9 research outputs found

    Sub-lethal concentrations of specific chemotherapy drugs enhance reovirus-induced apoptosis.

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    <p>Cell viability of HCT116 p53+/+ cells after exposure to various concentrations of (<b>A</b>) ActD, (<b>B</b>) Dox or (<b>C</b>) Etp. MTS reagents were added at 48 h post treatment. Accumulation of p53 was determined by western blot analysis using p53-specific antibody. Representative data from two independent experiments (triplicate wells, <i>±s.e.m.</i>) is shown. <b>D</b>. Apoptosis induced by reovirus alone, sub-lethal concentrations of ActD/Etp/Dox alone (Nutlin-3 as control), or the combination of reovirus and the drug. HCT116 p53+/+ and p53−/− cells were infected by reovirus at an MOI of 1 for 1 h and then supplemented with media with or without low concentrations of Dox, Etp or ActD. 1/10,000 dilution of DMSO in cell culture media was used as vehicle control. Percentage of apoptotic cells was determined by double-staining with Annexin V and 7-AAD at 24 hpi. Student’s <i>t</i>-test was used to compare percentage of cell death between reovirus infected cells and cells treated by the combination of reovirus and drugs; <i>*p</i><0.05; <i>**p</i><0.001; <i>***p</i><0.0001.</p

    NF-κB activation is important forenhanced apoptosis induced by reovirus and ActD/Etp.

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    <p><b>A. <i>NF-κB activation was determined by NF-κB p65 subunit nuclear translocation.</i></b> HCT116 p53+/+ and p53−/− cells were infected with reovirus at an MOI of 1 in the presence or absence of ActD (0.63 nM) or Etp (1.6 µM) treatment. Cells were fixed at 12-h post ActD/Etp treatment and cells were stained with anti-p65, anti-reo antibody and To-pro-3 for nucleus staining. <b>B. </b><b><i>NF-κB inhibitor N significantly reduces cell death induced by the combination of reovirus and ActD/Etp.</i></b> HC116 p53+/+ cells were treated with NF-κB inhibitor N for 1 h before reovirus infection in the absence or presence of either ActD (0.63 nM) or Etp (1.6 µM) treatment. Cells were collected at 24 hpi for PI staining and the percentage of sub-G1 for each sample was normalized to reovirus infection alone (±<i>s.e.m.</i>, n = 3). Student’s <i>t</i>-test was used to compare two groups of data; <i>*p</i><0.05.</p

    Enhanced apoptosis induced by reovirus and ActD/Etp depends on virus replication and caspase activities.

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    <p><b>A.</b> Percentage of cells that were infected by reovirus with or without the treatment of Dox, Etp, or ActD at 24 hpi, determined by FACS analysis using anti-reovirus antibody. <b>B. </b><b><i>Caspase inhibitor ZVAD blocks the enhancement of cell death induced by the combination of reovirus and a low concentration of ActD or Etp.</i></b> HCT116 p53+/+ and p53−/− cells were treated with ZVAD for 1 h before reovirus infection. Following reovirus infection, ZVAD and a low concentration of ActD or Etp were added to culture media of reovirus-infected cells. Cell death was determined by Annexin V and 7-AAD staining at 24 hpi. Student’s <i>t</i>-test was used to compare two groups of data; <i>*p</i><0.05 and <i>***p</i><0.0001. <b>C</b>. Immunohistochemical staining of the plaques formed on reovirus-infected HCT116 cells in the presence or absence of ActD (0.63 nM) or Etp (1.6 µM).</p

    Reovirus combined with ActD/Etp significantly enhances p53-target genes expression.

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    <p>(<b>A</b>) <i>noxa</i>, (<b>B</b>) <i>puma</i>, (<b>C</b>) <i>bax</i> and (<b>D</b>) <i>p21</i> expression levels in HCT116 p53+/+ and p53−/− cells treated with reovirus (reo), Etp/ActD or the combination of Etp/ActD and reovirus. RNA samples were collected at 24 hpi and processed for real-time q-PCR analysis using gene-specific primers (<i>±s.e.m.</i>, n = 3). Student’s <i>t</i>-test was used to compare expression levels between reovirus-infected cells and cells treated with the combination of reovirus and a drug; <i>*p</i><0.05, <i>**p</i><0.001 and <i>***p</i><0.0001.</p

    Marie-Claire / dir. Jean Prouvost

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    15 janvier 19441944/01/15 (A0,N304)-1944/01/15.Appartient à l’ensemble documentaire : UnivJeun

    Oncogenic RAS-induced downregulation of ATG12 is required for survival of malignant intestinal epithelial cells

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    <p>Activating mutations of RAS GTPase contribute to the progression of many cancers, including colorectal carcinoma. So far, attempts to develop treatments of mutant RAS-carrying cancers have been unsuccessful due to insufficient understanding of the salient mechanisms of RAS signaling. We found that RAS downregulates the protein ATG12 in colon cancer cells. ATG12 is a mediator of autophagy, a process of degradation and reutilization of cellular components. In addition, ATG12 can kill cells via autophagy-independent mechanisms. We established that RAS reduces ATG12 levels in cancer cells by accelerating its proteasomal degradation. We further observed that RAS-dependent ATG12 loss in these cells is mediated by protein kinases MAP2K/MEK and MAPK1/ERK2-MAPK3/ERK1, known effectors of RAS. We also demonstrated that the reversal of the effect of RAS on ATG12 achieved by the expression of exogenous ATG12 in cancer cells triggers both apoptotic and nonapoptotic signals and efficiently kills the cells. ATG12 is known to promote autophagy by forming covalent complexes with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is driven by RAS-dependent loss of ATG12 in these cells.</p

    Additional file 2: Figure. S1. of Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis

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    a,b. Surfen affects viability of cultured bone marrow derived macrophages (BMDMs) at higher doses (a as assessed by 7-ADD staining, b as assessed by MTT assay, doses indicated). c. Surfen (5 μM) binding to BMDMs is reduced by co-application of heparitinase-III and chondroitinase ABC, alone or in combination. Data is shown as mean ± SEM from 4 independent experiments. Significance compares surfen with vehicle unless otherwise indicated by cross bars (* = P < 0.05) (TIFF 1367 kb

    DataSheet_2_After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.docx

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    CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.</p

    DataSheet_1_After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.pdf

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    CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.</p
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