we develop a fundamental understanding of the causal absorption and charge generation mechanisms in organic homo-tandem solar cells. A 3-terminal tandem device architecture is used to measure the optoelectronic properties of both subcells individually. The analysis of the angle dependent external quantum efficiencies of the subcells and the tandem device reveal an internal balancing of the wavelength dependent subcell currents elucidating the low sensitivity of the tandem device properties on the angle of incidence. PMID:27409962

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

    PubMed Central

    2014-01-01

    Background 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. Results 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

  19. 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

  20. c-Myc protein expression is not an independent prognostic predictor in cervical squamous cell carcinoma.

    PubMed

    Brenna, S M F; Zeferino, L C; Pinto, G A; Souza, R A; Andrade, L A L; Vassalo, J; Martinez, E Z; Syrjanen, K J

    2002-04-01

    The c-myc protein is known to regulate the cell cycle, and its down-regulation can lead to cell death by apoptosis. The role of c-myc protein as an independent prognostic determinant in cervical cancer is controversial. In the present study, a cohort of 220 Brazilian women (mean age 53.4 years) with FIGO stage I, II and III (21, 28 and 51%, respectively) cervical squamous cell carcinomas was analyzed for c-myc protein expression using immunohistochemistry. The disease-free survival and relapse-rate were analyzed using univariate (Kaplan-Meier) survival analysis for 116 women who completed the standard FIGO treatment and were followed up for 5 years. Positive c-myc staining was detected in 40% of carcinomas, 29% being grade 1, 9% grade 2, and 2% grade 3. The distribution of positive c-myc according to FIGO stage was 19% (17 women) in stage I, 33% (29) in stage II, and 48% (43) in stage III of disease. During the 60-month follow-up, disease-free survival in univariate (Kaplan-Meier) survival analysis (116 women) was lower for women with c-myc-positive tumors, i.e., 60.5, 47.5 and 36.6% at 12, 36, and 60 months, respectively (not significant). The present data suggest that immunohistochemical demonstration of c-myc does not possess any prognostic value independent of FIGO stage, and as such is unlikely to be a useful prognostic marker in cervical squamous cell carcinoma.

  1. Independent Synchronized Control and Visualization of Interactions between Living Cells and Organisms

    PubMed Central

    Rouger, Vincent; Bordet, Guillaume; Couillault, Carole; Monneret, Serge; Mailfert, Sébastien; Ewbank, Jonathan J.; Pujol, Nathalie; Marguet, Didier

    2014-01-01

    To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode’s surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion. PMID:24853738

  2. Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma.

    PubMed

    Woollard, Wesley J; Kalaivani, Nithyha P; Jones, Christine L; Roper, Catherine; Tung, Lam; Lee, Jae Jin; Thomas, Bjorn R; Tosi, Isabella; Ferreira, Silvia; Beyers, Carl Z; McKenzie, Robert C T; Butler, Rosie M; Lorenc, Anna; Whittaker, Sean J; Mitchell, Tracey J

    2016-06-01

    Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment.

  3. Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma.

    PubMed

    Woollard, Wesley J; Kalaivani, Nithyha P; Jones, Christine L; Roper, Catherine; Tung, Lam; Lee, Jae Jin; Thomas, Bjorn R; Tosi, Isabella; Ferreira, Silvia; Beyers, Carl Z; McKenzie, Robert C T; Butler, Rosie M; Lorenc, Anna; Whittaker, Sean J; Mitchell, Tracey J

    2016-06-01

    Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment. PMID:26872600

  4. Endoplasmic reticulum stress induces ligand-independent TNFR1-mediated necroptosis in L929 cells

    PubMed Central

    Saveljeva, S; Mc Laughlin, S L; Vandenabeele, P; Samali, A; Bertrand, M J M

    2015-01-01

    Endoplasmic reticulum (ER) stress-induced cellular dysfunction and death is associated with several human diseases. It has been widely reported that ER stress kills through activation of the intrinsic mitochondrial apoptotic pathway. Here we demonstrate that ER stress can also induce necroptosis, an receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL)-dependent form of necrosis. Remarkably, we observed that necroptosis induced by various ER stressors in L929 cells is dependent on tumor necrosis factor receptor 1 (TNFR1), but occurs independently of autocrine TNF or lymphotoxin α production. Moreover, we found that repression of either TNFR1, RIPK1 or MLKL did not protect the cells from death but instead allowed a switch to ER stress-induced apoptosis. Interestingly, while caspase inhibition was sufficient to protect TNFR1- or MLKL-deficient cells from death, rescue of the RIPK1-deficient cells additionally required RIPK3 depletion, indicating a switch back to RIPK3-dependent necroptosis in caspase-inhibited conditions. The finding that ER stress also induces necroptosis may open new therapeutic opportunities for the treatment of pathologies resulting from unresolved ER stress. PMID:25569104

  5. BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

    PubMed

    Ambrosini, Grazia; Sawle, Ashley D; Musi, Elgilda; Schwartz, Gary K

    2015-10-20

    Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations. PMID:26397223

  6. 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.

  7. Smac mimetic LBW242 sensitizes XIAP-overexpressing neuroblastoma cells for TNF-α-independent apoptosis.

    PubMed

    Eschenburg, Georg; Eggert, Angelika; Schramm, Alexander; Lode, Holger N; Hundsdoerfer, Patrick

    2012-05-15

    Despite intensive treatment regimens, high-risk and late-stage neuroblastoma tends to have a poor survival outcome. Overexpression of the apoptotic regulator, X-linked inhibitor of apoptosis protein (XIAP), has been associated with chemotherapy resistance in several cancers including neuroblastoma. Here, we report preclinical evidence that XIAP offers an effective therapeutic target in neuroblastoma. Human and murine neuroblastoma cells were treated with the Smac mimetic LBW242 alone or in combination with cytotoxic drugs used clinically to treat neuroblastoma. Expression of XIAP protein, but not mRNA, was highly increased in neuroblastoma cells compared to healthy adrenal gland tissue, consistent with a posttranscriptional regulation of XIAP expression. Treatment with LBW242 sensitized human and murine neuroblastoma cells to chemotherapy-induced apoptosis, which was mediated by activation of both the intrinsic and extrinsic apoptosis pathways. Although Smac mimetics have been reported to stimulate TNF-α-induced apoptosis by degradation of cellular IAP (cIAP)-1/2, we found that LBW242-mediated sensitization in neuroblastoma cells occurred in a TNF-α-independent manner, despite induction of cIAP-1/2 degradation and TNF-α expression. Together, our findings show that XIAP targeting sensitizes neuroblastoma to chemotherapy-induced apoptosis, suggesting a novel therapeutic approach to treat this childhood malignancy.

  8. Discovery of New Cargo Proteins that enter Cells through Clathrin-Independent Endocytosis

    PubMed Central

    Eyster, Craig A.; Higginson, Jason D.; Huebner, Robert; Porat-Shliom, Natalie; Weigert, Roberto; Wu, Wells W.; Shen, Rong-Fong; Donaldson, Julie G.

    2010-01-01

    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. PMID:19302270

  9. Recognition of apoptotic cells by viable cells is specific, ubiquitous, and species independent: analysis using photonic crystal biosensors.

    PubMed

    Pattabiraman, Goutham; Lidstone, Erich A; Palasiewicz, Karol; Cunningham, Brian T; Ucker, David S

    2014-06-01

    Apoptotic recognition is innate and linked to a profound immune regulation (innate apoptotic immunity [IAI]) involving anti-inflammatory and immunosuppressive responses. Many of the molecular and mechanistic details of this response remain elusive. Although immune outcomes can be quantified readily, the initial specific recognition events have been difficult to assess. We developed a sensitive, real-time method to detect the recognition of apoptotic cells by viable adherent responder cells, using a photonic crystal biosensor approach. The method relies on characteristic spectral shifts resulting from the specific recognition and dose-dependent interaction of adherent responder cells with nonadherent apoptotic targets. Of note, the biosensor provides a readout of early recognition-specific events in responder cells that occur distal to the biosensor surface. We find that innate apoptotic cell recognition occurs in a strikingly species-independent manner, consistent with our previous work and inferences drawn from indirect assays. Our studies indicate obligate cytoskeletal involvement, although apoptotic cell phagocytosis is not involved. Because it is a direct, objective, and quantitative readout of recognition exclusively, this biosensor approach affords a methodology with which to dissect the early recognition events associated with IAI and immunosuppression.

  10. 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. PMID:22223356

  11. 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.

  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. MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Alam, Maroof; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal. PMID:25245423

  14. 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.

  15. Pharmacology of the skate electroretinogram indicates independent ON and OFF bipolar cell pathways

    PubMed Central

    1996-01-01

    Organization of afferent information into parallel ON and OFF pathways is a critical feature of the vertebrate visual system. All afferent visual information in the vertebrate retina reaches the inner plexiform layer (IPL) via bipolar cells. It is at the bipolar cell level that separation of ON and OFF information first appears for afferent information from cones. This may also hold true for the rod pathway of cold-blooded vertebrates, but not for mammals. The all-rod retina of the skate presents an opportunity to examine such pathways in a retina having but a single class of photoreceptor. Immunocytochemical evidence suggests that both ON and OFF bipolar cells are present in the skate retina. We examined the pharmacology of the skate electroretinogram (ERG) to test the hypothesis that independent ON and OFF bipolar cell pathways are functional as rod afferent pathways from outer to inner plexiform layer in the skate. 100 microM 2-amino-4-phosphonobutyric acid (APB) reversibly blocked the skate ERG b-wave. A small d-wave-like OFF component of the ERG revealed by DC recording of response to a prolonged (10 s) flash of light was reduced or blocked by 5 mM kynurenic acid (KYN). We found that addition of 200 microM picrotoxin to the Ringer's solution revealed prominent ON and OFF components of the skate ERG while reducing the c-wave. These ON and OFF components were reversibly blocked by 100 microM APB and 5 mM KYN, respectively. Reversible block of the OFF component by KYN was also accomplished in the presence of 500 microM N-methyl-DL-aspartate. From these findings, we conclude that ON and OFF bipolar cells are likely to be functional as parallel afferent interplexiform pathways in the all-rod retina of the skate. PMID:8722565

  16. Pdr12p-dependent and -independent fluorescein extrusion from baker's yeast cells.

    PubMed

    Lushchak, Volodymyr; Abrat, Oleksandra; Miedzobrodzki, Jacek; Semchyshyn, Halyna

    2008-01-01

    Fluorescein efflux from S. cerevisiae cells was measured to study the peculiarities of fluorescein transport system, which is important for yeast resistance to certain drugs and weak organic acid preservatives. Glucose-independent and glucose-stimulated fluorescein effluxes were characterized using iodoacetate, cyanide and orthovanadate, inhibitors of glycolysis, electron transport chain, and ATPases, respectively. It is supposed that in glucose-free medium fluorescein extrusion is ATP-dependent and the energy for this efflux is mainly provided by respiration. In glucose-containing medium, glycolysis plays a critical role for extrusion of fluorescein. The results indicate that acetic acid inhibits the fluorescein efflux from yeast cells. The inhibition constant of glucose-stimulated fluorescein efflux is significantly lower in parental strain than in two mutants defective in PDR12 (ABC-transporter Pdr12p) or WAR1 (transcription factor of Pdr12p). It can be suggested that the membrane protein Pdr12 is involved in fluorescein extrusion from the yeast cells, but component(s) other than Pdr12p is (are) also important.

  17. Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity.

    PubMed

    Li, Qiuxia; Shakya, Arvind; Guo, Xiaohua; Zhang, Hongbo; Tantin, Dean; Jensen, Peter E; Chen, Xinjian

    2012-05-01

    Foxp3 plays an essential role in conferring suppressive functionality to CD4(+)/Foxp3(+) regulatory T cells (Tregs). Although studies showed that Foxp3 has to form cooperative complexes with NFAT to bind to target genes, it remains unclear whether NFAT is available in the nucleus of primary Tregs for Foxp3 access. It is generally believed that NFAT in resting cells resides in the cytoplasm, and its nuclear translocation depends on calcineurin (CN) activation. We report that a fraction of NFAT protein constitutively localizes in the nucleus of primary Tregs, where it selectively binds to Foxp3 target genes. Treating Tregs with CN inhibitor does not induce export of NFAT from the nucleus, indicating that its nuclear translocation is independent of CN activity. Consistently, Tregs are resistant to CN inhibitors in the presence of IL-2 and continue to proliferate in response to anti-CD3 stimulation, whereas proliferation of non-Tregs is abrogated by CN inhibitors. In addition, PMA, which activates other transcription factors required for T cell activation but not NFAT, selectively induces Treg proliferation in the absence of ionomycin. TCR interaction with self-MHC class II is not required for PMA-induced Treg proliferation. Tregs expanded by PMA or in the presence of CN inhibitors maintain Treg phenotype and functionality. These findings shed light on Treg biology, paving the way for strategies to selectively activate Tregs.

  18. TRIM31 promotes Atg5/Atg7-independent autophagy in intestinal cells

    PubMed Central

    Ra, Eun A.; Lee, Taeyun A.; Won Kim, Seung; Park, Areum; Choi, Hyun jin; Jang, Insook; Kang, Sujin; Hee Cheon, Jae; Cho, Jin Won; Eun Lee, Ji; Lee, Sungwook; Park, Boyoun

    2016-01-01

    Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host–bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection. PMID:27216961

  19. 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.

  20. Tuning 3D Collagen Matrix Stiffness Independently of Collagen Concentration Modulates Endothelial Cell Behavior

    PubMed Central

    Mason, Brooke N.; Starchenko, Alina; Williams, Rebecca M.; Bonassar, Lawrence J.; Reinhart-King, Cynthia A.

    2012-01-01

    Numerous studies have described the effects of matrix stiffening on cell behavior using two dimensional (2D) synthetic surfaces; however less is known about the effects of matrix stiffening on cells embedded in three dimensional (3D) in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in 3D is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a 3-fold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation endproducts is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth. PMID:22902816

  1. 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. PMID:24289578

  2. U0126 protects cells against oxidative stress independent of its function as a MEK inhibitor.

    PubMed

    Ong, Qunxiang; Guo, Shunling; Zhang, Kai; Cui, Bianxiao

    2015-01-21

    U0126 is a potent and selective inhibitor of MEK1 and MEK2 kinases. It has been widely used as an inhibitor for the Ras/Raf/MEK/ERK signaling pathway with over 5000 references on the NCBI PubMed database. In particular, U0126 has been used in a number of studies to show that inhibition of the Raf/MEK/ERK pathway protects neuronal cells against oxidative stress. Here, we report that U0126 can function as an antioxidant that protects PC12 cells against a number of different oxidative-stress inducers. This protective effect of U0126 is independent of its function as a MEK inhibitor, as several other MEK inhibitors failed to show similar protective effects. U0126 reduces reactive oxygen species (ROS) in cells. We further demonstrate that U0126 is a direct ROS scavenger in vitro, and the oxidation products of U0126 exhibit fluorescence. Our finding that U0126 is a strong antioxidant signals caution for its future usage as a MEK inhibitor and for interpreting some previous results. PMID:25544156

  3. 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

  4. ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.

    PubMed

    Adams, Bret R; Hawkins, Amy J; Povirk, Lawrence F; Valerie, Kristoffer

    2010-09-01

    We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

  5. 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

  6. Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos.

    PubMed

    Hirate, Yoshikazu; Hirahara, Shino; Inoue, Ken-Ichi; Kiyonari, Hiroshi; Niwa, Hiroshi; Sasaki, Hiroshi

    2015-10-01

    In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.

  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. PMID:23812037

  8. 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

  9. Infection of XC cells by MLVs and Ebola virus is endosome-dependent but acidification-independent.

    PubMed

    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.

  10. Clostridium difficile toxin A elicits Ca(2+)-independent cytotoxic effects in cultured normal rat intestinal crypt cells.

    PubMed Central

    Fiorentini, C; Donelli, G; Nicotera, P; Thelestam, M

    1993-01-01

    In rat intestinal crypt cells, Clostridium difficile toxin A induces (i) early cytoskeletal alterations involving the whole population and (ii) late effects in 30 to 40% of the cells, consisting mainly of surface blebbing and nuclear fragmentation. All these effects were Ca2+ independent and were not abolished by protein synthesis inhibitors. Images PMID:8359922

  11. Goniothalamin-induced oxidative stress, DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

    PubMed Central

    Inayat-Hussain, S.H.; Chan, K.M.; Rajab, N.F.; Din, L.B.; Chow, S.C.; Kizilors, A.; Farzaneh, F.; Williams, G.T.

    2010-01-01

    Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4 h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1 h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2. PMID:20026395

  12. 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.

  13. 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.

  14. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis

    PubMed Central

    Kollmann, Karoline; Heller, Gerwin; Schneckenleithner, Christine; Warsch, Wolfgang; Scheicher, Ruth; Ott, Rene G.; Schäfer, Markus; Fajmann, Sabine; Schlederer, Michaela; Schiefer, Ana-Iris; Reichart, Ursula; Mayerhofer, Matthias; Hoeller, Christoph; Zöchbauer-Müller, Sabine; Kerjaschki, Dontscho; Bock, Christoph; Kenner, Lukas; Hoefler, Gerald; Freissmuth, Michael; Green, Anthony R.; Moriggl, Richard; Busslinger, Meinrad; Malumbres, Marcos; Sexl, Veronika

    2013-01-01

    Summary In contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a. PMID:23948297

  15. Differential infection of polarized epithelial cell lines by sialic acid-dependent and sialic acid-independent rotavirus strains.

    PubMed

    Ciarlet, M; Crawford, S E; Estes, M K

    2001-12-01

    Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence of N-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-microm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [(3)H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the

  16. 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

  17. 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.

  18. Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells.

    PubMed

    Chu, Chia; Bottaro, Donald P; Betenbaugh, Michael J; Shiloach, Joseph

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a complex cancer progression that can boost the metastatic potential of transformed cells by inducing migration, loss of cell adhesion, and promoting proliferation under anchorage-independent conditions. A DNA microarray analysis was performed comparing parental anchorage-dependent MDCK cells and anchorage-independent MDCK cells that were engineered to express human siat7e (ST6GALNAC5). The comparison identified several genes involved in the EMT process that were differentially expressed between the anchorage-dependent and the anchorage-independent MDCK cell lines. The hepatocyte growth factor gene (hgf) was found to be over-expressed in the engineered MDCK-siat7e cells at both transcription and protein expression levels. Phosphorylation analysis of the MET receptor tyrosine kinase confirmed the activation of an autocrine loop of the HGF/ MET signaling pathway in the MDCK-siat7e cells. When MET activities were suppressed by using the small-molecular inhibitor drug PF-02341066 (Crizotinib), the anchorage-independent MDCK-siat7e cells reverted to the cellular morphology of the parental anchorage-dependent MDCK cells. These observations indicate that the MET receptor plays a central role in the growth properties of the MDCK cells and its phosphorylation status is likely dependent on sialylation. Further investigation of the downstream signaling targets in the MET network showed that the degree of MDCK cell adhesion correlated with secretion levels of a matrix metalloproteinase, MMP1, suggesting a role of metalloproteinases in the EMT process. These results demonstrate that in addition to its application in biotechnology processes, MDCK-siat7e may serve as a model cell for metastasis studies to decipher the sequence of events leading up to the activation of EMT. PMID:26848584

  19. Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells

    PubMed Central

    Chu, Chia; Bottaro, Donald P.; Betenbaugh, Michael J.; Shiloach, Joseph

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a complex cancer progression that can boost the metastatic potential of transformed cells by inducing migration, loss of cell adhesion, and promoting proliferation under anchorage-independent conditions. A DNA microarray analysis was performed comparing parental anchorage-dependent MDCK cells and anchorage-independent MDCK cells that were engineered to express human siat7e (ST6GALNAC5). The comparison identified several genes involved in the EMT process that were differentially expressed between the anchorage-dependent and the anchorage-independent MDCK cell lines. The hepatocyte growth factor gene (hgf) was found to be over-expressed in the engineered MDCK-siat7e cells at both transcription and protein expression levels. Phosphorylation analysis of the MET receptor tyrosine kinase confirmed the activation of an autocrine loop of the HGF/ MET signaling pathway in the MDCK-siat7e cells. When MET activities were suppressed by using the small-molecular inhibitor drug PF-02341066 (Crizotinib), the anchorage-independent MDCK-siat7e cells reverted to the cellular morphology of the parental anchorage-dependent MDCK cells. These observations indicate that the MET receptor plays a central role in the growth properties of the MDCK cells and its phosphorylation status is likely dependent on sialylation. Further investigation of the downstream signaling targets in the MET network showed that the degree of MDCK cell adhesion correlated with secretion levels of a matrix metalloproteinase, MMP1, suggesting a role of metalloproteinases in the EMT process. These results demonstrate that in addition to its application in biotechnology processes, MDCK-siat7e may serve as a model cell for metastasis studies to decipher the sequence of events leading up to the activation of EMT. PMID:26848584

  20. A Trypanosoma cruzi alkaline antigen induces polyclonal B-cell activation of normal murine spleen cells by T-cell-independent, BCR-directed stimulation.

    PubMed

    Montes, C L; Zuñiga, E; Minoprio, P; Vottero-Cima, E; Gruppi, A

    1999-08-01

    We have previously reported that a cytosolic alkaline fraction (FI) obtained from epimastigotes of Trypanosoma cruzi promotes the activation, proliferation and differentiation of normal murine B cells into antibody-secreting plasmocytes. Neither the mechanism nor the cells involved in the FI-induced polyclonal B-cell activation were established. In this work we report that accessory cells are required for FI-induced polyclonal B-cell activation as no proliferative responses were obtained following treatment of normal spleen mononuclear cells (NSMC) with L-leucine methyl ester. Furthermore, FI did not induce the expression of CD25 on T cells and it promoted the proliferation of a T-cell-depleted population, indicating that it acts in a T-independent manner. We observed that NSMC were stimulated in vitro by FI-released cytokines, such as interleukin (IL)-4, IL-6 and IL-10, which are involved in B-cell proliferation and differentiation. Interestingly, while significant amounts of interferon-gamma (IFN-gamma) were found in culture supernatants we did not observe detectable levels of IL-2. Additionally, we found that B-cell receptor (BCR) and major histocompatibility complex (MHC) class II antigens were involved in the proliferative response induced by FI because antibodies directed against cell-surface immunoglobulin M (IgM), CD45 and MHC class II molecules inhibited the FI-induced B-cell proliferation. CD40 ligand (CD40L) did not participate in such a phenomenon.

  1. Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line.

    PubMed Central

    Ruscetti, S K; Janesch, N J; Chakraborti, A; Sawyer, S T; Hankins, W D

    1990-01-01

    Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor. Images PMID:2154592

  2. 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}.

  3. Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

    SciTech Connect

    Jokilehto, Terhi; Hoegel, Heidi; Heikkinen, Pekka; Rantanen, Krista; Elenius, Klaus; Sundstroem, Jari; Jaakkola, Panu M.

    2010-04-15

    Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1{alpha} and -2{alpha}). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1{alpha} or -2{alpha} expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.

  4. 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. PMID:26920061

  5. 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. PMID:22733631

  6. 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

  7. Propylthiouracil, independent of its antithyroid effect, promotes vascular smooth muscle cells differentiation via PTEN induction.

    PubMed

    Chen, Wei-Jan; Pang, Jong-Hwei S; Lin, Kwang-Huei; Lee, Dany-Young; Hsu, Lung-An; Kuo, Chi-Tai

    2010-01-01

    Propylthiouracil (PTU), independent of its antithyroid effect, is recently found to have an antiatherosclerotic effect. The aim of this study is to determine the impact of PTU on phenotypic modulation of vascular smooth muscle cells (VSMCs), as phenotypic modulation may contribute to the growth of atherosclerotic lesions and neointimal formation after arterial injury. Propylthiouracil reduced neointimal formation in balloon-injured rat carotid arteries. In vitro, PTU may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of SMC differentiation marker contractile proteins, including calponin and smooth muscle (SM)-myosin heavy chain (SM-MHC). Transient transfection studies in VSMCs demonstrated that PTU induced the activity of SMC marker genes (calponin and SM-MHC) promoters, indicating that PTU up-regulates these genes expression predominantly at the transcriptional level. Furthermore, PTU enhanced the expression of PTEN and inhibition of PTEN by siRNA knockdown blocked PTU-induced activation of contractile proteins expression and promoter activity. In the rat carotid injury model, PTU reversed the down-regulation of contractile proteins and up-regulated PTEN in the neointima induced by balloon injury. Propylthiouracil promotes VSMC differentiation, at lest in part, via induction of the PTEN-mediated pathway. These findings suggest a possible mechanism by which PTU may contribute to its beneficial effects on atherogenesis and neointimal formation after arterial injury.

  8. FLOT-2 is an independent prognostic marker in oral squamous cell carcinoma.

    PubMed

    Wen, Qiuyuan; Alnemah, Mohannad Ma; Luo, Jiadi; Wang, Weiyuan; Chu, Shuzhou; Chen, Lingjiao; Li, Jiao; Xu, Lina; Li, Meirong; Zhou, Jianhua; Fan, Songqing

    2015-01-01

    Flotillin-2 (Flot-2) is an important component of cellular membrane, which involves in various cellular processes and recent studies have revealed that Flot-2 played important roles in cancer progression. The expression and prognostic impact of Flot-2 in oral squamous cell carcinoma (OSCC) have not been well studied. So, a tissue microarray (TMA) based on immunohistochemical analysis of surgical resection of tumor tissues of 78 cases of OSCC patients and 27 cases of adjacent non-cancerous squamous epithelium tissues was conducted. This study focused on detecting Flot-2 expression and analyzing its prognostic impact on OSCC. The result showed that the positive percentage of Flot-2 expression in OSCC (74.4%, 58/78) was significantly higher than that in adjacent non-cancerous squamous epithelium tissues (25.9%, 7/27) (P<0.001). Additionally, the positive expression of Flot-2 in OSCC patients with a history of alcohol consumption was significantly higher than those nonusers (P=0.027). Both univariate and multivariate survival analysis indicated that increased expression Flot-2 protein was significantly correlated inversely with overall survival rates in OSCC patients (P=0.046, P=0.002). Taken together, positive expression of Flot-2 protein may be an independent biomarker for poor prognosis in OSCC. PMID:26339392

  9. Testosterone alters iron metabolism and stimulates red blood cell production independently of dihydrotestosterone

    PubMed Central

    Beggs, Luke A.; Yarrow, Joshua F.; Conover, Christine F.; Meuleman, John R.; Beck, Darren T.; Morrow, Matthew; Zou, Baiming; Shuster, Jonathan J.

    2014-01-01

    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. PMID:25074984

  10. 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.

  11. Cell Length Independent PBRB Model for Simulations of HE Reaction Initiation, Growth, and Detonation

    NASA Astrophysics Data System (ADS)

    Dwivedi, Sunil

    2015-06-01

    It has been our focus to use the Physics Based Reaction Burn (PBRB) model to simulate reaction initiation, growth, and detonation of HE composites at the mesoscale. The idealization of hot spots as planar surfaces reduces the 3D model to a 1D hot spot cell (1DHSC) model. The idealization also renders the model dependent on the 1DHSC length and mesh size. New developments are presented making the PBRB model 1DHSC length independent. First, the accurate prediction of the gas-solid interface temperature and thermal gradient are essential, achieved through a finite difference scheme with 500-2000 thermal grid points. Second, keeping the burn mass constant while varying the 1DHSC length is essential, achieved by varying the hot spot specific surface area. 1D and 2D simulation results are presented for shock response of RDX at 1 km/s and 2 km/s impact velocities. The 5, 10, and 50 micro meters 1DHSC lengths yield near identical run-to-detonation, time-to-detonation, and detonation velocity in agreement with experimental data. It is concluded that the new developments make the PBRB model well suited as a generic EOS model for HE composites. - Dr. John Brennan, ARL is acknowledged for his interactions and support. This work is supported in part by ARL Grant W911NF-12-2-0053 and DTRA Grant HDTRA1-12-1-0004.

  12. 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.

  13. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival

    PubMed Central

    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  14. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

    PubMed

    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  15. Role of manganese superoxide dismutase on growth and invasive properties of human estrogen-independent breast cancer cells.

    PubMed

    Kattan, Zilal; Minig, Vanessa; Leroy, Pierre; Dauça, Michel; Becuwe, Philippe

    2008-03-01

    Manganese superoxide dismutase (MnSOD) is known to play a role in cancer. MnSOD exerts a tumor suppressive effect in estrogen-dependent human breast cancer cells. In the present study we investigated the in vitro role of MnSOD in the growth of some aggressive and highly metastatic estrogen-independent breast cancer cells, i.e., MDA-MB231 and SKBR3 cells. We show that estrogen-independent cells expressed a significantly higher basal MnSOD level compared to estrogen-dependent human breast cancer cell lines (MCF-7 and T47D). For MDA-MB231 cells, the high-MnSOD level was accompanied by an overproduction of intracellular hydrogen peroxide (H2O2) and by a low expression of the major H2O2-detoxifying enzymes, catalase, and peroxiredoxin 3, compared to MCF-7 cells. Suppression of MnSOD expression by antisense RNA was associated with a decrease of H2O2 content and caused a stimulation of growth with a reduced cell doubling time but induced a decrease of colony formation. Furthermore, treatment of MDA-MB231 cells with H2O2 scavengers markedly reduced tumor cell growth and colony formation. In addition, MnSOD suppression or treatment with H2O2 scavengers reduced the invasive properties of MDA-MB231 cells up to 43%, with a concomitant decrease of metalloproteinase-9 activity. We conclude that MnSOD plays a role in regulating tumor cell growth and invasive properties of estrogen-independent metastatic breast cancer cells. These action are mediated by MnSOD-dependent H2O2 production. In addition, these results suggest that MnSOD up-regulation may be one mechanism that contributes to the development of metastatic breast cancers.

  16. 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

  17. 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.

  18. Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

    PubMed

    Ino, Yoko; Arakawa, Noriaki; Ishiguro, Hitoshi; Uemura, Hiroji; Kubota, Yoshinobu; Hirano, Hisashi; Toda, Tosifusa

    2016-04-01

    Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

  19. 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.

  20. 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.

  1. The Human NK Cell Response to Yellow Fever Virus 17D Is Primarily Governed by NK Cell Differentiation Independently of NK Cell Education.

    PubMed

    Marquardt, Nicole; Ivarsson, Martin A; Blom, Kim; Gonzalez, Veronica D; Braun, Monika; Falconer, Karolin; Gustafsson, Rasmus; Fogdell-Hahn, Anna; Sandberg, Johan K; Michaëlsson, Jakob

    2015-10-01

    NK cells play an important role in the defense against viral infections. However, little is known about the regulation of NK cell responses during the first days of acute viral infections in humans. In this study, we used the live attenuated yellow fever virus (YFV) vaccine 17D as a human in vivo model to study the temporal dynamics and regulation of NK cell responses in an acute viral infection. YFV induced a robust NK cell response in vivo, with an early activation and peak in NK cell function at day 6, followed by a delayed peak in Ki67 expression, which was indicative of proliferation, at day 10. The in vivo NK cell response correlated positively with plasma type I/III IFN levels at day 6, as well as with the viral load. YFV induced an increased functional responsiveness to IL-12 and IL-18, as well as to K562 cells, indicating that the NK cells were primed in vivo. The NK cell responses were associated primarily with the stage of differentiation, because the magnitude of induced Ki67 and CD69 expression was distinctly higher in CD57(-) NK cells. In contrast, NK cells expressing self- and nonself-HLA class I-binding inhibitory killer cell Ig-like receptors contributed, to a similar degree, to the response. Taken together, our results indicate that NK cells are primed by type I/III IFN in vivo early after YFV infection and that their response is governed primarily by the differentiation stage, independently of killer cell Ig-like receptor/HLA class I-mediated inhibition or education.

  2. 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

  3. 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.

  4. Docetaxel induces Bcl-2- and pro-apoptotic caspase-independent death of human prostate cancer DU145 cells

    PubMed Central

    OGURA, TAKEHARU; TANAKA, YOSHIYUKI; TAMAKI, HIROKI; HARADA, MAMORU

    2016-01-01

    Docetaxel is a useful chemotherapeutic agent for the first-line treatment of hormone-refractory prostate cancer. Abnormal expression of Bcl-2 is commonly found in cancer cells, which increases their anti-apoptotic potency and chemo-resistance. We investigated the effects of Bcl-2 expression status on the susceptibility of DU145 cells, an androgen-independent human prostate cancer cell line, to docetaxel and other anticancer agents. A panel of Bcl-2-expressing DU145 cell lines was established. Bcl-2 expression levels were unrelated to the susceptibility of DU145 cells to docetaxel. The sensitivity of DU145 cells to cisplatin fluctuated, and the sensitivity to tumor necrosis factor (TNF)-α was decreased by Bcl-2 overexpression. In a xenograft mouse model, overexpression of Bcl-2 drastically decreased the sensitivity of DU145 cells to cisplatin and TNF-α; however, there was no change in the response to docetaxel. Fluorescent microscopy revealed that Bcl-2-overexpression had no effect on the docetaxel-induced death of DU145 cells, but significantly decreased DU145 cell death induced by cisplatin or TNF-α. Interestingly, docetaxel hardly induced caspase-3/7 activation in control or Bcl-2-overexpressing DU145 cells, but did at a low level in LNCaP cells, another prostate cancer cell line. Moreover, in contrast to LNCaP cells, the reduced viabilities of docetaxel-treated control and Bcl-2-overexpressing DU145 cells were not restored by the addition of either a Bid inhibitor or a panel of pro-apoptotic caspase inhibitors. These findings indicate that the antitumor effects of docetaxel on DU145 cells are independent of both Bcl-2 and pro-apoptotic caspases. PMID:27082738

  5. Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity.

    PubMed

    Fernández-Escobar, Mercedes; Nájera, José Luis; Baldanta, Sara; Rodriguez, Dolores; Way, Michael; Esteban, Mariano; Guerra, Susana

    2015-12-09

    Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity.

  6. (−)-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

  7. Sarcopenia is an independent prognostic factor in male patients with diffuse large B-cell lymphoma.

    PubMed

    Nakamura, Nobuhiko; Hara, Takeshi; Shibata, Yuhei; Matsumoto, Takuro; Nakamura, Hiroshi; Ninomiya, Soranobu; Kito, Yusuke; Kitagawa, Junichi; Kanemura, Nobuhiro; Goto, Naoe; Shiraki, Makoto; Miyazaki, Tatsuhiko; Takeuchi, Tamotsu; Shimizu, Masahito; Tsurumi, Hisashi

    2015-12-01

    Sarcopenia reportedly predicts poor outcomes in elderly patients with diffuse large B-cell lymphoma (DLBCL). However, because previous studies only involved elderly patients, it is difficult to generalize these results to all patients with DLBCL. We retrospectively analyzed 207 patients with DLBCL who received the R-CHOP or R-THP-COP regimen between June 2004 and May 2014. Sarcopenia was measured by the analysis of CT images at the L3 level before treatment. The surface of muscular tissues was selected according to the CT Hounsfield unit. This value was normalized for stature in order to calculate the L3 skeletal muscle index (L3 SMI, cm(2)/m(2)). Median age at diagnosis in the 121 males and 86 females was 67 years (range, 19-86 years). The sex-specific cutoffs for the L3 SMI were determined by receiver operator curve (ROC) analysis. Sarcopenic patients were older than non-sarcopenic patients, with a median age of 70 and 65 years, respectively (p < 0.001). Other International Prognostic Index factors were not significantly different when comparing sarcopenic and non-sarcopenic patients. With a median follow-up of 50.4 months, the 3-year overall survival (OS) was 70 % in the sarcopenic group and 85 % in the non-sarcopenic group (p = 0.0260). In a subgroup analysis by gender, there was a significant difference in the OS when comparing sarcopenic and non-sarcopenic patients in males but not in females (p = 0.0003, p = 0.4440, respectively). Sarcopenia is an independent prognostic factor in male patients with DLBCL. PMID:26385388

  8. 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.

  9. Sarcopenia is an independent prognostic factor in male patients with diffuse large B-cell lymphoma.

    PubMed

    Nakamura, Nobuhiko; Hara, Takeshi; Shibata, Yuhei; Matsumoto, Takuro; Nakamura, Hiroshi; Ninomiya, Soranobu; Kito, Yusuke; Kitagawa, Junichi; Kanemura, Nobuhiro; Goto, Naoe; Shiraki, Makoto; Miyazaki, Tatsuhiko; Takeuchi, Tamotsu; Shimizu, Masahito; Tsurumi, Hisashi

    2015-12-01

    Sarcopenia reportedly predicts poor outcomes in elderly patients with diffuse large B-cell lymphoma (DLBCL). However, because previous studies only involved elderly patients, it is difficult to generalize these results to all patients with DLBCL. We retrospectively analyzed 207 patients with DLBCL who received the R-CHOP or R-THP-COP regimen between June 2004 and May 2014. Sarcopenia was measured by the analysis of CT images at the L3 level before treatment. The surface of muscular tissues was selected according to the CT Hounsfield unit. This value was normalized for stature in order to calculate the L3 skeletal muscle index (L3 SMI, cm(2)/m(2)). Median age at diagnosis in the 121 males and 86 females was 67 years (range, 19-86 years). The sex-specific cutoffs for the L3 SMI were determined by receiver operator curve (ROC) analysis. Sarcopenic patients were older than non-sarcopenic patients, with a median age of 70 and 65 years, respectively (p < 0.001). Other International Prognostic Index factors were not significantly different when comparing sarcopenic and non-sarcopenic patients. With a median follow-up of 50.4 months, the 3-year overall survival (OS) was 70 % in the sarcopenic group and 85 % in the non-sarcopenic group (p = 0.0260). In a subgroup analysis by gender, there was a significant difference in the OS when comparing sarcopenic and non-sarcopenic patients in males but not in females (p = 0.0003, p = 0.4440, respectively). Sarcopenia is an independent prognostic factor in male patients with DLBCL.

  10. Expression of Gli1 correlates with the transition of breast cancer cells to estrogen-independent growth.

    PubMed

    Zhao, Jieying; Chen, Guangchun; Cao, Dongmei; Li, Yidong; Diao, Fei; Cai, Haoyu; Jin, Yiduo; Lu, Jian

    2010-01-01

    The failure of breast cancer treatment is largely due to the development of estrogen independence. Current data illustrate that Hedgehog (Hh) signaling may play an important role in breast cancer development. Here, we show that the expression of the Hh effector protein, Gli1 was significantly higher in estrogen-independent breast cancer cells than in estrogen-dependent cells. Our data showed for the first time that stable expression of Gli1 in ER positive breast cancer cell lines MCF-7 and T47D can induce estrogen-independent proliferation and promote G1/S phase transition, which associated with cyclin-Rb axi. Gli1 can also attenuate the response of proliferation to estrogenic stimulation, which was correlated with down-regulation of expression of ERalpha and PR, as well as down-regulation of transactivation of ERalpha. Our results suggest that up-regulation of Gli1 in breast cancer cells may be one of the mechanisms responsible for developing estrogen independence and this process may be regulated through down-regulation of expression and transactivation of ERalpha.

  11. Apical localisation of crumbs in the boundary cells of the Drosophila hindgut is independent of its canonical interaction partner stardust.

    PubMed

    Kumichel, Alexandra; Knust, Elisabeth

    2014-01-01

    The transmembrane protein Crumbs/Crb is a key regulator of apico-basal epithelial cell polarity, both in Drosophila and in vertebrates. In most cases studied so far, the apical localisation of Drosophila Crumbs depends on the interaction of its C-terminal amino acids with the scaffolding protein Stardust. Consequently, embryos lacking either Crumbs or Stardust develop a very similar phenotype, characterised by the loss of epithelial tissue integrity and cell polarity in many epithelia. An exception is the hindgut, which is not affected by the loss of either gene. The hindgut is a single layered epithelial tube composed of two cell populations, the boundary cells and the principal cells. Here we show that Crumbs localisation in the principal cells depends on Stardust, similarly to other embryonic epithelia. In contrast, localisation of Crumbs in the boundary cells does not require Stardust and is independent of its PDZ domain- and FERM-domain binding motifs. In line with this, the considerable upregulation of Crumbs in boundary cells is not followed by a corresponding upregulation of its canonical binding partners. Our data are the first to suggest a mechanism controlling apical Crumbs localisation, which is independent of its conserved FERM- and PDZ-domain binding motifs.

  12. 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.

  13. A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

    PubMed

    Muz, Barbara; de la Puente, Pilar; Azab, Feda; Luderer, Micah John; King, Justin; Vij, Ravi; Azab, Abdel Kareem

    2016-04-01

    CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

  14. Infection by mink cell focus-forming viruses confers interleukin 2 (IL-2) independence to an IL-2-dependent rat T-cell lymphoma line.

    PubMed Central

    Tsichlis, P N; Bear, S E

    1991-01-01

    The development of T-cell lymphomas in rodents infected with type C retroviruses has been linked to the generation of a class of envelope (env) recombinant viruses called mink cell focus-forming viruses (MCF viruses) in the preleukemic thymus. To determine whether infection by MCF viruses altered the growth phenotype of retrovirus-induced T-cell lymphomas, a Moloney murine leukemia virus-induced interleukin-2 (IL-2)-dependent rat T-cell lymphoma line (4437A) was infected with MCF-247, modified MCF-V33 (mMCF-V33), or NZB-xenotropic (NZB-X) virus. The effects of virus infection on the IL-2 dependence of these cells was examined by cultivating them in the absence of IL-2. After IL-2 withdrawal, the uninfected and NZB-X-infected cells went through a crisis period characterized by massive death. All the independently maintained cultures of MCF- and mMCF-V33-infected cells, on the other hand, became IL-2 independent without a crisis. All the polytropic virus-infected IL-2-independent cultures contained a population of cells that was polyclonal with regard to polytropic provirus integration. Over this polyclonal background each culture produced multiple clones of cells that were selected rapidly after IL-2 withdrawal. Furthermore, the resulting MCF- or mMCF-V33-infected IL-2-independent cells retained the expression of IL-2 receptor. These data show that MCF and mMCF-V33 viruses may alter the growth phenotype of a T-cell lymphoma line and suggest that their effect on cell growth may be due to the direct interaction of the MCF envelope glycoprotein with cellular components, perhaps the IL-2 receptor. Images PMID:2052545

  15. 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

  16. 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.

  17. Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos

    PubMed Central

    OGAWA, Hidehiko; TAKYU, Ryuichi; MORIMOTO, Hiromu; TOEI, Shuntaro; SAKON, Hiroshi; GOTO, Shiori; MORIYA, Shota; KONO, Tomohiro

    2015-01-01

    We previously established trophoblast stem cells from mouse androgenetic embryos (AGTS cells). In this study, to further characterize AGTS cells, we compared cell proliferation activity between trophoblast stem (TS) cells and AGTS cells under fibroblast growth factor 4 (FGF4) signaling. TS cells continued to proliferate and maintained mitotic cell division in the presence of FGF4. After FGF4 deprivation, the cell proliferation stopped, the rate of M-phase cells decreased, and trophoblast giant cells formed. In contrast, some of AGTS cells continued to proliferate, and the rate of M-phase cells did not decrease after FGF4 deprivation, although the other cells differentiated into giant cells. RO3306, an ATP competitor that selectively inhibits CDK1, inhibited the cell proliferation of both TS and AGTS cells. Under RO3306 treatment, cell death was induced in AGTS cells but not in TS cells. These results indicate that RO3306 caused TS cells to shift mitotic cell division to endoreduplication but that some of AGTS cells did not shift to endoreduplication and induced cell death. In conclusion, the paternal genome facilitated the proliferation of trophoblast cells without FGF4 signaling. PMID:26498204

  18. 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.

  19. Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer's patch M cells.

    PubMed

    Sato, S; Kaneto, S; Shibata, N; Takahashi, Y; Okura, H; Yuki, Y; Kunisawa, J; Kiyono, H

    2013-07-01

    Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell-specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B-deficient mice showed reduced expression of most, but not all, other M-cell-specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B-deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B-deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.

  20. 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.

  1. Homozygous mdm2 SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death.

    PubMed

    Rosso, Melissa; Polotskaia, Alla; Bargonetti, Jill

    2015-10-27

    A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma. Surprisingly, following etoposide treatment transcription initiation occurred at the compromised target genes in MANCA and A875 cells similar to the T/T ML-1 cell line. In all cell lines tested there was equally robust recruitment of total and initiated RNA polymerase II (Pol II). We found that knockdown of MDM2 in G/G cells moderately increased expression of subsets of p53 target genes without increasing p53 stability. Importantly, etoposide and actinomycin D treatments increased histone H3K36 trimethylation in T/T, but not G/G cells, suggesting a G/G correlated inhibition of transcription elongation. We therefore tested a chemotherapeutic agent (8-amino-adenosine) that induces p53-independent cell death for higher clinically relevant cytotoxicity. We demonstrated that T/T and G/G mdm2 SNP309 cells were equally sensitive to 8-amino-adenosine induced cell death. In conclusion for cancer cells overexpressing MDM2, targeting MDM2 may be less effective than inducing p53-independent cell death.

  2. Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

    PubMed

    van den Wollenberg, Diana J M; Dautzenberg, Iris J C; van den Hengel, Sanne K; Cramer, Steve J; de Groot, Raoul J; Hoeben, Rob C

    2012-01-01

    Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

  3. The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration.

    PubMed

    Mantuano, Elisabetta; Lam, Michael S; Shibayama, Masataka; Campana, W Marie; Gonias, Steven L

    2015-09-15

    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

  4. Isolation of Reovirus T3D Mutants Capable of Infecting Human Tumor Cells Independent of Junction Adhesion Molecule-A

    PubMed Central

    van den Hengel, Sanne K.; Cramer, Steve J.; de Groot, Raoul J.; Hoeben, Rob C.

    2012-01-01

    Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible. PMID:23110175

  5. 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

  6. Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression

    PubMed Central

    Ward, Kristy K.; Tancioni, Isabelle; Lawson, Christine; Miller, Nichol L.G.; Jean, Christine; Chen, Xiao Lei; Uryu, Sean; Kim, Josephine; Tarin, David; Stupack, Dwayne G.; Plaxe, Steven C.; Schlaepfer, David D.

    2013-01-01

    Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the periovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer. PMID:23275034

  7. 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.

  8. The imidazoline compound RX871024 promotes insulinoma cell death independent of AMP-activated protein kinase inhibition.

    PubMed

    Zaitseva, Irina I; Zaitsev, Sergei V; Berggren, Per-Olof

    2016-08-01

    We have previously shown that the insulinotropic imidazoline compound RX871024 induces death of insulinoma MIN6 cells, an effect involving stimulation of c-Jun N-terminal kinase (JNK) and caspase 3. It has also been reported that AMP-activated protein kinase (AMPK) activates JNK and induces β-cell death. Here we show that RX871024, but not another insulinotropic imidazoline compound (BL11282), suppressed AMPK activity in MIN6 cells. The inhibitory effect of RX871024 on AMPK was supported by the observation that the imidazoline induced lipid droplet formation in the cytoplasm of MIN6 cells. This reflects stimulation of anabolic pathways and inhibition of catabolic pathways in the cell that happen under conditions when AMPK is inhibited. Activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) elevated basal and cytokine-induced death in primary β-cells and in insulinoma MIN6 cells. RX871024 aggravated AICAR-induced insulinoma MIN6 cell death regardless of the presence of pro-inflammatory cytokines. The specific cytotoxic effect of imidazoline compound RX871024 on insulinoma cell death but not primary β-cell death is independent of its action on AMPK and may suggest the possibility of using this type of compound in the treatment of insulinomas. PMID:27221730

  9. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

    PubMed Central

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

  10. Activation of CD1d-independent NK1.1+ T cells in the large intestine by Lactobacilli.

    PubMed

    Takahashi, Satoshi; Kawamura, Toshihiko; Kanda, Yasuhiro; Taniguchi, Tomoyo; Nishizawa, Tetsuro; Iiai, Tsuneo; Hatakeyama, Katsuyoshi; Abo, Toru

    2006-01-15

    Among digestive organs, the liver and the large intestine are abundant in T cells expressing NK1.1. NK1.1+ T cells in the liver are mostly CD1d-dependent whereas those in the large intestine are CD1d-independent. In this study, we investigated the effects of Lactobacilli on NK1.1+ T cells in the digestive organs of mice. C57BL/6 mice were orally given a dietary supplement prepared from mixed cultures of eight strains of Lactobacilli. Oral administration of Lactobacilli to mice resulted in the selective expansion of NK1.1+ T cells in the large intestine. These colon NK1.1+ T cells activated by Lactobacilli were found to express IFN-gamma mRNA. The level of IFN-gamma in the serum was also elevated by the administration of Lactobacilli. Our results suggest that Lactobacilli selectively activate CD1d-independent NK1.1+ T cells in the large intestine to produce IFN-gamma and therefore modulate Th1 immune responses.

  11. Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax®23.

    PubMed

    Roth, Alena; Glaesener, Stephanie; Schütz, Katharina; Meyer-Bahlburg, Almut

    2016-01-01

    Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA+ but not IgM+ or IgG+ memory B cells and a predominant generation of PnPS-specific IgA+ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax®23. Our results demonstrate that a characteristic TI response induced by Pneumovax®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.

  12. Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax®23

    PubMed Central

    Roth, Alena; Glaesener, Stephanie; Schütz, Katharina; Meyer-Bahlburg, Almut

    2016-01-01

    Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA+ but not IgM+ or IgG+ memory B cells and a predominant generation of PnPS-specific IgA+ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax®23. Our results demonstrate that a characteristic TI response induced by Pneumovax®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response. PMID:27031098

  13. IFT88 plays a cilia- and PCP-independent role in controlling oriented cell divisions during vertebrate embryonic development.

    PubMed

    Borovina, Antonia; Ciruna, Brian

    2013-10-17

    The role for cilia in establishing planar cell polarity (PCP) is contentious. Although knockdown of genes known to function in ciliogenesis has been reported to cause PCP-related morphogenesis defects in zebrafish, genetic mutations affecting intraflagellar transport (IFT) do not show PCP phenotypes despite the requirement for IFT in cilia formation. This discrepancy has been attributed to off-target effects of antisense morpholino oligonucleotide (MO) injection, confounding maternal effects in zygotic mutant embryos, or an inability to distinguish between cilia-dependent versus cilia-independent protein functions. To determine the role of cilia in PCP, we generated maternal + zygotic IFT88 (MZift88) mutant zebrafish embryos, which never form cilia. We clearly demonstrate that cilia are not required to establish PCP. Rather, IFT88 plays a cilia-independent role in controlling oriented cell divisions at gastrulation and neurulation. Our results have important implications for the interpretation of cilia gene function in normal development and in disease.

  14. ROCK1 and ROCK2 are required for non-small cell lung cancer anchorage-independent growth and invasion.

    PubMed

    Vigil, Dominico; Kim, Tai Young; Plachco, Ana; Garton, Andrew J; Castaldo, Linda; Pachter, Jonathan A; Dong, Hanqing; Chen, Xin; Tokar, Brianna; Campbell, Sharon L; Der, Channing J

    2012-10-15

    Evidence is emerging that the closely related ROCK1 and ROCK2 serine/threonine kinases support the invasive and metastatic growth of a spectrum of human cancer types. Therefore, inhibitors of ROCK are under preclinical development. However, a key step in their development involves the identification of genetic biomarkers that will predict ROCK inhibitor antitumor activity. One identified mechanism for ROCK activation in cancer involves the loss of function of the DLC1 tumor suppressor gene, which encodes a GTPase activating protein (RhoGAP) for the RhoA and RhoC small GTPases. DLC-1 loss may lead to hyperactivation of RhoA/C and its downstream effectors, the ROCK kinases. We therefore determined whether loss of DLC-1 protein expression identifies non-small cell lung carcinoma (NSCLC) cell lines whose growth and invasion phenotypes are sensitive to ROCK inhibition. We identified and characterized a novel small molecule pharmacologic inhibitor of ROCK and additionally applied genetic approaches to impair ROCK1 and/or ROCK2 activity, and we determined that although NSCLC anchorage-dependent growth was ROCK-independent, both anchorage-independent growth and Matrigel invasion were ROCK-dependent. However, loss of DLC-1 expression did not correlate with ROCK activation or with OXA-06 sensitivity. Unexpectedly, suppression of ROCK1 or ROCK2 expression alone was sufficient to impair anchorage-independent growth, supporting their nonoverlapping roles in oncogenesis. Mechanistically, the block in anchorage-independent growth was associated with accumulation of cells in the G(0)-G(1) phase of the cell cycle, but not increased anoikis. We conclude that ROCK may be a useful therapeutic target for NSCLC.

  15. KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms.

    PubMed

    Chen, Min-Bin; Zhou, Zhen-Tao; Yang, Lan; Wei, Mu-Xin; Tang, Min; Ruan, Ting-Yan; Xu, Jun-Ying; Zhou, Xiao-Zhong; Chen, Gang; Lu, Pei-Hua

    2016-03-29

    Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.

  16. Identification of an anabolic selective androgen receptor modulator that actively induces death of androgen-independent prostate cancer cells.

    PubMed

    Schmidt, Azriel; Meissner, Robert S; Gentile, Michael A; Chisamore, Michael J; Opas, Evan E; Scafonas, Angela; Cusick, Tara E; Gambone, Carlo; Pennypacker, Brenda; Hodor, Paul; Perkins, James J; Bai, Chang; Ferraro, Damien; Bettoun, David J; Wilkinson, Hilary A; Alves, Stephen E; Flores, Osvaldo; Ray, William J

    2014-09-01

    Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5α-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens. PMID:24565564

  17. NJK14013, a novel synthetic estrogen receptor-α agonist, exhibits estrogen receptor-independent, tumor cell-specific cytotoxicity.

    PubMed

    Kim, Hye-In; Kim, Taelim; Kim, Ji-Eun; Lee, Jun; Heo, Jinyuk; Lee, Na-Rae; Kim, Nam-Jung; Inn, Kyung-Soo

    2015-07-01

    Estrogens act through interactions with estrogen receptors (ERs) to play diverse roles in various pathophysiological conditions. A number of synthetic selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, have been developed and used to treat ER-related diseases, including breast cancer and osteoporosis. Here, we identified a novel compound, bis(4-hydroxyphenyl)methanone-O-isopentyl oxime, designated NJK14013, as an ER agonist. NJK14013 activated ER-dependent transcription in a concentration-dependent manner, while suppressing androgen receptor-dependent transcriptional activity. It induced the activation-related phosphorylation of ER and enhanced the transcription of growth regulation by estrogen in breast cancer 1 (GREB1), further supporting its ER-stimulating activity. NJK14013 exerted anti-proliferative effects on various cancer cell lines, including an ER-negative breast cancer cell line, suggesting that it is capable of suppressing the growth of cancer cells independent of its ER-modulating activity. In addition, NJK14013 treatment resulted in significant apoptotic death of MCF7 and Ishikawa cancer cells, but did not induce apoptosis in non-cancer human umbilical vein endothelial cells. Collectively, our findings demonstrate that NJK14013 is a novel SERM that can activate ER-mediated transcription in MCF7 cells and suppress the proliferation of various cancer cells, including breast cancer cells and endometrial cancer cells. These results suggest that NJK14013 has potential as a novel SERM for anticancer or hormone-replacement therapy with reduced risk of carcinogenesis.

  18. Psoralidin, An Herbal Molecule Inhibits PI3K Mediated Akt Signaling In Androgen Independent Prostate Cancer (AIPC) Cells

    PubMed Central

    Kumar, Raj; Srinivasan, Sowmyalakshmi; Koduru, Srinivas; Pahari, Pallab; Rohr, Jürgen; Kyprianou, Natasha; Damodaran, Chendil

    2008-01-01

    The protein kinase Akt plays an important role in cell proliferation and survival in many cancers, including prostate cancer. Due to its kinase activity, it serves as a molecular conduit for inhibiting apoptosis and promoting angiogenesis in most cell types. In most of the prostate tumors, Akt signaling is constitutively activated due to the deletion or mutation of the tumor suppressor PTEN, which negatively regulates PI3K through lipid phosphatase activity. Recently, we identified a natural compound, psoralidin, which inhibits Akt phosphorylation and its consequent activation in androgen independent prostate cancer cells (AIPC). Furthermore, ectopic expression of Akt renders AIPC cells resistant chemotherapy; however, psoralidin overcomes Akt-mediated resistance and induces apoptosis in AIPC cells. While dissecting the molecular events, both upstream and downstream of Akt, we found that psoralidin inhibits PI3 kinase activation and transcriptionally represses the activation of NF-κB and its target genes (Bcl-2, Survivin, and Bcl-xL, etc.), which results in the inhibition of cell viability and induction of apoptosis in PC-3 and DU-145 cells. Interestingly, psoralidin selectively targets cancer cells, without causing any toxicity to normal prostate epithelial cells. In vivo xenograft assays substantiate these in vitro findings, and show psoralidin inhibits prostate tumor growth in nude mice. Our findings are of therapeutic significance in the management of prostate cancer patients with advanced or metastatic disease, as they provide new directions for the development of a phyotochemical-based platform for prevention and treatment strategies for AIPC. PMID:19223576

  19. Depression of p53-independent Akt survival signals after high-LET radiation in mutated p53 cells

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo; Takahashi, Akihisa; Nakagawa, Yosuke

    Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses the activities of serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signals were analyzed with Western blotting analysis 1 h, 2 h, 3 h and 6 h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis.Akt-related protein levels were decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G _{2}/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and depresses cell growth by suppressing Akt-related signals, even in the mp53 cells.

  20. Type I interferons activate apoptosis in a Jurkat cell variant by caspase-dependent and independent mechanisms

    PubMed Central

    Gamero, Ana M.; Potla, Ramesh; Sakamoto, Shuji; Baker, Darren P.; Abraham, Robert; Larner, Andrew C.

    2007-01-01

    Although the antiviral actions of interferons (IFNs) are observed in most types of cells, the antiproliferative effects of IFNα/β are variable as are the mechanisms of growth inhibition that may or may not be due to the induction of apoptosis. To understand more about the mechanisms that are responsible for IFNα/β -stimulated apoptosis, we have characterized a new human Jurkat T cell variant named H123 where IFNα activates programmed cell death (PCD). No differences in IFNα -stimulated, Stat-dependent gene expression were detected between H123 cells and the parental Jurkat cells, which are growth inhibited, but do not undergo apoptosis with IFNα. Although IFNα stimulates the activity of both caspase 3 and 9 in H123 cells, the general caspase inhibitor Z-VAD only partially reverses the apoptotic actions of IFNα. Induction of apoptosis by IFNα occurs through a mitochondrial-dependent pathway in H123 cells, as demonstrated by the release of cytochrome C from the mitochondria. Furthermore, IFNα treatment of H123 cells stimulates the release of the serine protease HtrA2/Omi from the mitochondria, suggesting that it plays a role in the apoptotic actions of this cytokine. These results provide evidence for a novel type 1 IFN-mediated pathway that regulates apoptosis of T cells through a mitochondrial-dependent and caspase-dependent and independent pathway. PMID:16337360

  1. Macrophage-mediated chronic lymphocytic leukemia cell survival is independent of APRIL signaling.

    PubMed

    van Attekum, Mha; Terpstra, S; Reinen, E; Kater, A P; Eldering, E

    2016-01-01

    Survival of chronic lymphocytic leukemia (CLL) cells is mainly driven by interactions within the lymph node (LN) microenvironment with bystander cells such as T cells or cells from the monocytic lineage. Although the survival effect by T cells is largely governed by the TNFR ligand family member CD40L, the exact mechanism of monocyte-derived cell-induced survival is not known. An important role has been attributed to the TNFR ligand, a proliferation-inducing ligand (APRIL), although the exact mechanism remained unclear. Since we detected that APRIL was expressed by CD68+ cells in CLL LN, we addressed its relevance in various aspects of CLL biology, using a novel APRIL overexpressing co-culture system, recombinant APRIL, and APRIL reporter cells. Unexpectedly, we found, that in these various systems, APRIL had no effect on survival of CLL cells, and activation of NF-κB was not enhanced on APRIL stimulation. Moreover, APRIL stity mulation did not affect CLL proliferation, neither as single stimulus nor in combination with known CLL proliferation stimuli. Furthermore, the survival effect conveyed by macrophages to CLL cells was not affected by transmembrane activator and CAML interactor-Fc, an APRIL decoy receptor. We conclude that the direct role ascribed to APRIL in CLL cell survival might be overestimated due to application of supraphysiological levels of recombinant APRIL. PMID:27551513

  2. Bone cell-independent benefits of raloxifene on the skeleton: a novel mechanism for improving bone material properties.

    PubMed

    Gallant, Maxime A; Brown, Drew M; Hammond, Max; Wallace, Joseph M; Du, Jiang; Deymier-Black, Alix C; Almer, Jonathan D; Stock, Stuart R; Allen, Matthew R; Burr, David B

    2014-04-01

    Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle X-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.

  3. Bone cell-independent benefits of raloxifene on the skeleton: A novel mechanism for improving bone material properties

    PubMed Central

    Gallant, Maxime A.; Brown, Drew M.; Hammond, Max; Wallace, Joseph M.; Du, Jiang; Deymier-Black, Alix C.; Almer, Jonathan D.; Stock, Stuart R.; Allen, Matthew R.; Burr, David B.

    2014-01-01

    Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (−OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle x-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength. PMID:24468719

  4. Kaempferol inhibits angiogenesis and VEGF expression through both HIF dependent and independent pathways in human ovarian cancer cells.

    PubMed

    Luo, Haitao; Rankin, Gary O; Liu, Lingzhi; Daddysman, Matthew K; Jiang, Bing-Hua; Chen, Yi Charlie

    2009-01-01

    Ovarian cancer is 1 of the most significant malignancies in the Western world, and the antiangiogenesis strategy has been postulated for prevention and treatment of ovarian cancers. Kaempferol is a natural flavonoid present in many fruits and vegetables. The antiangiogenesis potential of kaempferol and its underlying mechanisms were investigated in two ovarian cancer cell lines, OVCAR-3 and A2780/CP70. Kaempferol mildly inhibits cell viability but significantly reduces VEGF gene expression at mRNA and protein levels in both ovarian cancer cell lines. In chorioallantoic membranes of chicken embryos, kaempferol significantly inhibits OVCAR-3-induced angiogenesis and tumor growth. HIF-1alpha, a regulator of VEGF, is downregulated by kaempferol treatment in both ovarian cancer cell lines. Kaempferol also represses AKT phosphorylation dose dependently at 5 to 20 muM concentrations. ESRRA is a HIF-independent VEGF regulator, and it is also downregulated by kaempferol in a dose-dependent manner. Overall, this study demonstrated that kaempferol is low in cytotoxicity but inhibits angiogenesis and VEGF expression in human ovarian cancer cells through both HIF-dependent (Akt/HIF) and HIF-independent (ESRRA) pathways and deserves further studies for possible application in angio prevention and treatment of ovarian cancers.

  5. PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

    PubMed Central

    Errafiy, Rajaa; Aguado, Carmen; Ghislat, Ghita; Esteve, Juan M.; Gil, Anabel; Loutfi, Mohammed; Knecht, Erwin

    2013-01-01

    Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation. PMID:24349488

  6. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

    PubMed Central

    Chu, D H; Spits, H; Peyron, J F; Rowley, R B; Bolen, J B; Weiss, A

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling. Images PMID:8947048

  7. Arsenic-induced cancer cell phenotype in human breast epithelia is estrogen receptor-independent but involves aromatase activation

    PubMed Central

    Xu, Yuanyuan; Tokar, Erik J.; Waalkes, Michael P.

    2013-01-01

    Accumulating data suggest arsenic may be an endocrine disruptor, and tentatively linked to breast cancer by some studies. Therefore, we tested the effects of chronic inorganic arsenic exposure on the normal, estrogen receptor (ER)-negative breast epithelial cell line, MCF-10A. Cells were chronically exposed to a low-level arsenite (500 nM) for up to 24 weeks. Markers of cancer cell phenotype and expression of critical genes relevant to breast cancer or stem cells (SCs) were examined. After 24 weeks, chronic arsenic-exposed breast epithelial (CABE) cells showed increases in secreted MMP activity, colony formation, invasion and proliferation rate, indicating an acquired cancer cell phenotype. These CABE cells presented with basal-like breast cancer characteristics, including ER-α, HER-2 and progesterone receptor negativity, and overexpression of K5 and p63. Putative CD44+/CD24−/low breast SCs were increased to 80% over control in CABE cells. CABE cells also formed multilayer cell mounds, indicative of loss of contact inhibition. These mounds showed high levels of K5 and p63 indicating the potential presence of CSCs. Epithelial-to-mesenchymal transition occurred during arsenic exposure.. Overexpression of aromatase, a key rate-limiting enzyme in estrogen synthesis, occurred with arsenic starting early on in exposure. Levels of 17β-estradiol increased in CABE cells and their conditioned medium. The aromatase inhibitor, letrozole abolished arsenic-induced increases of 17β-estradiol production, and reversed cancer cell phenotype. Thus, chronic arsenic exposure drive human breast epithelia into a cancer cell phenotype with an apparent overabundance of putative CSCs. Arsenic appears to transform breast epithelia through overexpression of aromatase, thereby activating oncogenic processes independent of ER. PMID:24068038

  8. Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

    PubMed Central

    Grossenbacher, Steven K.; Hsiao, Hui-Hua; Zamora, Anthony E.; Mirsoian, Annie; Koehn, Brent; Blazar, Bruce R.; Weiss, Jonathan M.; Wiltrout, Robert H.; Sckisel, Gail D.; Murphy, William J.

    2014-01-01

    We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8+CD44high) T cells displaying a CD25−NKG2D+ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion. PMID:25119341

  9. Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

    PubMed Central

    LeBlanc, Isabelle; Blot, Vincent; Bouchaert, Isabelle; Salamero, Jean; Goud, Bruno; Rosenberg, Arielle R.; Dokhélar, Marie-Christine

    2002-01-01

    Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system. PMID:11752179

  10. Regions within a single epidermal cell of Drosophila can be planar polarised independently

    PubMed Central

    Rovira, Miguel; Saavedra, Pedro; Casal, José; Lawrence, Peter A

    2015-01-01

    Planar cell polarity (PCP), the coordinated and consistent orientation of cells in the plane of epithelial sheets, is a fundamental and conserved property of animals and plants. Up to now, the smallest unit expressing PCP has been considered to be an entire single cell. We report that, in the larval epidermis of Drosophila, different subdomains of one cell can have opposite polarities. In larvae, PCP is driven by the Dachsous/Fat system; we show that the polarity of a subdomain within one cell is its response to levels of Dachsous/Fat in the membranes of contacting cells. During larval development, cells rearrange (Saavedra et al., 2014) and when two subdomains of a single cell have different types of neighbouring cells, then these subdomains can become polarised in opposite directions. We conclude that polarisation depends on a local comparison of the amounts of Dachsous and Fat within opposing regions of a cell's membrane. DOI: http://dx.doi.org/10.7554/eLife.06303.001 PMID:25671242

  11. TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors

    PubMed Central

    Doorduijn, Elien M.; Sluijter, Marjolein; Querido, Bianca J.; Oliveira, Cláudia C.; Achour, Adnane; Ossendorp, Ferry; van der Burg, Sjoerd H.; van Hall, Thorbald

    2016-01-01

    Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies. PMID:26784543

  12. TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors.

    PubMed

    Doorduijn, Elien M; Sluijter, Marjolein; Querido, Bianca J; Oliveira, Cláudia C; Achour, Adnane; Ossendorp, Ferry; van der Burg, Sjoerd H; van Hall, Thorbald

    2016-02-01

    Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the "self" TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies. PMID:26784543

  13. Understanding the Regulatory Roles of Natural Killer T Cells in Rheumatoid Arthritis: T Helper Cell Differentiation Dependent or Independent?

    PubMed

    Chen, J; Yang, J; Qiao, Y; Li, X

    2016-10-01

    Rheumatoid arthritis (RA) is the most common chronic systemic autoimmune disease. This disease is thought to be caused by pathogenic T cells. Th1, Th2, Th17 and Treg cells have been implicated in the pathogenesis of RA. These Th cells differentiate from CD4+ T cells primarily due to the effects of cytokines. Natural killer T (NKT) cells are a distinct subset of lymphocytes that can rapidly secrete massive amount of cytokines, including IL-2, IL-4, IL-12 and IFN-γ. Numerous studies showed that NKT cells can influence the differentiation of CD4+ T cells via cytokines in vitro. These findings suggest that NKT cells play an important role in RA by polarizing Th1, Th2, Th17 and Treg cells. In view of the complexity of RA, we discussed whether NKT cells really influence the development of RA through regulating the differentiation of Th cells. PMID:27384545

  14. Salmonella translocates across an in vitro M cell model independently of SPI-1 and SPI-2.

    PubMed

    Martinez-Argudo, Isabel; Jepson, Mark A

    2008-12-01

    We have used an in vitro model of intestinal M cells to examine the mechanisms by which Salmonella enterica translocates across these specialized cells, which constitute a primary site of infection of the mammalian host. S. enterica can invade cultured cells by deploying a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI-1) to translocate effector proteins into the host cell cytoplasm that trigger cellular responses, including prominent cytoskeletal rearrangements. After Salmonella enters the host cell, a second TTSS encoded in SPI-2 modulates intracellular trafficking and enables the bacteria to replicate within a modified vacuolar compartment. Within the host intestine, specialized antigen-sampling M cells, which reside in the epithelium overlying lymphoid tissues in the gut, are a preferential site of Salmonella invasion. The mechanisms of infection of M cells remain poorly defined and it is not known whether either SPI-1 or SPI-2 is required for infection of these cells. To address these questions we have employed an in vitro M cell model involving co-culture of polarized Caco-2 intestinal epithelial cells with Raji B cells. S. enterica serovar Typhimurium translocated across Caco-2/Raji co-cultures to a much greater extent than they cross native Caco-2 cell monolayers. Salmonella translocation was greatly reduced by heat treatment or fixation, suggesting that processes distinct from the sampling of inert particles are the main determinants of bacterial translocation. Translocation across both mono-cultured and co-cultured Caco-2 cells was partially inhibited by treatment with the dynamin inhibitor dynasore, but resistant to EIPA, an inhibitor of macropinocytosis. There was no difference between the abilities of wild-type Salmonella Typhimurium and mutants lacking multiple SPI-1 effectors to translocate across the M cell model, although the SPI-1 effector mutants were somewhat attenuated for translocation across native Caco

  15. Cellular Metabolism and Dose Reveal Carnitine-Dependent and -Independent Mechanisms of Butyrate Oxidation in Colorectal Cancer Cells.

    PubMed

    Han, Anna; Bennett, Natalie; MacDonald, Amber; Johnstone, Megan; Whelan, Jay; Donohoe, Dallas R

    2016-08-01

    Dietary fiber has been suggested to suppress colorectal cancer development, although the mechanisms contributing to this beneficial effect remain elusive. Butyrate, a fermentation product of fiber, has been shown to have anti-proliferative and pro-apoptotic effects on colorectal cancer cells. The metabolic fate of butyrate in the cell is important in determining whether, it acts as an HDAC inhibitor or is consumed as a short-chain fatty acid. Non-cancerous colonocytes utilize butyrate as the primary energy source whereas cancerous colonocytes increase glucose utilization through the Warburg effect. In this study, we show that butyrate oxidation is decreased in cancerous colonocytes compared to non-cancerous colonocytes. We demonstrate that colorectal cancer cells utilize both a carnitine-dependent and carnitine-independent mechanism that contributes to butyrate oxidation. The carnitine-dependent mechanism is contingent on butyrate concentration. Knockdown of CPT1A in colorectal cancer cells abolishes butyrate oxidation. In terms of selectivity, the carnitine-dependent mechanism only regulated butyrate oxidation, as acetate and propionate oxidation were carnitine-independent. Carnitine decreased the action of butyrate as an HDAC inhibitor and suppressed induction of H3 acetylation by butyrate in colorectal cancer cells. Thus, diminished oxidation of butyrate is associated with decreased HDAC inhibition and histone acetylation. In relation to the mechanism, we find that dichloroacetate, which decreases phosphorylation of pyruvate dehydrogenase, increased butyrate oxidation and that this effect was carnitine-dependent. In conclusion, these data suggest that colorectal cancer cells decrease butyrate oxidation through inhibition of pyruvate dehydrogenase, which is carnitine-dependent, and provide insight into why butyrate shows selective effects toward colorectal cancer cells. J. Cell. Physiol. 231: 1804-1813, 2016. © 2015 Wiley Periodicals, Inc.

  16. Interactions, functions and independence of plasma membrane STIM1 and TRPC1 in vascular smooth muscle cells

    PubMed Central

    Li, Jing; Sukumar, Piruthivi; Milligan, Carol J; Kumar, Bhaskar; Ma, Zhi-yong; Munsch, Christopher M; Jiang, Lin-Hua; Porter, Karen E; Beech, David J

    2009-01-01

    Stromal interaction molecule 1 (STIM1) is a predicted single membrane-spanning protein involved in store-operated calcium-entry and interacting with ion channels including TRPC1. Here we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a non-selective cationic current in response to store-depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA (siRNA). Calcium entry evoked by store-depletion was partially inhibited by STIM1 siRNA, where as calcium-release was unaffected. STIM1 siRNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N-terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store-depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1 and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localisation studies revealed TRPC1 alone as well as with STIM1. The data suggest a complex situation where there is plasma membrane-spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current, but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation. PMID:18802022

  17. Lipid IVa incompletely activates MyD88-independent Toll-like receptor 4 signaling in mouse macrophage cell lines.

    PubMed

    Ogura, Norihiko; Muroi, Masashi; Sugiura, Yuka; Tanamoto, Ken-ichi

    2013-04-01

    We investigated the difference in the effect of synthetic lipid A compounds on MyD88-dependent and -independent Toll-like receptor 4 (TLR4) signaling in mouse macrophage cells. At higher concentrations, Escherichia coli-type hexa-acylated lipid A 506, Salmonella-type hepta-acylated lipid A 516, the lipid A precursor lipid IVa and monophosphoryl lipid A induced similar levels of production of the MyD88-dependent cytokine IL-1β although their potencies varied, whereas the maximum production of the MyD88-independent cytokine RANTES induced by lipid IVa was less than 50% that of other lipid A compounds. A maximum level of NF-κB activation, which is involved in IL-1β gene transcription, was also induced to a similar level by these four lipid A compounds, while the maximum level of IFN-β promoter activity induced during MyD88-independent signaling was also less than 50% for lipid IVa stimulation compared with other lipid A compounds. Early IκBα phosphorylation activated by MyD88-dependent signaling was similarly induced by 506 and lipid IVa, whereas lipid IVa barely stimulated the phosphorylation of IRF3, a MyD88-independent transcription factor, although efficient phosphorylation was observed with 506 stimulation. These results indicate that lipid IVa has limited activity toward MyD88-independent signaling of TLR4, in macrophage cell lines, despite having efficient activity in the MyD88-dependent pathway.

  18. CD4(+) T-Cell-Independent Secondary Immune Responses to Pneumocystis Pneumonia.

    PubMed

    de la Rua, Nicholas M; Samuelson, Derrick R; Charles, Tysheena P; Welsh, David A; Shellito, Judd E

    2016-01-01

    Pneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4(+) T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4(+) T-cells is mediated by a robust memory humoral response, CD8(+) T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8(+) T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8(+) T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4(+) T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8(+) T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8(+) T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8(+) T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ(+) CD8(+) T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8(+) T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG. PMID:27242785

  19. CD4+ T-Cell-Independent Secondary Immune Responses to Pneumocystis Pneumonia

    PubMed Central

    de la Rua, Nicholas M.; Samuelson, Derrick R.; Charles, Tysheena P.; Welsh, David A.; Shellito, Judd E.

    2016-01-01

    Pneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4+ T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4+ T-cells is mediated by a robust memory humoral response, CD8+ T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8+ T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8+ T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4+ T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8+ T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8+ T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8+ T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ+ CD8+ T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8+ T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG. PMID:27242785

  20. Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: A novel antiangiogenic therapy

    SciTech Connect

    Kamisasanuki, Taro; Tokushige, Saori; Terasaki, Hiroto; Khai, Ngin Cin; Wang, Yuqing; Sakamoto, Taiji; Kosai, Ken-ichiro

    2011-09-16

    Highlights: {yields} CD9 plays stimulus-independent roles in angiogenesis in vitro and in vivo. {yields} Targeting CD9 expression is effective in an angiogenic disease model. {yields} Targeting CD9 expression predominantly affects activated endothelial cells. {yields} CD9 is involved in endothelial cell proliferation, but not survival. {yields} CD9 is part of angiogenic machinery in endothelial cells during angiogenesis. -- Abstract: The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent

  1. S6K1 controls pancreatic β cell size independently of intrauterine growth restriction.

    PubMed

    Um, Sung Hee; Sticker-Jantscheff, Melanie; Chau, Gia Cac; Vintersten, Kristina; Mueller, Matthias; Gangloff, Yann-Gael; Adams, Ralf H; Spetz, Jean-Francois; Elghazi, Lynda; Pfluger, Paul T; Pende, Mario; Bernal-Mizrachi, Ernesto; Tauler, Albert; Tschöp, Matthias H; Thomas, George; Kozma, Sara C

    2015-07-01

    Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of β cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased β cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic β cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore β cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic β cell lesion. Consistent with this hypothesis, reexpression of S6K1 in β cells of S6K1-/- mice restored embryonic β cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic β cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced β cell growth and eventual development of T2DM later in life.

  2. S6K1 controls pancreatic β cell size independently of intrauterine growth restriction

    PubMed Central

    Um, Sung Hee; Sticker-Jantscheff, Melanie; Chau, Gia Cac; Vintersten, Kristina; Mueller, Matthias; Gangloff, Yann-Gael; Adams, Ralf H.; Spetz, Jean-Francois; Elghazi, Lynda; Pfluger, Paul T.; Pende, Mario; Bernal-Mizrachi, Ernesto; Tauler, Albert; Tschöp, Matthias H.; Thomas, George; Kozma, Sara C.

    2015-01-01

    Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of β cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased β cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic β cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore β cell size or insulin levels in S6K1–/– embryos, suggesting that loss of S6K1 leads to an intrinsic β cell lesion. Consistent with this hypothesis, reexpression of S6K1 in β cells of S6K1–/– mice restored embryonic β cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic β cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced β cell growth and eventual development of T2DM later in life. PMID:26075820

  3. Antigen-dependent and -independent contributions to primary memory CD8 T cell activation and protection following infection.

    PubMed

    Martin, Matthew D; Badovinac, Vladimir P

    2015-12-10

    Memory CD8 T-cell activation, including expression of IFN-γ and granzymeB, can be induced by antigen (Ag)-dependent signals through the T-cell-receptor, or by pathogen-derived inflammatory cytokines in an Ag-independent manner. Recent studies have come to conflicting results regarding the contributions of Ag and/or inflammation to memory CD8 T-cell activation. Additionally, research has indicated that inflammation-driven CD8 T-cell responses during un-related infections (bystander activation) have the potential to provide protection, but whether protection occurs in immuno-competent hosts is unclear. To investigate these questions, we examined activation of virus-specific memory CD8 T-cells following infection with L. monocytogenes either expressing or not cognate Ag. We show that Ag and inflammation act synergistically in vitro to induce memory activation. In vivo, we found that when memory CD8 T-cells significantly contribute to clearance of infection, early activation and continued responses by these cells are enhanced by cognate Ag recognition. Mechanistically, we show that bystander responses by memory are dependent upon the dose of infection and the amount of inflammation elicited following infection and are able to provide protection in IFN-γ deficient mice, but not in immuno-competent hosts. The data elucidate the requirements for memory CD8 T-cell activation and the protective role of bystander responses.

  4. Antigen-dependent and –independent contributions to primary memory CD8 T cell activation and protection following infection

    PubMed Central

    Martin, Matthew D.; Badovinac, Vladimir P.

    2015-01-01

    Memory CD8 T-cell activation, including expression of IFN-γ and granzymeB, can be induced by antigen (Ag)-dependent signals through the T-cell-receptor, or by pathogen-derived inflammatory cytokines in an Ag-independent manner. Recent studies have come to conflicting results regarding the contributions of Ag and/or inflammation to memory CD8 T-cell activation. Additionally, research has indicated that inflammation-driven CD8 T-cell responses during un-related infections (bystander activation) have the potential to provide protection, but whether protection occurs in immuno-competent hosts is unclear. To investigate these questions, we examined activation of virus-specific memory CD8 T-cells following infection with L. monocytogenes either expressing or not cognate Ag. We show that Ag and inflammation act synergistically in vitro to induce memory activation. In vivo, we found that when memory CD8 T-cells significantly contribute to clearance of infection, early activation and continued responses by these cells are enhanced by cognate Ag recognition. Mechanistically, we show that bystander responses by memory are dependent upon the dose of infection and the amount of inflammation elicited following infection and are able to provide protection in IFN-γ deficient mice, but not in immuno-competent hosts. The data elucidate the requirements for memory CD8 T-cell activation and the protective role of bystander responses. PMID:26658291

  5. Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.

    PubMed

    Luis, Nuno Miguel; Morey, Lluis; Mejetta, Stefania; Pascual, Gloria; Janich, Peggy; Kuebler, Bernd; Cozutto, Luca; Roma, Guglielmo; Nascimento, Elisabete; Frye, Michaela; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-09-01

    Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. PMID:21885019

  6. Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

    SciTech Connect

    Takaoka, Yuki; Kawamoto, Seiji; Katayama, Akiko; Nakano, Toshiaki; Yamanaka, Yasushi; Takahashi, Miki; Shimada, Yayoi; Chiang, Kuei-Chen; Ohmori, Naoya; Aki, Tsunehiro; Goto, Takeshi; Sato, Shuji; Goto, Shigeru; Chen, Chao-Long; Ono, Kazuhisa

    2013-02-08

    Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4{sup +}Foxp3{sup +} Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

  7. Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel

    PubMed Central

    Desarnaud, Frank; Geck, Peter; Parkin, Christopher; Carpinito, Gino

    2011-01-01

    The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation. PMID:21057205

  8. Piperine, a Bioactive Component of Pepper Spice Exerts Therapeutic Effects on Androgen Dependent and Androgen Independent Prostate Cancer Cells

    PubMed Central

    Dakshinamoorthy, Gajalakshmi; Bartik, Mary Margaret; Johnson, Gary Leon; Webb, Brian; Zheng, Guoxing; Chen, Aoshuang; Kalyanasundaram, Ramaswamy; Munirathinam, Gnanasekar

    2013-01-01

    Prostate cancer is the most common solid malignancy in men, with 32,000 deaths annually. Piperine, a major alkaloid constituent of black pepper, has previously been reported to have anti-cancer activity in variety of cancer cell lines. The effect of piperine against prostate cancer is not currently known. Therefore, in this study, we investigated the anti-tumor mechanisms of piperine on androgen dependent and androgen independent prostate cancer cells. Here, we show that piperine inhibited the proliferation of LNCaP, PC-3, 22RV1 and DU-145 prostate cancer cells in a dose dependent manner. Furthermore, Annexin-V staining demonstrated that piperine treatment induced apoptosis in hormone dependent prostate cancer cells (LNCaP). Using global caspase activation assay, we show that piperine-induced apoptosis resulted in caspase activation in LNCaP and PC-3 cells. Further studies revealed that piperine treatment resulted in the activation of caspase-3 and cleavage of PARP-1 proteins in LNCaP, PC-3 and DU-145 prostate cancer cells. Piperine treatment also disrupted androgen receptor (AR) expression in LNCaP prostate cancer cells. Our evaluations further show that there is a significant reduction of Prostate Specific Antigen (PSA) levels following piperine treatment in LNCaP cells. NF-kB and STAT-3 transcription factors have previously been shown to play a role in angiogenesis and invasion of prostate cancer cells. Interestingly, treatment of LNCaP, PC-3 and DU-145 prostate cancer cells with piperine resulted in reduced expression of phosphorylated STAT-3 and Nuclear factor-κB (NF-kB) transcription factors. These results correlated with the results of Boyden chamber assay, wherein piperine treatment reduced the cell migration of LNCaP and PC-3 cells. Finally, we show that piperine treatment significantly reduced the androgen dependent and androgen independent tumor growth in nude mice model xenotransplanted with prostate cancer cells. Taken together, these results support

  9. Methylarsonous acid causes oxidative DNA damage in cells independent of the ability to biomethylate inorganic arsenic.

    PubMed

    Tokar, Erik J; Kojima, Chikara; Waalkes, Michael P

    2014-02-01

    Inorganic arsenic (iAs) and its toxic methylated metabolite, methylarsonous acid (MMA(III)), both have carcinogenic potential. Prior study shows iAs-induced malignant transformation in both arsenic methylation-proficient (liver) and methylation-deficient (prostate) cells, but only methylation-proficient cells show oxidative DNA damage (ODD) during this transformation. To further define whether arsenic methylation is necessary for transformation or ODD induction, here we chronically exposed these same liver or prostate cell lines to MMA(III) (0.25-1.0 μM) and tested for acquired malignant phenotype. Various metrics of oncogenic transformation were periodically assessed along with ODD during chronic MMA(III) exposure. Methylation-deficient and methylation-proficient cells both acquired a cancer phenotype with MMA(III) exposure at about 20 weeks, based on increased matrix metalloproteinase secretion, colony formation, and invasion. In contrast, prior work showed iAs-induced transformation took longer in biomethylation-deficient cells (~30 weeks) than in biomethylation-proficient cells (~18 weeks). In the present study, MMA(III) caused similar peak ODD levels at similar concentrations and at similar exposure times (18-22 weeks) in both cell types. At the approximate peak of ODD production, both cell types showed similar alterations in arsenic and oxidative stress adaptation factors (i.e., ABCC1, ABCC2, GST-π, SOD-1). Thus, MMA(III) causes oncogenic transformation associated with ODD in methylation-deficient cells, indicating that further methylation is not required to induce ODD. Together, these results show that MMA(III) and iAs cause an acquired malignant phenotype in methylation-deficient cells, yet iAs does not induce ODD. This indicates iAs likely has both genotoxic and non-genotoxic mechanisms dictated by the target cell's ability to methylate arsenic. PMID:24091636

  10. Methylarsonous acid causes oxidative DNA damage in cells independent of the ability to biomethylate inorganic arsenic.

    PubMed

    Tokar, Erik J; Kojima, Chikara; Waalkes, Michael P

    2014-02-01

    Inorganic arsenic (iAs) and its toxic methylated metabolite, methylarsonous acid (MMA(III)), both have carcinogenic potential. Prior study shows iAs-induced malignant transformation in both arsenic methylation-proficient (liver) and methylation-deficient (prostate) cells, but only methylation-proficient cells show oxidative DNA damage (ODD) during this transformation. To further define whether arsenic methylation is necessary for transformation or ODD induction, here we chronically exposed these same liver or prostate cell lines to MMA(III) (0.25-1.0 μM) and tested for acquired malignant phenotype. Various metrics of oncogenic transformation were periodically assessed along with ODD during chronic MMA(III) exposure. Methylation-deficient and methylation-proficient cells both acquired a cancer phenotype with MMA(III) exposure at about 20 weeks, based on increased matrix metalloproteinase secretion, colony formation, and invasion. In contrast, prior work showed iAs-induced transformation took longer in biomethylation-deficient cells (~30 weeks) than in biomethylation-proficient cells (~18 weeks). In the present study, MMA(III) caused similar peak ODD levels at similar concentrations and at similar exposure times (18-22 weeks) in both cell types. At the approximate peak of ODD production, both cell types showed similar alterations in arsenic and oxidative stress adaptation factors (i.e., ABCC1, ABCC2, GST-π, SOD-1). Thus, MMA(III) causes oncogenic transformation associated with ODD in methylation-deficient cells, indicating that further methylation is not required to induce ODD. Together, these results show that MMA(III) and iAs cause an acquired malignant phenotype in methylation-deficient cells, yet iAs does not induce ODD. This indicates iAs likely has both genotoxic and non-genotoxic mechanisms dictated by the target cell's ability to methylate arsenic.

  11. Immature B Cell Egress from Bone Marrow Is SOCS3 Independent

    PubMed Central

    Pereira, João P.

    2015-01-01

    Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3fl/fl Mb1cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling. PMID:26274929

  12. Particle-induced erythropoietin-independent effects of erythroid precursor cells in murine bone marrow.

    PubMed

    Ploemacher, R E; van Soest, P L; Wagemaker, G; van 't Hull, E

    1979-09-01

    A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment. PMID:519701

  13. Cyclin B1 overexpression induces cell death independent of mitotic arrest.

    PubMed

    Eichhorn, Joshua M; Kothari, Anisha; Chambers, Timothy C

    2014-01-01

    Microtubule inhibitors are widely used in cancer chemotherapy. These drugs characteristically induce mitotic arrest and cell death but the mechanisms linking the two are not firmly established. One of the problems is that cancer cells vary widely in their sensitivity to these agents, and thus comparison of data from different systems is difficult. To alleviate this problem we sought to molecularly induce mitotic death and study its mechanisms, by expressing non-degradable cyclin B (R42A) in HeLa cells. However, this approach failed to induce significant mitotic arrest, Cdk1 activation, or phosphorylation of anti-apoptotic Bcl-2 proteins, all characteristics of cells treated with microtubule inhibitors. Furthermore, cyclin B1-R42A induced rapid cell death, and when expressed in synchronized cells, cell death occurred in G1 phase. Decreasing the plasmid concentration reduced transfection efficiency but restored mitotic arrest and eliminated non-specific death. These results show that inappropriate overexpression of cyclin B1 causes non-specific cell death and suggest caution in its use for the study of mitotic events.

  14. RalGPS2 Is Essential for Survival and Cell Cycle Progression of Lung Cancer Cells Independently of Its Established Substrates Ral GTPases

    PubMed Central

    Parrini, Maria Carla; Camonis, Jacques

    2016-01-01

    The human genome contains six genes coding for proteins validated in vitro as specific activators of the small GTPases “Ras-related protein Ral-A” and “Ras-related protein Ral-B”, generically named Ral-guanine nucleotide exchange factors (RalGEF). Ral proteins are important contributors to Ras oncogenic signaling, and RAS oncogenes are important in human Non-Small Cell Lung Carcinoma (NSCLC). Therefore in this work, RalGEF contribution to oncogenic and non-oncogenic features of human NSCLC cell lines, as anchorage-dependent and independent growth, cell survival, and proliferation, was investigated. Among all human RalGEF, silencing of RGL1 and RALGPS1 had no detectable effect. However, silencing of either RGL2, RGL3, RALGDS or, to a larger extent, RALGPS2 inhibited cell population growth in anchorage dependent and independent conditions (up to 90 and 80%, respectively). RALGPS2 silencing also caused an increase in the number of apoptotic cells, up to 45% of the cell population in transformed bronchial BZR cells. In H1299 and A549, two NSCLC cell lines, RALGPS2 silencing caused an arrest of cells in the G0/G1-phase of cell cycle. Furthermore, it was associated with the modulation of important cell cycle regulators: the E3 Ubiquitin Protein Ligase S-phase kinase-associated protein 2 (Skp2) was strongly down-regulated (both at mRNA and protein levels), and its targets, the cell cycle inhibitors p27 and p21, were up-regulated. These molecular effects were not mimicked by silencing RALA, RALB, or both. However, RALB silencing caused a modest inhibition of cell cycle progression, which in H1299 cells was associated with Cyclin D1 regulation. In conclusion, RALGPS2 is implicated in the control of cell cycle progression and survival in the in vitro growth of NSCLC cell lines. This function is largely independent of Ral GTPases and associated with modulation of Skp2, p27 and p21 cell cycle regulators. PMID:27149377

  15. Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

    PubMed

    Sabiiti, Wilber; May, Robin C

    2012-01-01

    Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB.

  16. Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

    PubMed

    Sabiiti, Wilber; May, Robin C

    2012-01-01

    Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB. PMID:22530025

  17. Methylarsonous acid causes oxidative DNA damage in cells independent of the ability to biomethylate inorganic arsenic

    PubMed Central

    Tokar, Erik J.; Kojima, Chikara; Waalkes, Michael P.

    2013-01-01

    Inorganic arsenic (iAs) and its toxic methylated metabolite, methylarsonous acid (MMAIII), both have carcinogenic potential. Prior study shows iAs induced malignant transformation in both arsenic methylation-proficient (liver) and methylation-deficient (prostate) cells, but only methylation-proficient cells show oxidative DNA damage (ODD) during this transformation. To further define if arsenic methylation is necessary for transformation or ODD induction, here we chronically exposed these same liver or prostate cell lines to MMAIII (0.25–1.0 μM) and tested for acquired malignant phenotype. Various metrics of oncogenic transformation were periodically assessed along with ODD during chronic MMAIII exposure. Methylation-deficient and methylation-proficient cells both acquired a cancer phenotype with MMAIII exposure at about 20 weeks, based on increased matrix metalloproteinase secretion, colony formation and invasion. In contrast, prior work showed iAs-induced transformation took longer in biomethylation-deficient cells (~30 weeks) than in biomethylation-proficient cells (~18 weeks). In the present study, MMAIII caused similar peak ODD levels at similar concentrations and at similar exposure times (18–22 weeks) in both cell types. At the approximate peak of ODD production both cell types showed similar alterations in arsenic and oxidative stress adaptation factors (i.e. ABCC1, ABCC2, GST-π, SOD-1). Thus, MMAIII causes oncogenic transformation associated with ODD in methylation-deficient cells, indicating further methylation is not required to induce ODD. Together, these results show that, MMAIII and iAs cause an acquired malignant phenotype in methylation-deficient cells, yet iAs does not induce ODD. This indicates iAs likely has both genotoxic and non-genotoxic mechanisms dictated by the target cell’s ability to methylate arsenic. PMID:24091636

  18. Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression.

    PubMed

    Gracanin, Ana; Timmermans-Sprang, Elpetra P M; van Wolferen, Monique E; Rao, Nagesha A S; Grizelj, Juraj; Vince, Silvijo; Hellmen, Eva; Mol, Jan A

    2014-01-01

    Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand-independent mechanisms.

  19. Ligand-Independent Canonical Wnt Activity in Canine Mammary Tumor Cell Lines Associated with Aberrant LEF1 Expression

    PubMed Central

    van Wolferen, Monique E.; Rao, Nagesha A. S.; Grizelj, Juraj; Vince, Silvijo; Hellmen, Eva; Mol, Jan A.

    2014-01-01

    Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms. PMID:24887235

  20. Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.

    PubMed Central

    Zisman, E; Dayan, M; Sela, M; Mozes, E

    1993-01-01

    Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen. Images PMID:8381541

  1. Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression.

    PubMed

    Gracanin, Ana; Timmermans-Sprang, Elpetra P M; van Wolferen, Monique E; Rao, Nagesha A S; Grizelj, Juraj; Vince, Silvijo; Hellmen, Eva; Mol, Jan A

    2014-01-01

    Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand-independent mechanisms. PMID:24887235

  2. Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway

    SciTech Connect

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Won, Seong Su; Kang, Wonku; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-05-15

    Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1 expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells. - Highlights: • Leptin increased 4-hydroxyoestradiol in MCF-7 breast cancer cells. • Leptin activated ERK and Akt kinases related to ERα phosphorylation. • Leptin induces phosphorylation of ERα at serine residues 118 and 167. • Leptin induces ERE-luciferase activity.

  3. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    SciTech Connect

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    2013-02-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.

  4. Acute myeloid leukemia cells polarize macrophages towards a leukemia supporting state in a Growth factor independence 1 dependent manner

    PubMed Central

    Al-Matary, Yahya S.; Botezatu, Lacramioara; Opalka, Bertram; Hönes, Judith M.; Lams, Robert F.; Thivakaran, Aniththa; Schütte, Judith; Köster, Renata; Lennartz, Klaus; Schroeder, Thomas; Haas, Rainer; Dührsen, Ulrich; Khandanpour, Cyrus

    2016-01-01

    The growth of malignant cells is not only driven by cell-intrinsic factors, but also by the surrounding stroma. Monocytes/Macrophages play an important role in the onset and progression of solid cancers. However, little is known about their role in the development of acute myeloid leukemia, a malignant disease characterized by an aberrant development of the myeloid compartment of the hematopoietic system. It is also unclear which factors are responsible for changing the status of macrophage polarization, thus supporting the growth of malignant cells instead of inhibiting it. We report herein that acute myeloid leukemia leads to the invasion of acute myeloid leukemia-associated macrophages into the bone marrow and spleen of leukemic patients and mice. In different leukemic mouse models, these macrophages support the in vitro expansion of acute myeloid leukemia cell lines better than macrophages from non-leukemic mice. The grade of macrophage infiltration correlates in vivo with the survival of the mice. We found that the transcriptional repressor Growth factor independence 1 is crucial in the process of macrophage polarization, since its absence impedes macrophage polarization towards a leukemia supporting state and favors an anti-tumor state both in vitro and in vivo. These results not only suggest that acute myeloid leukemia-associated macrophages play an important role in the progression of acute myeloid leukemia, but also implicate Growth factor independence 1 as a pivotal factor in macrophage polarization. These data may provide new insights and opportunities for novel therapies for acute myeloid leukemia. PMID:27390361

  5. Induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through HDAC-independent and IP3-mediated PRKAA activation.

    PubMed

    Ji, Meng-Meng; Wang, Li; Zhan, Qin; Xue, Wen; Zhao, Yan; Zhao, Xia; Xu, Peng-Peng; Shen, Yang; Liu, Han; Janin, Anne; Cheng, Shu; Zhao, Wei-Li

    2015-01-01

    Autophagy is closely related to tumor cell sensitivity to anticancer drugs. The HDAC (histone deacetylase) inhibitor valproic acid (VPA) interacted synergistically with chemotherapeutic agents to trigger lymphoma cell autophagy, which resulted from activation of AMPK (AMP-activated protein kinase) and inhibition of downstream MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling. In an HDAC-independent manner, VPA potentiated the effect of doxorubicin on lymphoma cell autophagy via reduction of cellular inositol 1,4,5 trisphosphate (IP3), blockade of calcium into mitochondria and modulation of PRKAA1/2-MTOR cascade. In murine xenograft models established with subcutaneous injection of lymphoma cells, dual treatment of VPA and doxorubicin initiated IP3-mediated calcium depletion and PRKAA1/2 activation, induced in situ autophagy and efficiently retarded tumor growth. Aberrant genes involving mitochondrial calcium transfer were frequently observed in primary tumors of lymphoma patients. Collectively, these findings suggested an HDAC-independent chemosensitizing activity of VPA and provided an insight into the clinical application of targeting autophagy in the treatment of lymphoma.

  6. Cytoplasmic p53 and Activated Bax Regulate p53-dependent, Transcription-independent Neural Precursor Cell Apoptosis

    PubMed Central

    Geng, Ying; Walls, K.C.; Ghosh, Arindam P.; Akhtar, Rizwan S.; Klocke, Barbara J.; Roth, Kevin A.

    2010-01-01

    The prodeath effects of p53 are typically mediated via its transcriptional upregulation of proapoptotic Bcl-2 family members, including PUMA, Noxa, and/or Bax. We previously reported that staurosporine (STS), a broad-spectrum kinase inhibitor and prototypical apoptosis-inducing agent, produced p53-dependent, Bax-dependent, neural precursor cell (NPC) apoptosis, but that this effect occurred independently of new gene transcription and PUMA expression. To further characterize the mechanism by which p53 regulates NPC death, we used primary cerebellar NPCs derived from wild-type, p53-deficient, and Bax-deficient neonatal mice and the mouse cerebellar neural stem cell line, C17.2. We found that STS rapidly increased p53 cytoplasmic immunoreactivity in neuritic-like processes in C17.2 cells, which preceded Bax activation and caspase-3 cleavage. Confocal microscopy analysis of STS-treated cells revealed partial colocalization of p53 with the mitochondrial marker pyruvate dehydrogenase as well as with conformationally altered “activated” Bax, suggesting an interaction between these proapoptotic molecules in triggering apoptotic death. Nucleophosmin (NPM), a CRM1-dependent nuclear chaperone, also exhibited partial colocalization with both activated Bax and p53 following STS treatment. These observations suggest that cytoplasmic p53 can trigger transcription-independent NPC apoptosis through its potential interaction with NPM and activated Bax. (J Histochem Cytochem 58:265–275, 2010) PMID:19901272

  7. FOXP4 modulates tumor growth and independently associates with miR-138 in non-small cell lung cancer cells.

    PubMed

    Yang, Tian; Li, Hong; Thakur, Asmitananda; Chen, Tianjun; Xue, Jing; Li, Dan; Chen, Mingwei

    2015-09-01

    Family of forkhead box transcription factors, including forkhead box P4 (FOXP4), plays an important role in oncogenesis. The current study is to evaluate the role of FOXP4 in regulating human non-small cell lung cancer (NSCLC). Quantitative RT-PCR and Western blot were performed to evaluate the gene and protein expressions of FOXP4 in six NSCLC cell lines and 55 NSCLC patients. Lentivirus of small hairpin RNA (FOXP4-shRNA) was used to downregulate FOXP4 in NSCLC cell lines A549 and H1703 cells. Its effect on NSCLC growth, invasion, and cell cycle were evaluated by cell proliferation assay, migration assay, and cell cycle assay, respectively. Dual luciferase assay and Western blot were used to examine whether microRNA-138 (miR-138) was an upstream regulator of FOXP4. The dependence of FOXP4 on miR-138 associated signaling pathway was evaluated by ectopically overexpressing enhancer of zeste homolog 2 (EZH2), a known miR-138 target in NSCLC. FOXP4 was highly expressed in both NSCLC cell lines and NSCLC patients. FOXP4 downregulation by FOXP4-shRNA markedly reduced cancer cell growth and invasion, as well as induced cell cycle arrest in A549 and H1703 cells. MiR-138 was confirmed to be an upstream regulator of FOXP4 and directly regulated FOXP4 expression in A549 and H1703 cells. FOXP4 downregulation-mediated inhibition on cancer cell growth and invasion was independent on overexpressing EZH2, another direct target of miR-138 in NSCLC. Our data demonstrated that FOXP4 was a critical regulator in NSCLC and independently associated with miR-138 regulation.

  8. NF-YC in glioma cell proliferation and tumor growth and its role as an independent predictor of patient survival.

    PubMed

    Cui, Hua; Zhang, Mingkun; Wang, Yanxia; Wang, Yong

    2016-09-19

    Gliomas are tumors affecting the central nervous system and affecting approximately 7/10,000 people with the median survival of only 14.6 months. As such, there is a need to uncover and explore alternative targets and pathways of gliomagenesis as well as a need to develop early and effective predictive markers of the disease. In this study we utilized a wide range of patient glioma sections to assess the characteristic expression of NF-YC and investigate whether NF-YC could serve as an independent predictor of patient survival. Additionally, an in vitro glioma model of manipulated NF-YC was used to investigate NF-YC's role in the glioma growth process and ultimately validated in an animal model of tumor growth. Here, we present evidence of the NF-YC subunit of the NF-Y transcription factor complex as an independent prognostic maker for glioma patient survival. We also describe that NF-YC is positively correlated with a universal marker of cellular proliferation. Mechanistic investigation into the role of NF-YC in gliomagenesis showed that NF-YC plays a role in cell cycle progression through the inhibition of the cyclin-dependent kinase inhibitor p21. Finally, NF-YC plays a role in the epithelial-mesenchymal transition preceding metastasis. We propose a novel target of glioma cell proliferation, growth and metastasis. Additionally, we identify NF-YC as a novel and independent predictor of patient survival to be subsequently trialed. PMID:27495011

  9. TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses.

    PubMed

    López, Carolina B; Moltedo, Bruno; Alexopoulou, Lena; Bonifaz, Laura; Flavell, Richard A; Moran, Thomas M

    2004-12-01

    TLR signaling leads to dendritic cell (DC) maturation and immunity to diverse pathogens. The stimulation of TLRs by conserved viral structures is the only described mechanism leading to DC maturation after a virus infection. In this report, we demonstrate that mouse myeloid DCs mature normally after in vivo and in vitro infection with Sendai virus (SeV) in the absence of TLR3, 7, 8, or 9 signaling. DC maturation by SeV requires virus replication not necessary for TLR-mediated triggering. Moreover, DCs deficient in TLR signaling efficiently prime for Th1 immunity after infection with influenza or SeV, generating IFN-gamma-producing T cells, CTLs and antiviral Abs. We have previously demonstrated that SeV induces DC maturation independently of the presence of type I IFN, which has been reported to mature DCs in a TLR-independent manner. The data presented here provide evidence for the existence of a novel intracellular pathway independent of TLR-mediated signaling responsible for live virus triggering of DC maturation and demonstrate its critical role in the onset of antiviral immunity. The revelation of this pathway should stimulate invigorating research into the mechanism for virus-induced DC maturation and immunity.

  10. Subcutaneous Adipose Tissue–Derived Stem Cell Utility Is Independent of Anatomical Harvest Site

    PubMed Central

    Choudhery, Mahmood S.; Badowski, Michael; Muise, Angela; Pierce, John; Harris, David T.

    2015-01-01

    Abstract One of the challenges for tissue engineering and regenerative medicine is to obtain suitably large cell numbers for therapy. Mesenchymal stem cells (MSCs) can easily be expanded in vitro to obtain large numbers of cells, but this approach may induce cellular senescence. The characteristics of cells are dependent on variables like age, body mass index (BMI), and disease conditions, however, and in the case of adipose tissue–derived stem cells (ASCs), anatomical harvest site is also an important variable that can affect the regenerative potential of isolated cells. We therefore had kept the parameters (age, BMI, disease conditions) constant in this study to specifically assess influence of anatomical sites of individual donors on utility of ASCs. Adipose tissue was obtained from multiple anatomical sites in individual donors, and viability and nucleated cell yield were determined. MSC frequency was enumerated using colony forming unit assay and cells were characterized by flow cytometry. Growth characteristics were determined by long-term population doubling analysis of each sample. Finally, MSCs were induced to undergo adipogenic, osteogenic, and chondrogenic differentiation. To validate the findings, these results were compared with similar single harvest sites from multiple individual patients. The results of the current study indicated that MSCs obtained from multiple harvest sites in a single donor have similar morphology and phenotype. All adipose depots in a single donor exhibited similar MSC yield, viability, frequency, and growth characteristics. Equivalent differentiation capacity into osteocytes, adipocytes, and chondrocytes was also observed. On the basis of results, we conclude that it is acceptable to combine MSCs obtained from various anatomical locations in a single donor to obtain suitably large cell numbers required for therapy, avoiding in vitro senescence and lengthy and expensive in vitro culturing and expansion steps. PMID:26309790

  11. Expression of PKC iota affects neuronal differentiation of PC12 cells at least partly independent of kinase function

    PubMed Central

    Doonachar, Alana; Schoenfeld, Alan R.

    2014-01-01

    Atypical PKC (aPKC) plays a role in establishing cell polarity and has been indicated in neuronal differentiation and polarization, including neurite formation in rat pheochromocytoma PC12 cells, albeit by unclear mechanisms. Here, the role of the aPKC isoform, PKC iota (PKCι), in the early neuronal differentiation of PC12 cells was investigated. NGF-treated PC12 cells with stably expressed exogenous wild-type PKCι showed decreased expression of a neuroendocrine marker, increased expression of a neuronal marker, and increased neurite formation. Stable expression of a kinase- inactive PKCι, but not constitutively active PKCι lacking a regulatory domain, had similar although less potent effects. Pharmacological inhibition of endogenous aPKC kinase activity in parental PC12 cells did not inhibit neurite formation, suggesting that some of the observed effects of PKCι expression on neuronal differentiation are kinase- independent. Interestingly, exogenous expression of wild-type and kinase-inactive PKCι had little effect on overall PKCι activity, but caused a decrease in PKC zeta (PKCζ) kinase activity, suggesting an interplay between the two isoforms that may underlie the observed results. Overall, these findings suggest that in PC12 and perhaps other neuroendocrine precursor cells, PKCι influences an early differentiation decision between the neuroendocrine (chromaffin) and sympathetic neuron cell lineages, potentially by affecting PKCζ function. PMID:24910851

  12. The absence of a mitochondrial genome in rho0 yeast cells extends lifespan independently of retrograde regulation.

    PubMed

    Woo, Dong Kyun; Poyton, Robert O

    2009-01-01

    The absence of mtDNA in rho0 yeast cells affects both respiration and mitochondrial-nuclear communication (e.g., retrograde regulation, intergenomic signaling, or pleiotropic drug resistance). Previously, it has been reported that some rho0 strains have increased replicative lifespans, attributable to the lack of respiration and retrograde regulation. Here, we have been able to confirm that rho0 cells exhibit increased replicative lifespans but have found that this is not associated with the lack of respiration or reduced oxidative stress but instead, is related to the lack of mtDNA per se in rho0 cells. Also, we find no correlation between the strength of retrograde regulation and lifespan. Furthermore, we find that pdr3- or rtg2- mutations are not responsible for lifespan extension in rho0 cells, ruling out a specific role for PDR3-pleiotropic drug resistance or RGT2-retrograde regulation pathways in the extended lifespans of rho0 cells. Surprisingly, Rtg3p, which acts downstream of Rtg2p, is required for lifespan increase in rho0 cells. Together, these findings indicate that the loss of mtDNA per se and not the lack of respiration lead to extended longevity in rho0 cells. They also suggest that Rtg3p, acting independently of retrograde regulation, mediates this effect, possibly via intergenomic signaling.

  13. Growth rate of late passage sarcoma cells is independent of epigenetic events but dependent on the amount of chromosomal aberrations

    SciTech Connect

    Becerikli, Mustafa; Jacobsen, Frank; Rittig, Andrea; Köhne, Wiebke; Nambiar, Sandeep; Mirmohammadsadegh, Alireza; Stricker, Ingo; Tannapfel, Andrea; Wieczorek, Stefan; Epplen, Joerg Thomas; Tilkorn, Daniel; Steinstraesser, Lars

    2013-07-15

    Soft tissue sarcomas (STS) are characterized by co-participation of several epigenetic and genetic events during tumorigenesis. Having bypassed cellular senescence barriers during oncogenic transformation, the factors further affecting growth rate of STS cells remain poorly understood. Therefore, we investigated the role of gene silencing (DNA promoter methylation of LINE-1, PTEN), genetic aberrations (karyotype, KRAS and BRAF mutations) as well as their contribution to the proliferation rate and migratory potential that underlies “initial” and “final” passage sarcoma cells. Three different cell lines were used, SW982 (synovial sarcoma), U2197 (malignant fibrous histiocytoma (MFH)) and HT1080 (fibrosarcoma). Increased proliferative potential of final passage STS cells was not associated with significant differences in methylation (LINE-1, PTEN) and mutation status (KRAS, BRAF), but it was dependent on the amount of chromosomal aberrations. Collectively, our data demonstrate that these fairly differentiated/advanced cancer cell lines have still the potential to gain an additional spontaneous growth benefit without external influences and that maintenance of increased proliferative potential towards longevity of STS cells (having crossed senescence barriers) may be independent of overt epigenetic alterations. -- Highlights: Increased proliferative potential of late passage STS cells was: • Not associated with epigenetic changes (methylation changes at LINE-1, PTEN). • Not associated with mutation status of KRAS, BRAF. • Dependent on presence/absence of chromosomal aberrations.

  14. Chk2 and p53 Are Haploinsufficient with Dependent and Independent Functions to Eliminate Cells after Telomere Loss

    PubMed Central

    Xie, Heng B.; Golic, Kent G.

    2011-01-01

    The mechanisms that cells use to monitor telomere integrity, and the array of responses that may be induced, are not fully defined. To date there have been no studies in animals describing the ability of cells to survive and contribute to adult organs following telomere loss. We developed assays to monitor the ability of somatic cells to proliferate and differentiate after telomere loss. Here we show that p53 and Chk2 limit the growth and differentiation of cells that lose a telomere. Furthermore, our results show that two copies of the genes encoding p53 and Chk2 are required for the cell to mount a rapid wildtype response to a missing telomere. Finally, our results show that, while Chk2 functions by activating the p53-dependent apoptotic cascade, Chk2 also functions independently of p53 to limit survival. In spite of these mechanisms to eliminate cells that have lost a telomere, we find that such cells can make a substantial contribution to differentiated adult tissues. PMID:21655087

  15. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  16. Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation.

    PubMed

    Jaber-Hijazi, Farah; Lo, Priscilla J K P; Mihaylova, Yuliana; Foster, Jeremy M; Benner, Jack S; Tejada Romero, Belen; Chen, Chen; Malla, Sunir; Solana, Jordi; Ruzov, Alexey; Aziz Aboobaker, A

    2013-12-01

    Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5(m)C) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.

  17. Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation☆

    PubMed Central

    Jaber-Hijazi, Farah; Lo, Priscilla J.K.P.; Mihaylova, Yuliana; Foster, Jeremy M.; Benner, Jack S.; Tejada Romero, Belen; Chen, Chen; Malla, Sunir; Solana, Jordi; Ruzov, Alexey; Aziz Aboobaker, A.

    2013-01-01

    Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation. PMID:24063805

  18. Discriminating the Independent Influence of Cell Adhesion and Spreading Area on Stem Cell Fate Determination Using Micropatterned Surfaces

    PubMed Central

    Wang, Xinlong; Hu, Xiaohong; Dulińska-Molak, Ida; Kawazoe, Naoki; Yang, Yingnan; Chen, Guoping

    2016-01-01

    Adhesion and spreading are essential processes of anchorage dependent cells involved in regulation of cell functions. Cells interact with their extracellular matrix (ECM) resulting in different degree of adhesion and spreading. However, it is not clear whether cell adhesion or cell spreading is more important for cell functions. In this study, 10 types of isotropical micropatterns that were composed of 2 μm microdots were prepared to precisely control the adhesion area and spreading area of human mesenchymal stem cells (MSCs). The respective influence of adhesion and spreading areas on stem cell functions was investigated. Adhesion area showed more significant influences on the focal adhesion formation, binding of myosin to actin fibers, cytoskeletal organization, cellular Young’s modulus, accumulation of YAP/TAZ in nuclei, osteogenic and adipogenic differentiation of MSCs than did the spreading area. The results indicated that adhesion area rather than spreading area played more important roles in regulating cell functions. This study should provide new insight of the influence of cell adhesion and spreading on cell functions and inspire the design of biomaterials to process in an effective manner for manipulation of cell functions. PMID:27349298

  19. Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

    SciTech Connect

    Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

    2012-07-15

    Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

  20. Independent Orbiter Assessment (IOA): Assessment of the electrical power generation/fuel cell powerplant subsystem FMEA/CIL

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

    Results of the Independent Orbiter Assessment (IOA) of the Failure Modes and Effects Analysis (FMEA) and Critical Items List (CIL) are presented. The IOA effort first completed an analysis of the Electrical Power Generation/Fuel Cell Powerplant (EPG/FCP) hardware, generating draft failure modes and potential critical items. To preserve independence, this analysis was accomplished without reliance upon the results contained within the NASA FMEA/CIL documentation. The IOA results were then compared to the proposed Post 51-L NASA FMEA/CIL baseline. A resolution of each discrepancy from the comparison was provided through additional analysis as required. This report documents the results of that comparison for the Orbiter EPG/FCP hardware.

  1. ANGPTL4 expression induced by butyrate and rosiglitazone in human intestinal epithelial cells utilizes independent pathways.

    PubMed

    Korecka, Agata; de Wouters, Tomas; Cultrone, Antonietta; Lapaque, Nicolas; Pettersson, Sven; Doré, Joël; Blottière, Hervé M; Arulampalam, Velmurugesan

    2013-06-01

    Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolic products of carbohydrate fermentation by the microbiota and constitute the main source of energy for host colonocytes. SCFAs are also important for gastrointestinal health, immunity, and host metabolism. Intestinally produced angiopoietin-like protein 4 (ANGPTL4) is a secreted protein with metabolism-altering properties and may offer a route by which microbiota can regulate host metabolism. Peroxisome proliferator-activated receptor (PPAR)-γ has previously been shown to be involved in microbiota-induced expression of intestinal ANGPTL4, but the role of bacterial metabolites in this process has remained elusive. Here, we show that the SCFA butyrate regulates intestinal ANGPTL4 expression in a PPAR-γ-independent manner. Although PPAR-γ is not required for butyrate-driven intestinal ANGPTL4 expression, costimulating with PPAR-γ ligands and SCFAs leads to additive increases in ANGPTL4 levels. We suggest that PPAR-γ and butyrate rely on two separate regulatory sites, a PPAR-responsive element downstream the transcription start site and a butyrate-responsive element(s) within the promoter region, 0.5 kb upstream of the transcription start site. Furthermore, butyrate gavage and colonization with Clostridium tyrobutyricum, a SCFA producer, can independently induce expression of intestinal ANGPTL4 in germ-free mice. Thus, oral administration of SCFA or use of SCFA-producing bacteria may be additional routes to maintain intestinal ANGPTL4 levels for preventive nutrition or therapeutic purposes.

  2. EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer

    PubMed Central

    Schneck, Helen; Gierke, Berthold; Uppenkamp, Frauke; Behrens, Bianca; Niederacher, Dieter; Stoecklein, Nikolas H.; Templin, Markus F.; Pawlak, Michael; Fehm, Tanja; Neubauer, Hans

    2015-01-01

    Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far–including the gold standard CellSearch—rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1–24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. Ep

  3. Satratoxin G–Induced Apoptosis in PC-12 Neuronal Cells is Mediated by PKR and Caspase Independent

    PubMed Central

    Islam, Zahidul; Hegg, Colleen C.; Bae, Hee Kyong; Pestka, James J.

    2008-01-01

    Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum, a mold suggested to play an etiologic role in damp building-related illnesses. Acute intranasal exposure of mice to SG specifically induces apoptosis in olfactory sensory neurons of the nose. The PC-12 rat pheochromocytoma cell model was used to elucidate potential mechanisms of SG-induced neuronal cell death. Agarose gel electrophoresis revealed that exposure to SG at 10 ng/ml or higher for 48-h induced DNA fragmentation characteristic of apoptosis in PC-12 cells. SG-induced apoptosis was confirmed by microscopic morphology, hypodiploid fluorescence and annexin V-fluorescein isothiocyanate (FITC) uptake. Messenger RNA expression of the proapoptotic genes p53, double-stranded RNA–activated protein kinase (PKR), BAX, and caspase-activated DNAse was significantly elevated from 6 to 48 h after SG treatment. SG also induced apoptosis and proapoptotic gene expression in neural growth factor-differentiated PC-12 cells. Although SG-induced caspase-3 activation, caspase inhibition did not impair apoptosis. Moreover, SG induced nuclear translocation of apoptosis-inducing factor (AIF), a known contributor to caspase-independent neuronal cell death. SG-induced apoptosis was not affected by inhibitors of oxidative stress or mitogen-activated protein kinases but was suppressed by the PKR inhibitor C16 and by PKR siRNA transfection. PKR inhibition also blocked SG-induced apoptotic gene expression and AIF translocation but not caspase-3 activation. Taken together, SG-induced apoptosis in PC-12 neuronal cells is mediated by PKR via a caspase-independent pathway possibly involving AIF translocation. PMID:18535002

  4. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

    PubMed

    Gupta, Shelly; Prasad, G V R Krishna; Mukhopadhaya, Arunika

    2015-12-25

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

  5. ING5 Is Phosphorylated by CDK2 and Controls Cell Proliferation Independently of p53

    PubMed Central

    Linzen, Ulrike; Lilischkis, Richard; Pandithage, Ruwin; Schilling, Britta; Ullius, Andrea; Lüscher-Firzlaff, Juliane; Kremmer, Elisabeth; Lüscher, Bernhard; Vervoorts, Jörg

    2015-01-01

    Inhibitor of growth (ING) proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2) in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53. PMID:25860957

  6. CD38 Expression in a Subset of Memory T Cells Is Independent of Cell Cycling as a Correlate of HIV Disease Progression

    PubMed Central

    Würsch, Daniela; Ormsby, Christopher E.; Romero-Rodríguez, Dámaris P.; Olvera-García, Gustavo; Zúñiga, Joaquín; Jiang, Wei; Pérez-Patrigeon, Santiago

    2016-01-01

    In order to determine if the expression of the activation marker CD38 can correlate with HIV disease progression independently of cycling, we performed a cluster-based multivariate correlation analysis of total circulating CD4+ T cell counts and viral loads with frequencies of CD38 and Ki67 expression on CD4+ lymphocytes from patients with untreated HIV infection, stratified in maturation subpopulations, and subpopulation subsets defined by the expression of CXCR5, CXCR3, and CCR4. The frequencies of the activated phenotypes %CD38+ Ki67− and %CD38+ Ki67+ of the CXCR5− CXCR3− CCR4+ (“pre-Th2”) central memory (TCM) cell subset clustered together, comprising a significant negative correlate of total circulating CD4+ T cell counts and a positive correlate of viral load in multivariate analysis. Frequency of cycling-uncoupled CD38 expression in “pre-Th2” TCM cells was a negative correlate of total circulating CD4+ T cell counts in univariate analysis, which was not the case of their %CD38+ Ki67+. CXCR5+ CXCR3− CCR4−  TCM cells were underrepresented in patients, and their absolute counts correlated negatively with their %CD38+ Ki67− but not with their % CD38+ Ki67+. Our results may imply that CD38 expression either reflects or participates in pathogenic mechanisms of HIV disease independently of cell cycling. PMID:27064238

  7. CD38 Expression in a Subset of Memory T Cells Is Independent of Cell Cycling as a Correlate of HIV Disease Progression.

    PubMed

    Würsch, Daniela; Ormsby, Christopher E; Romero-Rodríguez, Dámaris P; Olvera-García, Gustavo; Zúñiga, Joaquín; Jiang, Wei; Pérez-Patrigeon, Santiago; Espinosa, Enrique

    2016-01-01

    In order to determine if the expression of the activation marker CD38 can correlate with HIV disease progression independently of cycling, we performed a cluster-based multivariate correlation analysis of total circulating CD4(+) T cell counts and viral loads with frequencies of CD38 and Ki67 expression on CD4(+) lymphocytes from patients with untreated HIV infection, stratified in maturation subpopulations, and subpopulation subsets defined by the expression of CXCR5, CXCR3, and CCR4. The frequencies of the activated phenotypes %CD38(+) Ki67(-) and %CD38(+) Ki67(+) of the CXCR5(-) CXCR3(-) CCR4(+) ("pre-Th2") central memory (T(CM)) cell subset clustered together, comprising a significant negative correlate of total circulating CD4(+) T cell counts and a positive correlate of viral load in multivariate analysis. Frequency of cycling-uncoupled CD38 expression in "pre-Th2" T(CM) cells was a negative correlate of total circulating CD4(+) T cell counts in univariate analysis, which was not the case of their %CD38(+) Ki67(+). CXCR5(+) CXCR3(-) CCR4(-) T(CM) cells were underrepresented in patients, and their absolute counts correlated negatively with their %CD38(+) Ki67(-) but not with their % CD38(+) Ki67(+). Our results may imply that CD38 expression either reflects or participates in pathogenic mechanisms of HIV disease independently of cell cycling.

  8. The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content.

    PubMed

    Delain, E; Aouani, A; Vignal, A; Couture-Tosi, E; Hovsépian, S; Fayet, G

    1987-02-01

    The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin. Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy. In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule. When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found. These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol. Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape. The variability in size measurements remains in a 2% range for all thyroglobulin types. Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil. PMID:3556752

  9. A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells

    PubMed Central

    Gowen, Benjamin G; Chim, Bryan; Marceau, Caleb D; Greene, Trever T; Burr, Patrick; Gonzalez, Jeanmarie R; Hesser, Charles R; Dietzen, Peter A; Russell, Teal; Iannello, Alexandre; Coscoy, Laurent; Sentman, Charles L; Carette, Jan E; Muljo, Stefan A; Raulet, David H

    2015-01-01

    Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger. DOI: http://dx.doi.org/10.7554/eLife.08474.001 PMID:26565589

  10. Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells

    PubMed Central

    Cockbill, Louisa M. R.; Murk, Kai; Love, Seth; Hanley, Jonathan G.

    2015-01-01

    Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain–containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention. PMID:26466675

  11. Imatinib and Nilotinib increase glioblastoma cell invasion via Abl-independent stimulation of p130Cas and FAK signalling

    PubMed Central

    Frolov, Antonina; Evans, Ian M.; Li, Ningning; Sidlauskas, Kastytis; Paliashvili, Ketevan; Lockwood, Nicola; Barrett, Angela; Brandner, Sebastian; Zachary, Ian C.; Frankel, Paul

    2016-01-01

    Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRβ) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility. PMID:27293031

  12. Entry of aminoglycosides into renal tubular epithelial cells via endocytosis-dependent and endocytosis-independent pathways.

    PubMed

    Nagai, Junya; Takano, Mikihisa

    2014-08-15

    Aminoglycoside antibiotics such as gentamicin and amikacin are well recognized as a clinically important antibiotic class because of their reliable efficacy and low cost. However, the clinical use of aminoglycosides is limited by their nephrotoxicity and ototoxicity. Nephrotoxicity is induced mainly due to high accumulation of the antibiotics in renal proximal tubular cells. Therefore, a lot of studies on characterization of the renal transport system for aminoglycosides so far reported involved various in-vivo and in-vitro techniques. Early studies revealed that aminoglycosides are taken up through adsorptive endocytosis in renal epithelial cells. Subsequently, it was found that megalin, a multiligand endocytic receptor abundantly expressed on the apical side of renal proximal tubular cells, can bind aminoglycosides and that megalin-mediated endocytosis plays a crucial role in renal accumulation of aminoglycosides. Therefore, megalin has been suggested to be a promising molecular target for the prevention of aminoglycoside-induced nephrotoxicity. On the other hand, recently, some reports have indicated that aminoglycosides are transported via a pathway that does not require endocytosis, such as non-selective cation channel-mediated entry, in cultured renal tubular cells as well as cochlear outer hair cells. In this commentary article, we review the cellular transport of aminoglycosides in renal epithelial cells, focusing on endocytosis-dependent and -independent pathways.

  13. A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells.

    PubMed

    Gowen, Benjamin G; Chim, Bryan; Marceau, Caleb D; Greene, Trever T; Burr, Patrick; Gonzalez, Jeanmarie R; Hesser, Charles R; Dietzen, Peter A; Russell, Teal; Iannello, Alexandre; Coscoy, Laurent; Sentman, Charles L; Carette, Jan E; Muljo, Stefan A; Raulet, David H

    2015-01-01

    Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

  14. Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis.

    PubMed

    Boglev, Yeliz; Badrock, Andrew P; Trotter, Andrew J; Du, Qian; Richardson, Elsbeth J; Parslow, Adam C; Markmiller, Sebastian J; Hall, Nathan E; de Jong-Curtain, Tanya A; Ng, Annie Y; Verkade, Heather; Ober, Elke A; Field, Holly A; Shin, Donghun; Shin, Chong H; Hannan, Katherine M; Hannan, Ross D; Pearson, Richard B; Kim, Seok-Hyung; Ess, Kevin C; Lieschke, Graham J; Stainier, Didier Y R; Heath, Joan K

    2013-01-01

    Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death. PMID:23408911

  15. Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

    PubMed Central

    Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

    2010-01-01

    Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. PMID:20478262

  16. Interleukin-32 is progressively expressed in Mycosis Fungoides independent of helper T-cell 2 and helper T-cell 9 polarization

    PubMed Central

    Ohmatsu, Hanako; Humme, Daniel; Gulati, Nicholas; Gonzalez, Juana; Möbs, Markus; Suárez-Fariñas, Mayte; Cardinale, Irma; Mitsui, Hiroshi; Guttman-Yassky, Emma; Sterry, Wolfram; Krueger, James G.

    2014-01-01

    Mycosis Fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL) is characterized by a helper T cell 2 (Th2)-skewing with a mature CD4+ memory T-cell phenotype. Using skin samples from MF patients (n=21), healthy volunteers (n=17), individuals with atopic dermatitis (n=17) and psoriasis (n=9), we found interleukin (IL)-32 mRNA expression significantly higher in MF samples than in samples from benign inflammatory skin diseases, and its expression increases with disease progression. By immunohistochemistry and immunofluorescence, we confirmed IL-32 protein expression in many CD3+CD4+ T cells and some epidermotropic T cells in MF lesions. MyLa cells (a MF cell line) express IL-32, which in turn could promote cellular proliferation and viability in a dose-dependent fashion. IL-32-treated MyLa and CTCL HH cells up-regulated cell proliferation and survival genes. Of the major “polarizing” T-cell cytokines, only IFNγ mRNA increases with MF progression and positively correlates with IL-32 mRNA expression. Th2 cytokines do not positively correlate with IL-32 mRNA expression or MF progression. Furthermore, by flow cytometry, IL-32 production by circulating activated T-cells in healthy individuals was found in both IFNγ+ and IFNγ− cells but not in IL-4+ or IL-13+ cells. In conclusion, we have identified IL-32+ cells as the likely tumor cells in MF, and demonstrated that IL-32 mRNA expression increases with MF progression and is significantly higher than those in other skin diseases, and that some IL-32+ T cells are independent from the defined Th subsets. Thus IL-32 may play a unique role in MF progression as an autocrine cytokine. PMID:24938282

  17. IL32 is progressively expressed in mycosis fungoides independent of helper T-cell 2 and helper T-cell 9 polarization.

    PubMed

    Ohmatsu, Hanako; Humme, Daniel; Gulati, Nicholas; Gonzalez, Juana; Möbs, Markus; Suárez-Fariñas, Mayte; Cardinale, Irma; Mitsui, Hiroshi; Guttman-Yassky, Emma; Sterry, Wolfram; Krueger, James G

    2014-09-01

    Mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), is characterized by a helper T-cell 2 (Th2) skewing with a mature CD4(+) memory T-cell phenotype. Using skin samples from patients with mycosis fungoides (n = 21), healthy volunteers (n = 17), and individuals with atopic dermatitis (n = 17) and psoriasis (n = 9), we found IL32 mRNA expression significantly higher in mycosis fungoides samples than in samples from benign inflammatory skin diseases, and its expression increases with disease progression. By IHC and immunofluorescence, we confirmed IL32 protein expression in many CD3(+)CD4(+) T cells and some epidermotropic T cells in mycosis fungoides lesions. MyLa cells (a mycosis fungoides cell line) express IL32, which, in turn, could promote cellular proliferation and viability in a dose-dependent fashion. IL32-treated MyLa and CTCL HH cells upregulated cell proliferation and survival genes. Of the major "polarizing" T-cell cytokines, only IFNγ mRNA increases with mycosis fungoides progression and positively correlates with IL32 mRNA expression. Th2 cytokines do not positively correlate with IL32 mRNA expression or mycosis fungoides progression. Furthermore, by flow cytometry, IL32 production by circulating activated T cells in healthy individuals was found in both IFNγ(+) and IFNγ(-) cells but not in IL4(+) or IL13(+) cells. In conclusion, we have identified IL32(+) cells as the likely tumor cells in mycosis fungoides, and demonstrated that IL32 mRNA expression increases with mycosis fungoides progression and is significantly higher than mRNA expression in other skin diseases, and that some IL32(+) T cells are independent from the defined Th subsets. Thus, IL32 may play a unique role in mycosis fungoides progression as an autocrine cytokine.

  18. Phenotype-independent effects of retroviral transduction in human dental pulp stem cells.

    PubMed

    Egbuniwe, Obi; Grant, Andrew D; Renton, Tara; Di Silvio, Lucy

    2013-07-01

    An immortalized human dental pulp stem cell (DPSC) line of an odontoblastic phenotype is established to circumvent the normal programmed senescence and to maintain the cell line's usefulness as a tool for further study of cellular activity. DPSCs are isolated from human dental pulp tissues and transfected using hTERT. The influence of this process on the DPSC phenotype and the mRNA expression of oncogenes involved in cellular senescence is investigated. The results reveal an absence of altered DPSC morphology and phenotype following the exogenous introduction of the hTERT gene, which is coupled with a significant reduction in p16 mRNA expression. This provides insight into how to circumvent in vitro dental pulp stem cell death following the exogenous introduction of hTERT.

  19. TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

    PubMed

    Salerno, Fiamma; Guislain, Aurelie; Cansever, Dilay; Wolkers, Monika C

    2016-05-01

    CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections. PMID:27016606

  20. MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors are Therapeutically Susceptible to BH3 Mimetic Agents

    PubMed Central

    Fan, Weiwen; Tang, Zhe; Yin, Lihong; Morrison, Bei; Hafez-Khayyata, Said; Fu, Pingfu; Huang, Honglian; Bagai, Rakesh; Jiang, Shan; Kresak, Adam; Howell, Scott; Vasanji, Amit; Flask, Chris A.; Halmos, Balazs; Koon, Henry; Ma, Patrick C.

    2011-01-01

    Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible EGFR inhibitors, alone or in combination with MET kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, demonstrating BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial anti-tumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BH3 mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early resistant lung tumor cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive co-treatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung cancer treatment. PMID:21555370

  1. Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate.

    PubMed

    Motevaseli, Elahe; Shirzad, Mahdieh; Akrami, Seyed Mohammad; Mousavi, Azam-Sadat; Mirsalehian, Akbar; Modarressi, Mohammad Hossein

    2013-07-01

    Cervical cancer is a human papilloma virus (HPV)-related cancer, but most HPV infections are transient or intermittent and resolve spontaneously. Thus, other factors, such as cervical microflora, which are dominated by lactobacilli, must be involved in invasive cervical carcinoma development after HPV infection. Previous studies have demonstrated that lactobacilli have antitumour effects, and it is possible that vaginal lactobacilli prevent cervical cancer. Here we examined the proliferative and apoptotic responses of normal and tumour cervical cells to common vaginal lactobacilli components by investigating human normal fibroblast-like cervical (normal cervical) and HeLa (cervical tumour) cell responses to Lactobacillus gasseri and Lactobacillus crispatus. The effects of different lactobacilli components, such as culture supernatants, cytoplasmic extracts, cell-wall extracts and live cells, were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, trypan blue staining, lactate dehydrogenase assay and colorimetric caspase-3 activity assay. Changes in caspase-3 and human chorionic gonadotropin β (hCGβ) expression were analysed by quantitative RT-PCR. Tumour cell growth inhibition by culture supernatants was higher than that by pH- and lactate-adjusted controls. However, the effects of the supernatants on normal cells were similar to those of lactate-adjusted controls. Apoptosis was inhibited by supernatants, which was consistent with higher hCGβ expression since hCG inhibits apoptosis. Our study demonstrated that common vaginal lactobacilli exert cytotoxic effects on cervical tumour cells, but not on normal cells, and that this cytotoxicity is independent of pH and lactate. Our results encourage further studies on the interaction between lactobacilli and cervical cells, and administration of common vaginal lactobacilli as probiotics.

  2. HDAC inhibitor treatment of hepatoma cells induces both TRAIL-independent apoptosis and restoration of sensitivity to TRAIL.

    PubMed

    Pathil, Anita; Armeanu, Sorin; Venturelli, Sascha; Mascagni, Paolo; Weiss, Thomas S; Gregor, Michael; Lauer, Ulrich M; Bitzer, Michael

    2006-03-01

    Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs or innovative tumor cell apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Recently, we found 2 histone deacetylase inhibitors (HDAC-I), valproic acid and ITF2357, exhibiting inherent therapeutic activity against HCC. In TRAIL-sensitive cancer cells, the mechanism of HDAC-I-induced cell death has been identified to be TRAIL-dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC-derived cells, a prototype of TRAIL-resistant tumor cells, we found a HDAC-I-mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC-I and TRAIL, increasing the fraction of apoptotic cells two- to threefold compared with HDAC-I treatment alone, whereas any premature HDAC-I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell-specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC-I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC-I. Importantly, combination HDAC-I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC-I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC. PMID:16583461

  3. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    SciTech Connect

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline; Sun, Jianmin; Jögi, Annika; Neumann, Drorit; Rönnstrand, Lars; Påhlman, Sven

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  4. Genetic analysis reveals that FLO11 upregulation and cell polarization independently regulate invasive growth in Saccharomyces cerevisiae.

    PubMed Central

    Palecek, S P; Parikh, A S; Kron, S J

    2000-01-01

    Under inducing conditions, haploid Saccharomyces cerevisiae perform a dimorphic transition from yeast-form growth on the agar surface to invasive growth, where chains of cells dig into the solid growth medium. Previous work on signaling cascades that promote agar invasion has demonstrated upregulation of FLO11, a cell-surface flocculin involved in cell-cell adhesion. We find that increasing FLO11 transcription is sufficient to induce both invasive and filamentous growth. A genetic screen for repressors of FLO11 isolated mutant strains that dig into agar (dia) and identified mutations in 35 different genes: ELM1, HSL1, HSL7, BUD3, BUD4, BUD10, AXL1, SIR2, SIR4, BEM2, PGI1, GND1, YDJ1, ARO7, GRR1, CDC53, HSC82, ZUO1, ADH1, CSE2, GCR1, IRA1, MSN5, SRB8, SSN3, SSN8, BPL1, GTR1, MED1, SKN7, TAF25, DIA1, DIA2, DIA3, and DIA4. Indeed, agar invasion in 20 dia mutants requires upregulation of the endogenous FLO11 promoter. However, 13 mutants promote agar invasion even with FLO11 clamped at a constitutive low-expression level. These FLO11 promoter-independent dia mutants establish distinct invasive growth pathways due to polarized bud site selection and/or cell elongation. Epistasis with the STE MAP kinase cascade and cytokinesis/budding checkpoint shows these pathways are targets of DIA genes that repress agar invasion by FLO11 promoter-dependent and -independent mechanisms, respectively. PMID:11063681

  5. Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis.

    PubMed

    Najafinobar, Neda; Mellander, Lisa J; Kurczy, Michael E; Dunevall, Johan; Angerer, Tina B; Fletcher, John S; Cans, Ann-Sofie

    2016-01-01

    Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release. PMID:27650365

  6. Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis

    PubMed Central

    Najafinobar, Neda; Mellander, Lisa J.; Kurczy, Michael E.; Dunevall, Johan; Angerer, Tina B.; Fletcher, John S.; Cans, Ann-Sofie

    2016-01-01

    Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release. PMID:27650365

  7. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening. PMID:24395411

  8. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.

  9. FtsZ-independent septal recruitment and function of cell wall remodelling enzymes in chlamydial pathogens

    PubMed Central

    Frandi, Antonio; Jacquier, Nicolas; Théraulaz, Laurence; Greub, Gilbert; Viollier, Patrick H.

    2014-01-01

    The nature and assembly of the chlamydial division septum is poorly defined due to the paucity of a detectable peptidoglycan (PG)-based cell wall, the inhibition of constriction by penicillin and the presence of coding sequences for cell wall precursor and remodelling enzymes in the reduced chlamydial (pan-)genome. Here we show that the chlamydial amidase (AmiA) is active and remodels PG in Escherichia coli. Moreover, forward genetics using an E. coli amidase mutant as entry point reveals that the chlamydial LysM-domain protein NlpD is active in an E. coli reporter strain for PG endopeptidase activity (ΔnlpI). Immunolocalization unveils NlpD as the first septal (cell-wall-binding) protein in Chlamydiae and we show that its septal sequestration depends on prior cell wall synthesis. Since AmiA assembles into peripheral clusters, trimming of a PG-like polymer or precursors occurs throughout the chlamydial envelope, while NlpD targets PG-like peptide crosslinks at the chlamydial septum during constriction. PMID:24953095

  10. FtsZ-independent septal recruitment and function of cell wall remodelling enzymes in chlamydial pathogens.

    PubMed

    Frandi, Antonio; Jacquier, Nicolas; Théraulaz, Laurence; Greub, Gilbert; Viollier, Patrick H

    2014-06-23

    The nature and assembly of the chlamydial division septum is poorly defined due to the paucity of a detectable peptidoglycan (PG)-based cell wall, the inhibition of constriction by penicillin and the presence of coding sequences for cell wall precursor and remodelling enzymes in the reduced chlamydial (pan-)genome. Here we show that the chlamydial amidase (AmiA) is active and remodels PG in Escherichia coli. Moreover, forward genetics using an E. coli amidase mutant as entry point reveals that the chlamydial LysM-domain protein NlpD is active in an E. coli reporter strain for PG endopeptidase activity (ΔnlpI). Immunolocalization unveils NlpD as the first septal (cell-wall-binding) protein in Chlamydiae and we show that its septal sequestration depends on prior cell wall synthesis. Since AmiA assembles into peripheral clusters, trimming of a PG-like polymer or precursors occurs throughout the chlamydial envelope, while NlpD targets PG-like peptide crosslinks at the chlamydial septum during constriction.

  11. Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance.

    PubMed

    Laws, Kaitlin M; Sampson, Leesa L; Drummond-Barbosa, Daniela

    2015-03-15

    Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate.

  12. Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism

    PubMed Central

    Rotin, Lianne E.; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L.; Minden, Mark D.; Slassi, Malik; Schimmer, Aaron D.

    2016-01-01

    Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease. PMID:26624983

  13. Deubiquitylating enzyme USP9x regulates radiosensitivity in glioblastoma cells by Mcl-1-dependent and -independent mechanisms

    PubMed Central

    Wolfsperger, F; Hogh-Binder, S A; Schittenhelm, J; Psaras, T; Ritter, V; Bornes, L; Huber, S M; Jendrossek, V; Rudner, J

    2016-01-01

    that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms. PMID:26775694

  14. The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism.

    PubMed

    Lakatos, Petra; Hegedűs, Csaba; Salazar Ayestarán, Nerea; Juarranz, Ángeles; Kövér, Katalin E; Szabó, Éva; Virág, László

    2016-08-01

    A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5J/cm(2)) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ-34+UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and -8. In conclusion, PJ-34 is a photosensitizer and PJ-34+UVA causes DNA damage and caspase-mediated cell death independently of PARP-1 inhibition. PMID:27427773

  15. Receptor-independent, direct membrane binding leads to cell surface lipid sorting and Syk kinase activation in dendritic cells

    PubMed Central

    Ng, Gilbert; Sharma, Karan; Ward, Sandra M.; Desrosiers, Melanie D.; Stephens, Leslie A.; Schoel, W. Michael; Li, Tonglei; Lowell, Clifford A.; Ling, Chang-Chun; Amrein, Matthias W.; Shi, Yan

    2008-01-01

    Summary Binding of particulate antigens by antigen presenting cells (APC) is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. Using atomic force microscopy as a tool for real time single cell activation analysis, we report that uric acid crystals can directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein based cellular contacts. Binding of particulate substances activates Syk kinase-dependent signaling in dendritic cells (DCs). These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation and adjuvanticity. PMID:18993083

  16. NIK regulates MT1-MMP activity and promotes glioma cell invasion independently of the canonical NF-κB pathway

    PubMed Central

    Duran, C L; Lee, D W; Jung, J-U; Ravi, S; Pogue, C B; Toussaint, L G; Bayless, K J; Sitcheran, R

    2016-01-01

    A growing body of evidence implicates the noncanonical NF-κB pathway as a key driver of glioma invasiveness and a major factor underlying poor patient prognoses. Here, we show that NF-κB-inducing kinase (NIK/MAP3K14), a critical upstream regulator of the noncanonical NF-κB pathway, is both necessary and sufficient for cell-intrinsic invasion, as well as invasion induced by the cytokine TWEAK, which is strongly associated with tumor pathogenicity. NIK promotes dramatic alterations in glioma cell morphology that are characterized by extensive membrane branching and elongated pseudopodial protrusions. Correspondingly, NIK increases the phosphorylation, enzymatic activity and pseudopodial localization of membrane type-1 matrix metalloproteinase (MT1-MMP/MMP14), which is associated with enhanced tumor cell invasion of three-dimensional collagen matrices. Moreover, NIK regulates MT1-MMP activity in cells lacking the canonical NF-κB p65 and cRel proteins. Finally, increased expression of NIK is associated with elevated MT1-MMP phosphorylation in orthotopic xenografts and co-expression of NIK and MT1-MMP in human tumors is associated with poor glioma patient survival. These data reveal a novel role of NIK to enhance pseudopodia formation, MT1-MMP enzymatic activity and tumor cell invasion independently of p65. Collectively, our findings underscore the therapeutic potential of approaches targeting NIK in highly invasive tumors. PMID:27270613

  17. Celecoxib and LLW-3-6 reduce survival of human glioma cells independently and synergistically with sulfasalazine

    PubMed Central

    Yerokun, Tokunbo; Winfield, Leyte L.

    2016-01-01

    Gliomas are among the most commonly diagnosed central nervous system tumors. Celecoxib has been utilized with success in the treatment of several types of cancer, including gliomas. The present study examined the antiproliferative effects of celecoxib and its benzimidazole-based analog, LLW-3-6, when used as co-treatments with sulfasalazine against human glioma LN18 cells. At 48-hour treatment, the glioma cells maintained 60% viability in the presence of celecoxib or LLW-3-6 at the maximum concentration tested (40 μM). Co-treatment of the glioma cells with a non-lethal dose (50 μM) of sulfasalazine and either celecoxib or LLW-3-6 (administered at different concentrations) resulted in improved inhibition of cell viability. The concentration of the molecules needed to reduce cell growth in the combined treatments was significantly less than that needed when either molecule was administered independently. Based on computational values, LLW-3-6 has physiochemical characteristics that should allow for improved bioavailability in comparison to that of celecoxib. PMID:26637851

  18. IL-2-independent and TNFα-dependent Expansion of Vβ5+ Natural Regulatory T Cells During Retrovirus Infection

    PubMed Central

    Myers, Lara; Joedicke, Jara J.; Carmody, Aaron B.; Messer, Ronald J.; Kassiotis, George; Dudley, Jaquelin P.; Dittmer, Ulf; Hasenkrug, Kim J.

    2013-01-01

    Friend virus (FV) infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of Neuropilin 1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4+ T cells that converted into induced Tregs. Analysis of Treg TCR Vβ chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vβ5+ Treg subset, which are known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vβ5+ subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8+ T cell response rather than the level of FV infection. Surprisingly, the expansion and accumulation of the Vβ5+ Tregs was IL-2 independent but dependent upon TNFα. These experiments reveal a subset-specific Treg induction by a new pathway. PMID:23645880

  19. Affinity-based Selection of Regulatory T Cells Occurs Independent of Agonist-mediated Induction of Foxp3 Expression1

    PubMed Central

    Relland, Lance M.; Mishra, Manoj K.; Haribhai, Dipica; Edwards, Brandon; Ziegelbauer, Jennifer; Williams, Calvin B.

    2010-01-01

    Natural regulatory T (nTreg)3 cells recognize self-peptides with high affinity, yet the understanding of how affinity influences their selection in the thymus is incomplete. We use altered peptide ligands in transgenic mice and in organ culture to create thymic environments spanning a broad range of ligand affinity. We demonstrate that the nTreg TCR repertoire is shaped by affinity-based selection, similar to conventional T (Tconv) cells. The effect of each ligand on the two populations is distinct, consistent with early nTreg cell lineage specification. Foxp3 expression is an independent process that does not rely on “high affinity” binding per se, but requires a high-potency agonistic interaction for its induction. The timing of ligand exposure, TGFβ signaling, and the organization of the thymic architecture are also important. The development of nTreg cells is therefore a multi-step process where ligand affinity, potency and timing of presentation all play roles in determining cell fate. PMID:19155480

  20. PTEN regulates angiogenesis and VEGF expression through phosphatase-dependent and -independent mechanisms in HepG2 cells.

    PubMed

    Tian, Tao; Nan, Ke-Jun; Wang, Shu-Hong; Liang, Xuan; Lu, Chuang-Xin; Guo, Hui; Wang, Wen-Juan; Ruan, Zhi-Ping

    2010-07-01

    Hepatocellular carcinoma (HCC) is a typical hypervascular tumor, and increased levels of vascular endothelial growth factor (VEGF) are associated with progression of HCC. Tumor suppression gene PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important antagonist of the phosphoinositide-3-kinase (PI3K)/adenosine triphosphate-dependent tyrosine kinase (Akt) pathway, is also commonly lost or mutated in HCC. However, the effect of PTEN on VEGF-mediated angiogenesis in HCC remains unknown. To explore this relationship, we expressed a panel of PTEN mutants in human HCC cells with low expression of PTEN (HepG2 cells). Overexpression of PTEN in HepG2 cells resulted in the downregulation of proliferation and migration of cocultured endothelial cells and decreased expression of hypoxia-inducible factor 1 (HIF-1) and VEGF. Similarly, using a nude mouse model, we demonstrated that PTEN decreased expression of HIF-1 and VEGF and suppressed HepG2-induced angiogenesis. This inhibitory effect was not observed in cells expressing a phosphatase-deficient PTEN mutant, suggesting that PTEN inhibits angiogenesis and VEGF through a phosphatase-dependent pathway. Strikingly, reintroducing the C2 domain of PTEN also resulted in a significant decrease in angiogenesis and VEGF expression, although it did not affect Akt phosphorylation or HIF-1 expression. In summary, this study suggests the novel viewpoint that PTEN suppresses angiogenesis and VEGF expression in HCC through both phosphatase-dependent and -independent mechanisms.

  1. Residual powders from Shochu distillation remnants induce apoptosis in human hepatoma cells via the caspase-independent pathway.

    PubMed

    Ohgidani, Masahiro; Komizu, Yuji; Goto, Koichi; Ueoka, Ryuichi

    2012-07-01

    Shochu distillation remnants (SDR) are by-products in the manufacturing process of the Japanese liquor Shochu and include various useful organic compounds derived from the fermentation of grains. We have obtained valuable powder (PSDR) from freeze-dried SDR by the treatment with ethanol. In this study, we examined the anticancer effects of barley-, rice-, and sweet potato-PSDR against HepG2 and HuH-7 cells of human hepatocellular carcinoma (HCC) in vitro. All PSDR inhibited the growth of both these HCC cells through the induction of apoptosis. Especially, barley-PSDR was the most effective for the growth inhibition and apoptosis induction of HCC cells of all PSDR. We next examined the apoptotic mechanisms induced by barley-PSDR. Decrease in mitochondrial membrane potential and release of cytochrome c from mitochondria were observed in HCC cells after the treatment with barley-PSDR. Furthermore, barley-PSDR induced the nuclear translocation of apoptosis-inducing factor (AIF) from mitochondria, while it did not significantly affect the activities of caspase-3, -8, and -9. The results suggested that barley-PSDR induced apoptosis against HCC cells via the caspase-independent mitochondrial pathway. The findings in this study suggest that PSDR has the possibility of therapeutic and/or preventive agents of HCC.

  2. An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity

    PubMed Central

    Gazzaniga, Francesca S.

    2014-01-01

    Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease. PMID:25320237

  3. Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells

    PubMed Central

    Ozkumur, Emre; Shah, Ajay M.; Ciciliano, Jordan C.; Emmink, Benjamin L.; Miyamoto, David T.; Brachtel, Elena; Yu, Min; Chen, Pin-i; Morgan, Bailey; Trautwein, Julie; Kimura, Anya; Sengupta, Sudarshana; Stott, Shannon L.; Karabacak, Nezihi Murat; Barber, Thomas A.; Walsh, John R.; Smith, Kyle; Spuhler, Philipp S.; Sullivan, James P.; Lee, Richard J.; Ting, David T.; Luo, Xi; Shaw, Alice T.; Bardia, Aditya; Sequist, Lecia V.; Louis, David N.; Maheswaran, Shyamala; Kapur, Ravi; Haber, Daniel A.; Toner, Mehmet

    2013-01-01

    Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing–enhanced microfluidic CTC capture platform, termed “CTC-iChip,” that is capable of sorting rare CTCs from whole blood at 107 cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer. PMID:23552373

  4. Chlamydia Induces Anchorage Independence in 3T3 Cells and Detrimental Cytological Defects in an Infection Model

    PubMed Central

    Knowlton, Andrea E.; Fowler, Larry J.; Patel, Rahul K.; Wallet, Shannon M.; Grieshaber, Scott S.

    2013-01-01

    Chlamydia are Gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI) Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV). We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect. PMID:23308295

  5. Development and evaluation of an anchorage-independent agar-based clonal assay for human primary breast carcinoma cells

    SciTech Connect

    Besch, G.J.

    1985-01-01

    The development and evaluation of an anchorage-independent clonal cytotoxic assay for primary human breast carcinoma cells is described in this thesis. This assay was developed in three stages which include: (1) the optimization of the production of a monodispersed cell suspension from solid breast carcinomas, (2) the systematic development of a growth medium for the clonal growth of these cells, and (3) the adaptation of these methods for use in the quantitation of cytotoxicity. The results of these studies indicated that hydrocortisone, fetal bovine serum and red blood cells stimulated the clonal growth of breast carcinoma cells. The optimal concentrations of these three factors were simultaneously determined using response surface methodology. These culture conditions were then used to develop radiation-cytotoxicity assays for both primary and recurrent breast carcinomas. The methodology developed and evaluated in this thesis may be useful to: (1) study the biology and radiobiology of human breast cancer, (2) customize the treatment of individual breast cancer patients, and (3) identify and/or develop new drugs and/or other treatment modalities for breast cancer.

  6. Estrogens decrease {gamma}-ray-induced senescence and maintain cell cycle progression in breast cancer cells independently of p53

    SciTech Connect

    Toillon, Robert-Alain . E-mail: robert.toillon@univ-lille1.fr; Magne, Nicolas; Laios, Ioanna; Castadot, Pierre; Kinnaert, Eric; Van Houtte, Paul; Desmedt, Christine B.Sc.; Leclercq, Guy; Lacroix, Marc

    2007-03-15

    Purpose: Sequential administration of radiotherapy and endocrine therapy is considered to be a standard adjuvant treatment of breast cancer. Recent clinical reports suggest that radiotherapy could be more efficient in association with endocrine therapy. The aim of this study was to evaluate the estrogen effects on irradiated breast cancer cells (IR-cells). Methods and Materials: Using functional genomic analysis, we examined the effects of 17-{beta}-estradiol (E{sub 2}, a natural estrogen) on MCF-7 breast cancer cells. Results: Our results showed that E{sub 2} sustained the growth of IR-cells. Specifically, estrogens prevented cell cycle blockade induced by {gamma}-rays, and no modification of apoptotic rate was detected. In IR-cells we observed the induction of genes involved in premature senescence and cell cycle progression and investigated the effects of E{sub 2} on the p53/p21{sup waf1/cip1}/Rb pathways. We found that E{sub 2} did not affect p53 activation but it decreased cyclin E binding to p21{sup waf1/cip1} and sustained downstream Rb hyperphosphorylation by functional inactivation of p21{sup waf1/cip1}. We suggest that Rb inactivation could decrease senescence and allow cell cycle progression in IR-cells. Conclusion: These results may help to elucidate the molecular mechanism underlying the maintenance of breast cancer cell growth by E{sub 2} after irradiation-induced damage. They also offer clinicians a rational basis for the sequential administration of ionizing radiation and endocrine therapies.

  7. System modeling of an air-independent solid oxide fuel cell system for unmanned undersea vehicles

    NASA Astrophysics Data System (ADS)

    Burke, A. Alan; Carreiro, Louis G.

    To examine the feasibility of a solid oxide fuel cell (SOFC)-powered unmanned undersea vehicle (UUV), a system level analysis is presented that projects a possible integration of the SOFC stack, fuel steam reformer, fuel/oxidant storage and balance of plant components into a 21-in. diameter UUV platform. Heavy hydrocarbon fuel (dodecane) and liquid oxygen (LOX) are chosen as the preferred reactants. A maximum efficiency of 45% based on the lower heating value of dodecane was calculated for a system that provides 2.5 kW for 40 h. Heat sources and sinks have been coupled to show viable means of thermal management. The critical design issues involve proper recycling of exhaust steam from the fuel cell back into the reformer and effective use of the SOFC stack radiant heat for steam reformation of the hydrocarbon fuel.

  8. Metformin inhibits proliferation and migration of glioblastoma cells independently of TGF-β2.

    PubMed

    Seliger, Corinna; Meyer, Anne-Louise; Renner, Kathrin; Leidgens, Verena; Moeckel, Sylvia; Jachnik, Birgit; Dettmer, Katja; Tischler, Ulrike; Gerthofer, Valeria; Rauer, Lisa; Uhl, Martin; Proescholdt, Martin; Bogdahn, Ulrich; Riemenschneider, Markus J; Oefner, Peter J; Kreutz, Marina; Vollmann-Zwerenz, Arabel; Hau, Peter

    2016-07-01

    To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor β 2 (TGF-β2) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-β2-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/-TGF-β2 revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-β2 and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-β2 mRNA expression (qRT-PCR), TGF-β2 protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-β2-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.

  9. MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points

    PubMed Central

    Dahlmans, Vivian E. H.; Spaapen, Frank; Salvaing, Juliette; Vanhove, Jolien; Geijselaers, Claudia; Bartels, Stefanie J. J.; Partouns, Iris; Neumann, Dietbert; Speel, Ernst-Jan; Takihara, Yoshihiro; Wouters, Bradly G.; Voncken, Jan Willem

    2015-01-01

    Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent. PMID:25853770

  10. Cell death-independent functions of granzymes: hit viruses where it hurts.

    PubMed

    van Domselaar, Robert; Bovenschen, Niels

    2011-09-01

    Granule exocytosis by cytotoxic lymphocytes is the key mechanism of our immune response to eliminate virus-infected cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes (GrA, GrB, GrH, GrK, GrM) that display distinct substrate specificities. Granzymes have mostly been studied for their ability to induce cell death. However, viruses have evolved many inhibitors to effectively block apoptosis. Evidence is emerging that granzymes also use noncytotoxic strategies to inhibit viral replication and potential viral reactivation from latency. Granzymes directly cleave viral or host cell proteins that are required in the viral life cycle. Furthermore, granzymes induce a pro-inflammatory cytokine response to create an antiviral environment. In this review, we summarize and discuss these novel strategies by which the immune system counteracts viral infections, and we will address the potential therapeutic applications that could emerge from this intriguing mechanism. PMID:21714121

  11. Steam Methane Reformation Testing for Air-Independent Solid Oxide Fuel Cell Systems

    NASA Technical Reports Server (NTRS)

    Mwara, Kamwana N.

    2015-01-01

    Recently, NASA has been looking into utilizing landers that can be propelled by LOX-CH (sub 4), to be used for long duration missions. Using landers that utilize such propellants, also provides the opportunity to use solid oxide fuel cells as a power option, especially since they are able to process methane into a reactant through fuel reformation. One type of reformation, called steam methane reformation, is a process to reform methane into a hydrogen-rich product by reacting methane and steam (fuel cell exhaust) over a catalyst. A steam methane reformation system could potentially use the fuel cell's own exhaust to create a reactant stream that is hydrogen-rich, and requires less internal reforming of the incoming methane. Also, steam reformation may hold some advantages over other types of reforming, such as partial oxidation (PROX) reformation. Steam reformation does not require oxygen, while up to 25 percent can be lost in PROX reformation due to unusable CO (sub 2) reformation. NASA's Johnson Space Center has conducted various phases of steam methane reformation testing, as a viable solution for in-space reformation. This has included using two different types of catalysts, developing a custom reformer, and optimizing the test system to find the optimal performance parameters and operating conditions.

  12. The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways

    PubMed Central

    Ishikawa, Fumihiko; Niiro, Hiroaki; Iino, Tadafumi; Yoshida, Shuro; Saito, Noriyuki; Onohara, Shinya; Miyamoto, Toshihiro; Minagawa, Hiroko; Fujii, Shin-ichiro; Shultz, Leonard D.; Harada, Mine

    2007-01-01

    Two distinct dendritic cell (DC) subsets, conventional DCs (cDCs) and plasmacytoid DCs (pDCs), have been shown to develop via either the myeloid or the lymphoid pathway in murine hematopoiesis. Lineage-specific phenotypes or functions of “myeloid” and “lymphoid” DCs, however, still remain elusive. Furthermore, such analysis has been particularly difficult in humans, due to lack of an assay system appropriate for the analysis of human stem and progenitor cell differentiation. Here, using a highly efficient xenotransplantation model, we extensively analyze the origin and the molecular signature of human DCs. Purified human common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) were intravenously transplanted into nonobese diabetic–severe combined immunodeficiency (NOD-scid)/IL2rγnull newborn mice. CMPs and CLPs displayed significant expansion in the xenogeneic host, and human cDC and pDC progeny were isolatable. Strikingly, each human DC subset possessed indistinguishable expression patterns of surface phenotype and gene transcripts regardless of their CMP or CLP origin, even at the genome-wide level. Thus, cDC and pDC normally develop after cells have committed to the myeloid or the lymphoid lineage in human hematopoiesis, while their transcriptional signatures are well preserved irrespective of their lineage origin. We propose that human DCs use unique and flexible developmental programs that cannot be categorized into the conventional myeloid or lymphoid pathway. PMID:17664352

  13. Molecular design and synthesis of a pH independent and cell permeant fluorescent dye and its applications.

    PubMed

    Jiao, Xiaojie; Liu, Chang; Huang, Kun; Zhang, Siwen; He, Song; Zhao, Liancheng; Zeng, Xianshun

    2015-06-21

    Fluorescent dyes have played crucial roles in the field of molecular imaging as fluorescent fluorophores. In this work, a novel water-soluble and pH-independent fluorescent xanthene dye, a hydroxyl regioisomeric 3',4'-benzorhodol, has been designed and synthesized. Compared with those of rhodol dyes, the absorption (ca. 570 nm) and maximum emission (ca. 620 nm) of the dye are largely red-shifted. Due to its ring-opened zwitterion structure in water media, the dye showed good membrane permeability and distributed in the whole cell cytoplasm upon incubation with live cells. Meanwhile, the dye could be easily modified to probes. The hydrazide derivative of the dye exhibited an excellent Hg(2+) selectivity over other relevant metal ions with a detection limit down to 3 nM. Thus, the excellent fluorescence properties and chemical properties of the dye allow it to be designed as a fluorescent chemosensor and biomarker for biological applications. PMID:25990913

  14. Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca(2+)-independent phenomenon

    PubMed Central

    1995-01-01

    MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KCl, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca2+ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KCl increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth. PMID:7622569

  15. Mountain cedar pollen induces IgE-independent mast cell degranulation, IL-4 production, and intracellular reactive oxygen species generation

    PubMed Central

    Endo, Shuichiro; Hochman, Daniel J.; Midoro-Horiuti, Terumi; Goldblum, Randall M.; Brooks, Edward G.

    2011-01-01

    Cedar pollens cause severe allergic disease throughout the world. We have previously characterized allergenic pollen glycoproteins from mountain cedar (Juniperus ashei) that bind to allergen-specific immunoglobulin E (IgE). In the present report, we investigated an alternative pathway of mast cell activation by mountain cedar pollen extract through IgE-independent mechanisms. We show that mountain cedar pollen directly induces mast cell serotonin and IL-4 release and enhances release induced by IgE cross-linking. Concomitant with mediator release, high levels of intracellular reactive oxygen species (ROS) were generated, and both ROS and serotonin release were inhibited by anti-oxidants. These findings suggest that alternative mechanisms exist whereby pollen exposure enhances allergic inflammatory mediator release through mechanisms that involve ROS. These mechanisms have the potential for enhancing the allergenic potency of pollens. PMID:21944563

  16. NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective, PAR-independent metabolic catastrophe and cell death induced by β-lapachone

    PubMed Central

    Moore, Z; Chakrabarti, G; Luo, X; Ali, A; Hu, Z; Fattah, F J; Vemireddy, R; DeBerardinis, R J; Brekken, R A; Boothman, D A

    2015-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD+ synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD+ pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, β-lapachone (β-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. β-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD+ consumption. Synergy with FK866+β-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD+ synthesis while increasing NAD+ consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)+ depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, μ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD+-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD+ synthesis and β-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD+ pools in cancer cells to sensitize them to ROS-mediated cell death by β-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors. PMID:25590809

  17. Ca2+-independent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell.

    PubMed

    Homann, U; Tester, M

    1997-06-10

    Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca2+ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 microM) free Ca2+ and nonhydrolyzable guanine nucleotides guanosine 5'-gamma-thio]triphosphate (GTP[gammaS]) or guanosine 5'-[beta-thio]diphosphate (GDP[betaS]). With less than 2 nM cytoplasmic free Ca2+, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[gammaS] or GDP[betaS]. In contrast, dialyzing protoplasts with 1 microM free Ca2+ resulted in a large increase in membrane capacitance that was slightly reduced by GTP[gammaS] and strongly inhibited by GDP[betaS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca2+-independent and whose regulation is currently not known and another that is stimulated by Ca2+ and modulated by GTP-binding proteins. We suggest that Ca2+-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca2+-stimulated exocytosis may play a role in gibberellic acid-stimulated alpha-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.

  18. Mitochondrial biogenesis is required for the anchorage-independent survival and propagation of stem-like cancer cells

    PubMed Central

    Peiris-Pagès, Maria; Ozsvari, Bela; Smith, Duncan L.; Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E.; Cappello, Anna Rita; Pezzi, Vincenzo; Lisanti, Michael P.; Sotgia, Federica

    2015-01-01

    Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFβ-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/β-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a ‘safe’ mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse

  19. MITF-independent pro-survival role of BRG1-containing SWI/SNF complex in melanoma cells.

    PubMed

    Ondrušová, Lubica; Vachtenheim, Jiri; Réda, Jiri; Záková, Petra; Benková, Kamila

    2013-01-01

    Metastasized malignant melanoma has a poor prognosis because of its intrinsic resistance to chemotherapy and radiotherapy. The central role in the melanoma transcriptional network has the transcription factor MITF (microphthalmia-associated transcription factor). It has been shown recently that the expression of MITF and some of its target genes require the SWI/SNF chromatin remodeling complex. Here we demonstrate that survival of melanoma cells requires functional SWI/SNF complex not only by supporting expression of MITF and its targets and but also by activating expression of prosurvival proteins not directly regulated by MITF. Microarray analysis revealed that besides the MITF-driven genes, expression of proteins like osteopontin, IGF1, TGFß2 and survivin, the factors known to be generally associated with progression of tumors and the antiapoptotic properties, were reduced in acute BRG1-depleted 501mel cells. Western blots and RT-PCR confirmed the microarray findings. These proteins have been verified to be expressed independently of MITF, because MITF depletion did not impair their expression. Because these genes are not regulated by MITF, the data suggests that loss of BRG1-based SWI/SNF complexes negatively affects survival pathways beyond the MITF cascade. Immunohistochemistry showed high expression of both BRM and BRG1 in primary melanomas. Exogenous CDK2, osteopontin, or IGF1 each alone partly relieved the block of proliferation imposed by BRG1 depletion, implicating that more factors, besides the MITF target genes, are involved in melanoma cell survival. Together these results demonstrate an essential role of SWI/SNF for the expression of MITF-dependent and MITF-independent prosurvival factors in melanoma cells and suggest that SWI/SNF may be a potential and effective target in melanoma therapy.

  20. Impact of thiamine deficiency on T-cell dependent and T-cell independent antibody production in lake trout

    USGS Publications Warehouse

    Ottinger, Christopher A.; Honeyfield, Dale C.; Densmore, Christine L.; Iwanowicz, Luke R.

    2012-01-01

    Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

  1. Pomegranate Juice Metabolites, Ellagic Acid and Urolithin A, Synergistically Inhibit Androgen-Independent Prostate Cancer Cell Growth via Distinct Effects on Cell Cycle Control and Apoptosis

    PubMed Central

    Vicinanza, Roberto; Henning, Susanne M.; Heber, David

    2013-01-01

    Ellagitannins (ETs) from pomegranate juice (PJ) are bioactive polyphenols with chemopreventive potential against prostate cancer (PCa). ETs are not absorbed intact but are partially hydrolyzed in the gut to ellagic acid (EA). Colonic microflora can convert EA to urolithin A (UA), and EA and UA enter the circulation after PJ consumption. Here, we studied the effects of EA and UA on cell proliferation, cell cycle, and apoptosis in DU-145 and PC-3 androgen-independent PCa cells and whether combinations of EA and UA affected cell proliferation. EA demonstrated greater dose-dependent antiproliferative effects in both cell lines compared to UA. EA induced cell cycle arrest in S phase associated with decreased cyclin B1 and cyclin D1 levels. UA induced a G2/M arrest and increased cyclin B1 and cdc2 phosphorylation at tyrosine-15, suggesting inactivation of the cyclin B1/cdc2 kinase complex. EA induced apoptosis in both cell lines, while UA had a less pronounced proapoptotic effect only in DU-145. Cotreatment with low concentrations of EA and UA dramatically decreased cell proliferation, exhibiting synergism in PC-3 cells evaluated by isobolographic analysis and combination index. These data provide information on pomegranate metabolites for the prevention of PCa recurrence, supporting the role of gut flora-derived metabolites for cancer prevention. PMID:23710216

  2. Acute Endoplasmic Reticulum Stress-Independent Unconventional Splicing of XBP1 mRNA in the Nucleus of Mammalian Cells.

    PubMed

    Wang, Yuanyuan; Xing, Pan; Cui, Wenjing; Wang, Wenwen; Cui, Yanfen; Ying, Guoguang; Wang, Xin; Li, Binghui

    2015-06-10

    The regulation of expression of X-box-binding protein-1 (XBP1), a transcriptional factor, involves an unconventional mRNA splicing that removes the 26 nucleotides intron. In contrast to the conventional splicing that exclusively takes place in the nucleus, determining the location of unconventional splicing still remains controversial. This study was designed to examine whether the unconventional spicing of XBP1 mRNA could occur in the nucleus and its possible biological relevance. We use RT-PCR reverse transcription system and the expand high fidelity PCR system to detect spliced XBP1 mRNA, and fraction cells to determine the location of the unconventional splicing of XBP1 mRNA. We employ reporter constructs to show the presence of unconventional splicing machinery in mammal cells independently of acute endoplasmic reticulum (ER) stress. Our results reveal the presence of basal unconventional splicing of XBP1 mRNA in the nucleus that also requires inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α) and can occur independently of acute ER stress. Furthermore, we confirm that acute ER stress induces the splicing of XBP1 mRNA predominantly occurring in the cytoplasm, but it also promotes the splicing in the nucleus. The deletion of 5'-nucleotides in XBP1 mRNA significantly increases its basal unconventional splicing, suggesting that the secondary structure of XBP1 mRNA may determine the location of unconventional splicing. These results suggest that the unconventional splicing of XBP1 mRNA can take place in the nucleus and/or cytoplasm, which possibly depends on the elaborate regulation. The acute ER stress-independent unconventional splicing in the nucleus is most likely required for the maintaining of day-to-day folding protein homeostasis.

  3. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

    PubMed Central

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung

    2016-01-01

    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions. PMID:27783042

  4. Curcumin induces senescence of primary human cells building the vasculature in a DNA damage and ATM-independent manner.

    PubMed

    Grabowska, Wioleta; Kucharewicz, Karolina; Wnuk, Maciej; Lewinska, Anna; Suszek, Małgorzata; Przybylska, Dorota; Mosieniak, Grazyna; Sikora, Ewa; Bielak-Zmijewska, Anna

    2015-02-01

    Curcumin is considered not only as a supplement of the diet but also as a drug in many types of diseases and even as a potential anti-aging compound. It can reduce inflammation that increases with age and accompanies almost all age-related diseases. It has been suggested that curcumin can play a beneficial role in the cardiovascular system. However, there are also data showing that curcumin can induce senescence in cancer cells, which is a beneficial effect in cancer therapy but an undesirable one in the case of normal cells. It is believed that cellular senescence accompanies age-related changes in the cardiovascular system. The aim of this study was to check if curcumin, in a certain range of concentrations, can induce senescence in cells building the vasculature. We have found that human vascular smooth muscle and endothelial cells derived from aorta are very sensitive to curcumin treatment and can senesce upon treatment with cytostatic doses. We observed characteristic senescence markers but the number of DNA damage foci decreased. Surprisingly, in vascular smooth muscle cell (VSMC) activation of DNA damage response pathway downstream of ataxia-telangiectasia mutated (ATM) was observed. ATM silencing and the supplementation of antioxidants, N-acetyl-L-cysteine (NAC) or trolox, did not reduce the number of senescent cells. Thus, we have shown that curcumin can induce senescence of cells building the vasculature, which is DNA damage and ATM independent and is not induced by increased reactive oxygen species (ROS) level. We postulate that an increase in the bioavailability of curcumin should be introduced very carefully considering senescence induction as a side effect.

  5. Epstein–Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation

    PubMed Central

    Hernando, Henar; Islam, Abul B. M. M. K.; Rodríguez-Ubreva, Javier; Forné, Ignasi; Ciudad, Laura; Imhof, Axel; Shannon-Lowe, Claire; Ballestar, Esteban

    2014-01-01

    Epstein–Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation. PMID:24097438

  6. Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2

    PubMed Central

    Gurgis, FMS; Åkerfeldt, MC; Heng, B; Wong, C; Adams, S; Guillemin, GJ; Johns, TG; Chircop, M; Munoz, L

    2015-01-01

    MAPK-activated protein kinase 2 (MK2) is a checkpoint kinase involved in the DNA damage response. MK2 inhibition enhances the efficacy of chemotherapeutic agents; however, whether MK2 inhibition alone, without concurrent chemotherapy, would attenuate survival of cancer cells has not been investigated. CMPD1 is a widely used non-ATP competitive inhibitor that prevents MK2 phosphorylation. We employed CMPD1 together with MK2 knock-down and ATP-competitive MK2 inhibitor III (MK2i) in a panel of glioblastoma cells to assess whether MK2 inhibition could induce cancer cell death. While CMPD1 was effective at selective killing of cancer cells, MK2i and MK2 knock-down had no effect on viability of glioblastoma cells. CMPD1 treatment induced a significant G2/M arrest but MK2i-treated cells were only minimally arrested at G1 phase. Intriguingly, at doses that were cytotoxic to glioblastoma cells, CMPD1 did not inhibit phosphorylation of MK2 and of its downstream substrate Hsp27. These results suggest that CMPD1 exhibits cytotoxic activity independently of MK2 inhibition. Indeed, we identified tubulin as a primary target of the CMPD1 cytotoxic activity. This study demonstrates how functional and mechanistic studies with appropriate selection of test compounds, combining genetic knock-down and pharmacological inhibition, coordinating timing and dose levels enabled us to uncover the primary target of an MK2 inhibitor commonly used in the research community. Tubulin is emerging as one of the most common non-kinase targets for kinase inhibitors and we propose that potential tubulin-targeting activity should be assessed in preclinical pharmacology studies of all novel kinase inhibitors. PMID:27551460

  7. Mechanisms and distribution of ion channels in retinal ganglion cells: using temperature as an independent variable.

    PubMed

    Fohlmeister, Jürgen F; Cohen, Ethan D; Newman, Eric A

    2010-03-01

    Trains of action potentials of rat and cat retinal ganglion cells (RGCs) were recorded intracellularly across a temperature range of 7-37 degrees C. Phase plots of the experimental impulse trains were precision fit using multicompartment simulations of anatomically reconstructed rat and cat RGCs. Action potential excitation was simulated with a "Five-channel model" [Na, K(delayed rectifier), Ca, K(A), and K(Ca-activated) channels] and the nonspace-clamped condition of the whole cell recording was exploited to determine the channels' distribution on the dendrites, soma, and proximal axon. At each temperature, optimal phase-plot fits for RGCs occurred with the same unique channel distribution. The "waveform" of the electrotonic current was found to be temperature dependent, which reflected the shape changes in the experimental action potentials and confirmed the channel distributions. The distributions are cell-type specific and adequate for soma and dendritic excitation with a safety margin. The highest Na-channel density was found on an axonal segment some 50-130 microm distal to the soma, as determined from the temperature-dependent "initial segment-somadendritic (IS-SD) break." The voltage dependence of the gating rate constants remains invariant between 7 and 23 degrees C and between 30 and 37 degrees C, but undergoes a transition between 23 and 30 degrees C. Both gating-kinetic and ion-permeability Q10s remain virtually constant between 23 and 37 degrees C (kinetic Q10s = 1.9-1.95; permeability Q10s = 1.49-1.64). The Q10s systematically increase for T <23 degrees C (kinetic Q10 = 8 at T = 8 degrees C). The Na channels were consistently "sleepy" (non-Arrhenius) for T <8 degrees C, with a loss of spiking for T <7 degrees C.

  8. Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways

    PubMed Central

    Willett, Julia L. E.; Gucinski, Grant C.; Fatherree, Jackson P.; Low, David A.; Hayes, Christopher S.

    2015-01-01

    Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal “translocation domains.” These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria. PMID:26305955

  9. Neuronal cell adhesion genes and antidepressant response in three independent samples.

    PubMed

    Fabbri, C; Crisafulli, C; Gurwitz, D; Stingl, J; Calati, R; Albani, D; Forloni, G; Calabrò, M; Martines, R; Kasper, S; Zohar, J; Juven-Wetzler, A; Souery, D; Montgomery, S; Mendlewicz, J; Girolamo, G D; Serretti, A

    2015-12-01

    Drug-effect phenotypes in human lymphoblastoid cell lines recently allowed to identify CHL1 (cell adhesion molecule with homology to L1CAM), GAP43 (growth-associated protein 43) and ITGB3 (integrin beta 3) as new candidates for involvement in the antidepressant effect. CHL1 and ITGB3 code for adhesion molecules, while GAP43 codes for a neuron-specific cytosolic protein expressed in neuronal growth cones; all the three gene products are involved in synaptic plasticity. Sixteen polymorphisms in these genes were genotyped in two samples (n=369 and 90) with diagnosis of major depressive episode who were treated with antidepressants in a naturalistic setting. Phenotypes were response, remission and treatment-resistant depression. Logistic regression including appropriate covariates was performed. Genes associated with outcomes were investigated in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) genome-wide study (n=1861) as both individual genes and through a pathway analysis (Reactome and String databases). Gene-based analysis suggested CHL1 rs4003413, GAP43 rs283393 and rs9860828, ITGB3 rs3809865 as the top candidates due to their replication across the largest original sample and the STAR*D cohort. GAP43 molecular pathway was associated with both response and remission in the STAR*D, with ELAVL4 representing the gene with the highest percentage of single nucleotide polymorphisms (SNPs) associated with outcomes. Other promising genes emerging from the pathway analysis were ITGB1 and NRP1. The present study was the first to analyze cell adhesion genes and their molecular pathways in antidepressant response. Genes and biomarkers involved in neuronal adhesion should be considered by further studies aimed to identify predictors of antidepressant response. PMID:25850031

  10. Cytokine-Independent Detection of Antigen-Specific Germinal Center T Follicular Helper Cells in Immunized Nonhuman Primates Using a Live Cell Activation-Induced Marker Technique.

    PubMed

    Havenar-Daughton, Colin; Reiss, Samantha M; Carnathan, Diane G; Wu, Jennifer E; Kendric, Kayla; Torrents de la Peña, Alba; Kasturi, Sudhir Pai; Dan, Jennifer M; Bothwell, Marcella; Sanders, Rogier W; Pulendran, Bali; Silvestri, Guido; Crotty, Shane

    2016-08-01

    A range of current candidate AIDS vaccine regimens are focused on generating protective HIV-neutralizing Ab responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective Ab responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GCs) are the engines of Ab affinity maturation. GC T follicular helper (Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as Ag-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining. Cytokine production by GC Tfh cells may be intrinsically limited in comparison with other Th effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify Ag-specific GC Tfh cells. RNA sequencing was performed using TCR-stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL-2Rα) and OX40 to be highly upregulated activation-induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison with intracellular cytokine staining, the AIM assay identified >10-fold more Ag-specific GC Tfh cells in HIV Env protein-immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In summary, AIM demonstrates that Ag-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function. PMID:27335502

  11. CD8 T Cell-Independent Antitumor Response and Its Potential for Treatment of Malignant Gliomas.

    PubMed

    Murphy, Katherine A; Griffith, Thomas S

    2016-01-01

    Malignant brain tumors continue to represent a devastating diagnosis with no real chance for cure. Despite an increasing list of potential salvage therapies, standard-of-care for these patients has not changed in over a decade. Immunotherapy has been seen as an exciting option, with the potential to offer specific and long lasting tumor clearance. The "gold standard" in immunotherapy has been the development of a tumor-specific CD8 T cell response to potentiate tumor clearance and immunological memory. While many advances have been made in the field of immunotherapy, few therapies have seen true success. Many of the same principles used to develop immunotherapy in tumors of the peripheral organs have been applied to brain tumor immunotherapy. The immune-specialized nature of the brain should call into question whether this approach is appropriate. Recent results from our own experiments require a rethinking of current dogma. Perhaps a CD8 T cell response is not sufficient for an organ as immunologically unique as the brain. Examination of previously elucidated principles of the brain's immune-specialized status and known immunological preferences should generate discussion and experimentation to address the failure of current therapies. PMID:27472363

  12. Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification

    PubMed Central

    Moudjou, Mohammed; Chapuis, Jérôme; Mekrouti, Mériem; Reine, Fabienne; Herzog, Laetitia; Sibille, Pierre; Laude, Hubert; Vilette, Didier; Andréoletti, Olivier; Rezaei, Human; Dron, Michel; Béringue, Vincent

    2016-01-01

    Prions are formed of misfolded assemblies (PrPSc) of the variably N-glycosylated cellular prion protein (PrPC). In infected species, prions replicate by seeding the conversion and polymerization of host PrPC. Distinct prion strains can be recognized, exhibiting defined PrPSc biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrPSc assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrPC glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrPC species of interest as substrate. Applying the technique to PrPC glycosylation mutants expressing cells revealed that neither PrPC nor PrPSc glycoform stoichiometry was instrumental to PrPSc formation and strainness perpetuation. Our study supports the view that strain properties, including PrPSc glycotype are enciphered within PrPSc structural backbone, not in the attached glycans. PMID:27384922

  13. α5β1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3

    PubMed Central

    Paul, Nikki R.; Allen, Jennifer L.; Chapman, Anna; Morlan-Mairal, Maria; Zindy, Egor; Jacquemet, Guillaume; Fernandez del Ama, Laura; Ferizovic, Nermina; Green, David M.; Howe, Jonathan D.; Ehler, Elisabeth; Hurlstone, Adam

    2015-01-01

    Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo. PMID:26370503

  14. Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis.

    PubMed

    Button, Robert W; Vincent, Joseph H; Strang, Conor J; Luo, Shouqing

    2016-02-01

    The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy.

  15. CD4+ T-cell-independent mechanisms suppress reactivation of latent tuberculosis in a macaque model of HIV coinfection.

    PubMed

    Foreman, Taylor W; Mehra, Smriti; LoBato, Denae N; Malek, Adel; Alvarez, Xavier; Golden, Nadia A; Bucşan, Allison N; Didier, Peter J; Doyle-Meyers, Lara A; Russell-Lodrigue, Kasi E; Roy, Chad J; Blanchard, James; Kuroda, Marcelo J; Lackner, Andrew A; Chan, John; Khader, Shabaana A; Jacobs, William R; Kaushal, Deepak

    2016-09-20

    The synergy between Mycobacterium tuberculosis (Mtb) and HIV in coinfected patients has profoundly impacted global mortality because of tuberculosis (TB) and AIDS. HIV significantly increases rates of reactivation of latent TB infection (LTBI) to active disease, with the decline in CD4(+) T cells believed to be the major causality. In this study, nonhuman primates were coinfected with Mtb and simian immunodeficiency virus (SIV), recapitulating human coinfection. A majority of animals exhibited rapid reactivation of Mtb replication, progressing to disseminated TB and increased SIV-associated pathology. Although a severe loss of pulmonary CD4(+) T cells was observed in all coinfected macaques, a subpopulation of the animals was still able to prevent reactivation and maintain LTBI. Investigation of pulmonary immune responses and pathology in this cohort demonstrated that increased CD8(+) memory T-cell proliferation, higher granzyme B production, and expanded B-cell follicles correlated with protection from reactivation. Our findings reveal mechanisms that control SIV- and TB-associated pathology. These CD4-independent protective immune responses warrant further studies in HIV coinfected humans able to control their TB infection. Moreover, these findings will provide insight into natural immunity to Mtb and will guide development of novel vaccine strategies and immunotherapies. PMID:27601645

  16. Retinoids induce cellular senescence in breast cancer cells by RAR-β dependent and independent pathways: Potential clinical implications (Review)

    PubMed Central

    SHILKAITIS, ANNE; GREEN, ALBERT; CHRISTOV, KONSTANTIN

    2015-01-01

    Most studies on cellular senescence (CS) have been performed in vitro by employing cytotoxic agents, irradiation, chromatin and telomerase modulators or by activating certain oncogenes. All these approaches usually lead to DNA damage, gene instability and/or chromatin alterations that primarily affect p53-p21 signaling. Little is known on whether retinoids and rexinoids, which are cell differentiation agents, can also induce CS in vitro and in vivo, and which molecular mechanisms are involved in promoting the senescent phenotype. We reviewed the recent publications on CS induced by retinoids and rexinoids in ER+ and ER− breast cancer cell lines and in corresponding animal models of mammary carcinogenesis which simulate those of human breast cancer. The role of retinoic acid receptors β2 and 5 (RARβ2 and RARβ5) and of receptor independent genes involved in mediating the senescence program of retinoids and rexinoids in ER+ and ER− breast cancer cells is discussed. Potential strategists for clinical implication of CS as biomarker of prognosis and of response to treatment with retinoids, rexinoids and with other cell differentiation and antitumor agents are outlined. PMID:25997921

  17. Growth hormone expression in murine bone marrow cells is independent of the pituitary transcription factor Pit-1.

    PubMed

    Kooijman, R; Malur, A; Van Buul-Offers, S C; Hooghe-Peters, E L

    1997-09-01

    GH has been shown to promote the development and function of leukocytes. The expression of both GH and GH-receptors in lymphoid cells has led to the hypothesis that GH acts in an autocrine or paracrine fashion. The described effects of GH on hematopoiesis and B cell development, led us to investigate GH expression in bone marrow cells. By immunocytochemistry, we show that bone marrow-derived granulocytes and macrophages contain immunoreactive GH. We found that 65 +/- 24% of the granulocytes were stained with anti-GH, whereas 5.8 +/- 1.5% of the granulocytes contained detectable amounts of GH mRNA as assessed by in situ hybridization. To address a possible alternative regulation mechanism in bone marrow and to establish whether locally derived GH might still play a role in pituitary-deficient dwarf mice, we also addressed GH expression in bone marrow from hypopituitary Snell dwarf mice. These mice have a mutated gene for the pituitary transcription factor Pit-1 that is deficient in DNA binding. Our finding that GH expression (immunoreactive protein and mRNA) in bone marrow cells from dwarf mice is similar to that in normal mice points to a Pit-1 independent regulation of GH in mouse bone marrow.

  18. A SET-domain-independent role of WRAD complex in cell-cycle regulatory function of mixed lineage leukemia

    PubMed Central

    Ali, Aamir; Veeranki, Sailaja Naga; Tyagi, Shweta

    2014-01-01

    MLL, the trithorax ortholog, is a well-characterized histone 3 lysine 4 methyltransferase that is crucial for proper regulation of the Hox genes during embryonic development. Chromosomal translocations, disrupting the Mll gene, lead to aggressive leukemia with poor prognosis. However, the functions of MLL in cellular processes like cell-cycle regulation are not well studied. Here we show that the MLL has a regulatory role during multiple phases of the cell cycle. RNAi-mediated knockdown reveals that MLL regulates S-phase progression and, proper segregation and cytokinesis during M phase. Using deletions and mutations, we narrow the cell-cycle regulatory role to the C subunit of MLL. Our analysis reveals that the transactivation domain and not the SET domain is important for the S-phase function of MLL. Surprisingly, disruption of MLL–WRAD interaction is sufficient to disrupt proper mitotic progression. These mitotic functions of WRAD are independent of SET domain of MLL and, therefore, define a new role of WRAD in subset of MLL functions. Finally, we address the overlapping and unique roles of the different SET family members in the cell cycle. PMID:24880690

  19. A SET-domain-independent role of WRAD complex in cell-cycle regulatory function of mixed lineage leukemia.

    PubMed

    Ali, Aamir; Veeranki, Sailaja Naga; Tyagi, Shweta

    2014-07-01

    MLL, the trithorax ortholog, is a well-characterized histone 3 lysine 4 methyltransferase that is crucial for proper regulation of the Hox genes during embryonic development. Chromosomal translocations, disrupting the Mll gene, lead to aggressive leukemia with poor prognosis. However, the functions of MLL in cellular processes like cell-cycle regulation are not well studied. Here we show that the MLL has a regulatory role during multiple phases of the cell cycle. RNAi-mediated knockdown reveals that MLL regulates S-phase progression and, proper segregation and cytokinesis during M phase. Using deletions and mutations, we narrow the cell-cycle regulatory role to the C subunit of MLL. Our analysis reveals that the transactivation domain and not the SET domain is important for the S-phase function of MLL. Surprisingly, disruption of MLL-WRAD interaction is sufficient to disrupt proper mitotic progression. These mitotic functions of WRAD are independent of SET domain of MLL and, therefore, define a new role of WRAD in subset of MLL functions. Finally, we address the overlapping and unique roles of the different SET family members in the cell cycle. PMID:24880690

  20. Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis

    PubMed Central

    Button, Robert W.; Vincent, Joseph H.; Strang, Conor J.; Luo, Shouqing

    2016-01-01

    The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy. PMID:26814436

  1. Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence

    PubMed Central

    Oladipupo, Sunday S.; Hu, Song; Santeford, Andrea C.; Yao, Junjie; Kovalski, Joanna R.; Shohet, Ralph V.; Maslov, Konstantin

    2011-01-01

    Neovascularization is a crucial component of tumor growth and ischemia. Although prior work primarily used disease models, delineation of neovascularization in the absence of disease can reveal intrinsic mechanisms of microvessel regulation amenable to manipulation in illness. We created a conditional model of epithelial HIF-1 induction in adult mice (TetON-HIF-1 mice). Longitudinal photoacoustic microscopy (L-PAM) was coincidentally developed for noninvasive, label-free serial imaging of red blood cell-perfused vasculature in the same mouse for weeks to months. TetON-HIF-1 mice evidenced 3 stages of neovascularization: development, maintenance, and transgene-dependent regression. Regression occurred despite extensive and tight pericyte coverage. L-PAM mapped microvascular architecture and quantified volumetric changes in neocapillary morphogenesis, arteriovenous remodeling, and microvessel regression. Developmental stage endothelial proliferation down-regulation was associated with a DNA damage checkpoint consisting of p53, p21, and endothelial γ-H2AX induction. The neovasculature was temporally responsive to VEGFR2 immuno-blockade, with the developmental stage sensitive, and the maintenance stage resistant, to DC101 treatment. L-PAM analysis also pinpointed microvessels ablated or resistant to VEGFR2 immuno-blockade. HIF-1–recruited myeloid cells did not mediate VEGFR2 inhibitor resistance. Thus, HIF-1 neovascularization in the absence of disease is self-regulated via cell autonomous endothelial checkpoints, and resistant to angiogenesis inhibitors independent of myeloid cells. PMID:21307392

  2. Mechanisms and Distribution of Ion Channels in Retinal Ganglion Cells: Using Temperature as an Independent Variable

    PubMed Central

    Cohen, Ethan D.; Newman, Eric A.

    2010-01-01

    Trains of action potentials of rat and cat retinal ganglion cells (RGCs) were recorded intracellularly across a temperature range of 7–37°C. Phase plots of the experimental impulse trains were precision fit using multicompartment simulations of anatomically reconstructed rat and cat RGCs. Action potential excitation was simulated with a “Five-channel model” [Na, K(delayed rectifier), Ca, K(A), and K(Ca-activated) channels] and the nonspace-clamped condition of the whole cell recording was exploited to determine the channels' distribution on the dendrites, soma, and proximal axon. At each temperature, optimal phase-plot fits for RGCs occurred with the same unique channel distribution. The “waveform” of the electrotonic current was found to be temperature dependent, which reflected the shape changes in the experimental action potentials and confirmed the channel distributions. The distributions are cell-type specific and adequate for soma and dendritic excitation with a safety margin. The highest Na-channel density was found on an axonal segment some 50–130 μm distal to the soma, as determined from the temperature-dependent “initial segment–somadendritic (IS-SD) break.” The voltage dependence of the gating rate constants remains invariant between 7 and 23°C and between 30 and 37°C, but undergoes a transition between 23 and 30°C. Both gating-kinetic and ion-permeability Q10s remain virtually constant between 23 and 37°C (kinetic Q10s = 1.9–1.95; permeability Q10s = 1.49–1.64). The Q10s systematically increase for T <23°C (kinetic Q10 = 8 at T = 8°C). The Na channels were consistently “sleepy” (non-Arrhenius) for T <8°C, with a loss of spiking for T <7°C. PMID:20053849

  3. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.

    PubMed

    Chan, O T; Hannum, L G; Haberman, A M; Madaio, M P; Shlomchik, M J

    1999-05-17

    The precise role of B cells in systemic autoimmunity is incompletely understood. Although B cells are necessary for expression of disease (Chan, O., and M.J. Shlomchik. 1998. J. Immunol. 160:51-59, and Shlomchik, M.J., M.P. Madaio, D. Ni, M. Trounstine, and D. Huszar. 1994. J. Exp. Med. 180:1295-1306), it is unclear whether autoantibody production, antigen presentation, and/or other B cell functions are required for the complete pathologic phenotype. To address this issue, two experimental approaches were used. In the first, the individual contributions of circulating antibodies and B cells were analyzed using MRL/MpJ-Faslpr (MRL/lpr) mice that expressed a mutant transgene encoding surface immunoglobulin (Ig), but which did not permit the secretion of circulating Ig. These mice developed nephritis, characterized by cellular infiltration within the kidney, indicating that B cells themselves, without soluble autoantibody production, exert a pathogenic role. The results indicate that, independent of serum autoantibody, functional B cells expressing surface Ig are essential for disease expression, either by serving as antigen-presenting cells for antigen-specific, autoreactive T cells, or by contributing directly to local inflammation.

  4. BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

    SciTech Connect

    Hinitt, C.A.M.; Wood, J.; Lee, S.S.; Williams, A.C.; Howarth, J.L.; Glover, C.P.; Uney, J.B.; Hague, A.

    2010-08-01

    Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

  5. 40.8% Efficient Inverted Triple-Junction Solar Cell with Two Independently Metamorphic Junctions

    SciTech Connect

    Geisz, J. F.; Friedman, D. J.; Ward, J. S.; Duda, A.; Olavarria, W. J.; Moriarty, T. E.; Kiehl, J. T.; Romero, M. J.; Norman, A. G.; Jones, K. M.

    2008-01-01

    A photovoltaic conversion efficiency of 40.8% at 326 suns concentration is demonstrated in a monolithically grown, triple-junction III-V solar cell structure in which each active junction is composed of an alloy with a different lattice constant chosen to maximize the theoretical efficiency. The semiconductor structure was grown by organometallic vapor phase epitaxy in an inverted configuration with a 1.83 eV Ga{sub .51}In{sub .49}P top junction lattice-matched to the GaAs substrate, a metamorphic 1.34 eV In{sub .04}Ga{sub .96}As middle junction, and a metamorphic 0.89 eV In{sub .37}Ga{sub .63}As bottom junction. The two metamorphic junctions contained approximately 1 x 10{sup 5} cm{sup -2} and 2-3 x 10{sup 6} cm{sup -2} threading dislocations, respectively.

  6. FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon

    PubMed Central

    Dover, Katarzyna; Marra, Christopher; Solinas, Sergio; Popovic, Marko; Subramaniyam, Sathyaa; Zecevic, Dejan; D'Angelo, Egidio; Goldfarb, Mitchell

    2016-01-01

    Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell's unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as <1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons. PMID:27666389

  7. MHC class II exacerbates demyelination in vivo independently of T cells.

    PubMed

    Hiremath, Meenaxi M; Chen, Vivian S; Suzuki, Kinuko; Ting, Jenny P Y; Matsushima, Glenn K

    2008-10-15

    We have shown previously the importance of MHC class II for central nervous system remyelination; however, the function of MHC class II during cuprizone-induced demyelination has not been examined. Here, we show that I-A(beta)-/- mice exhibit significantly reduced inflammation and demyelination. RAG-1(1/1) mice are indistinguishable from controls, indicating T cells may not play a role. The role of MHC class II depends on an intact cytoplasmic tail that leads to the production of IL-1beta, TNF-alpha, and nitric oxide, and oligodendrocyte apoptosis. Thus, the function of MHC class II cytoplasmic tail appears to increase microglial proliferation and activation that exacerbates demyelination. PMID:18805594

  8. Id-1 expression induces androgen-independent prostate cancer cell growth through activation of epidermal growth factor receptor (EGF-R).

    PubMed

    Ling, Ming-Tat; Wang, Xianghong; Lee, Davy T; Tam, P C; Tsao, Sai-Wah; Wong, Yong-Chuan

    2004-04-01

    The failure of prostate cancer treatment is largely due to the development of androgen independence, since the androgen depletion therapy remains the front-line option for this cancer. Previously, we reported that over-expression of the helix-loop-helix protein Id-1 was associated with progression of prostate cancer and ectopic expression of Id-1 induced serum-independent proliferation in prostate cancer cells. In the present study, we investigated if exogenous Id-1 expression in the androgen sensitive LNCaP cells had any effect on androgen-dependent cell growth and studied the molecular mechanisms involved. Using stable Id-1 transfectants, we found that expression of Id-1 was able to reduce androgen-stimulated growth and S phase fraction of the cell cycle in LNCaP cells, indicating that Id-1 may be involved in the development of androgen independence in these cells. The Id-1-induced androgen-independent prostate cancer cell growth was correlated with up-regulation of EGF-R (epidermal growth factor-receptor) and PSA (prostate specific antigen) expression, as confirmed by western blotting analysis and luciferase assays. In contrast, down-regulation of Id-1 in androgen-independent DU145 cells by its antisense oligonucleotides resulted in suppression of EGF-R expression at both transcriptional and protein levels. In addition, the results from immunohistochemistry study showed that Id-1 expression was significantly elevated in hormone refractory prostate cancer tissues when compared with the hormone-dependent tumours. Our results suggest that up-regulation of Id-1 in prostate cancer cells may be one of the mechanisms responsible for developing androgen independence and this process may be regulated through induction of EGF-R expression. Inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of androgen-independent prostate cancer cell growth.

  9. Impact of HIV-1 Membrane Cholesterol on Cell-Independent Lytic Inactivation and Cellular Infectivity.

    PubMed

    Kalyana Sundaram, Ramalingam Venkat; Li, Huiyuan; Bailey, Lauren; Rashad, Adel A; Aneja, Rachna; Weiss, Karl; Huynh, James; Bastian, Arangaserry Rosemary; Papazoglou, Elisabeth; Abrams, Cameron; Wrenn, Steven; Chaiken, Irwin

    2016-01-26

    Peptide triazole thiols (PTTs) have been found previously to bind to HIV-1 Env spike gp120 and cause irreversible virus inactivation by shedding gp120 and lytically releasing luminal capsid protein p24. Since the virions remain visually intact, lysis appears to occur via limited membrane destabilization. To better understand the PTT-triggered membrane transformation involved, we investigated the role of envelope cholesterol on p24 release by measuring the effect of cholesterol depletion using methyl beta-cyclodextrin (MβCD). An unexpected bell-shaped response of PTT-induced lysis to [MβCD] was observed, involving lysis enhancement at low [MβCD] vs loss of function at high [MβCD]. The impact of cholesterol depletion on PTT-induced lysis was reversed by adding exogenous cholesterol and other sterols that support membrane rafts, while sterols that do not support rafts induced only limited reversal. Cholesterol depletion appears to cause a reduced energy barrier to lysis as judged by decreased temperature dependence with MβCD. Enhancement/replenishment responses to [MβCD] also were observed for HIV-1 infectivity, consistent with a similar energy barrier effect in the membrane transformation of virus cell fusion. Overall, the results argue that cholesterol in the HIV-1 envelope is important for balancing virus stability and membrane transformation, and that partial depletion, while increasing infectivity, also makes the virus more fragile. The results also reinforce the argument that the lytic inactivation and infectivity processes are mechanistically related and that membrane transformations occurring during lysis can provide an experimental window to investigate membrane and protein factors important for HIV-1 cell entry.

  10. Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

    PubMed Central

    GUO, QIANQIAN; LIU, ZHIYAN; JIANG, LILI; LIU, MENGJIE; MA, JIEQUN; YANG, CHENGCHENG; HAN, LILI; NAN, KEJUN; LIANG, XUAN

    2016-01-01

    Metformin, the most widely administered oral anti-diabetic therapeutic agent, exerts its glucose-lowering effect predominantly via liver kinase B1 (LKB1)-dependent activation of adenosine monophosphate-activated protein kinase (AMPK). Accumulating evidence has demonstrated that metformin possesses potential antitumor effects. However, whether the antitumor effect of metformin is via the LKB1/AMPK signaling pathway remains to be determined. In the current study, the effects of metformin on proliferation, cell cycle progression, and apoptosis of human non-small cell lung cancer (NSCLC) H460 (LKB1-null) and H1299 (LKB1-positive) cells were assessed, and the role of LKB1/AMPK signaling in the anti-growth effects of metformin were investigated. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis were assessed by flow cytometry, and protein expression levels were measured by western blotting. Metformin inhibited proliferation, induced significant cell cycle arrest at the G0–G1 phase and increased apoptosis in NSCLC cells in a time- and concentration-dependent manner, regardless of the level of LKB1 protein expression. Furthermore, knockdown of LKB1 with short hairpin RNA (shRNA) did not affect the antiproliferative effect of metformin in the H1299 cells. Metformin stimulated AMPK phosphorylation and subsequently suppressed the phosphorylation of mammalian target of rapamycin and its downstream effector, 70-kDa ribosomal protein S6 kinase in the two cell lines. These effects were abrogated by silencing AMPK with small interfering RNA (siRNA). In addition, knockdown of AMPK with siRNA inhibited the effect of metformin on cell proliferation in the two cell lines. These results provide evidence that the growth inhibition of metformin in NSCLC cells is mediated by LKB1-independent activation of AMPK, indicating that metformin may be a potential therapeutic agent for the treatment of

  11. Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.).

    PubMed

    Thyssen, Gregory N; Song, Xianliang; Naoumkina, Marina; Kim, Hee-Jin; Fang, David D

    2014-07-01

    The mitochondrial genomes of flowering plants exist both as a "master circle" chromosome and as numerous subgenomic sublimons that are generated by intramolecular recombination. Differential stability or replication of these sublimons allows individual mitochondrial gene copy numbers to vary independently between different cell types and developmental stages. Our objective was to determine the relationship between mitochondrial gene copy number and transcript abundance in the elongating fiber cells of Upland cotton (Gossypium hirsutum L.). We compared RNA and DNA from cotton fiber cells at five developmental time points from early elongation through secondary cell wall thickening from the Ligon-lintless 2 (Li2) short fiber mutant and its wild type near isogenic line (NIL) DP5690. Mitochondrial gene copy number decreased from 3 to 8-DPA in the developing cotton fiber cells while transcript levels remained low. As secondary cell wall biosynthesis began in developing fibers, the expression levels and copy numbers of mitochondrial genes involved in energy production and respiration were up-regulated in wild type cotton DP5690. However, the short fiber mutant Li2, failed to increase expression of these genes, which include three subunits of ATP synthase, atp1, atp8 and atp9 and two cytochrome genes cox1 and cob. At the same time, Li2 failed to increase the copy numbers of these highly expressed genes. Surprisingly, we found that when mitochondrial genes were highly transcribed, they also had very high copy numbers. This observation suggests that in developing cotton fibers, increased mitochondrial sublimon replication may support increases in gene transcription.

  12. Senescent mesenchymal cells accumulate in human fibrosis by a telomere-independent mechanism and ameliorate fibrosis through matrix metalloproteinases.

    PubMed

    Pitiyage, Gayani Nadika; Slijepcevic, Predrag; Gabrani, Aliya; Chianea, Yaghoub Gozaly; Lim, Kue Peng; Prime, Stephen Stewart; Tilakaratne, Wanninayake Mudiyanselage; Fortune, Farida; Parkinson, Eric Kenneth

    2011-04-01

    Fibrosis can occur in many organs, where it is a debilitating and preneoplastic condition. The senescence of acti