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  • Cell target genes of Epstein-Barr virus transcription factor EBNA-2: induction of the p55alpha regulatory subunit of PI3-kinase and its role in survival of EREB2.5 cells. 16963743

    Microarray analysis covering most of the annotated RNAs in the human genome identified a panel of genes induced by the Epstein-Barr virus (EBV) EBNA-2 transcription factor in the EREB2.5 human B-lymphoblastoid cell line without the need for any intermediate protein synthesis. Previous data indicating that PIK3R1 RNA (the alpha regulatory subunit of PI3-kinase) was induced were confirmed, but it is now shown that it is the p55alpha regulatory subunit that is induced. Several EBV-immortalized lymphoblastoid cell lines were shown to express p55alpha. Expression of PI3-kinase p85 regulatory and p110 catalytic subunits was not regulated by EBNA-2. Proliferation of EREB2.5 lymphoblastoid cells was inhibited by RNAi knock-down of p55alpha protein expression, loss of p55alpha being accompanied by an increase in apoptosis. p55alpha is thus a functional target of EBNA2 in EREB2.5 cells and the specific regulation of p55alpha by EBV will provide an opportunity to investigate the physiological function of p55alpha in this human cell line.
    Document Type:
    Reference
    Product Catalog Number:
    06-195

  • Cell type-specific targeting dissociates the therapeutic from the adverse effects of protein kinase inhibition in allergic skin disease. 22615377

    The kinase p38α, originally identified because of its endotoxin- and cytokine-inducible activity and affinity for antiinflammatory compounds, has been posited as a promising therapeutic target for various immune-mediated disorders. In clinical trials, however, p38α inhibitors produced adverse skin reactions and other toxic effects that often outweighed their benefits. Such toxicity may arise from a perturbation of physiological functions unrelated to or even protective against the disease being treated. Here, we show that the effect of interfering with p38α signaling can be therapeutic or adverse depending on the targeted cell type. Using a panel of mutant mice devoid of p38α in distinct cell types and an experimental model of allergic skin disease, we find that dendritic cell (DC)-intrinsic p38α function is crucial for both antigen-specific T-cell priming and T-cell-mediated skin inflammation, two independent processes essential for the immunopathogenesis. By contrast, p38α in other cell types serves to prevent excessive inflammation or maintain naïve T-cell pools in the peripheral lymphoid tissues. These findings highlight a dilemma in the clinical use of p38α inhibitors, yet also suggest cell-selective targeting as a potential solution for improving their therapeutic index.
    Document Type:
    Reference
    Product Catalog Number:
    04-384

  • p-MAPK1/3 and DUSP6 regulate epididymal cell proliferation and survival in a region-specific manner in mice. 20650883

    A fully developed, functional epididymis is important for male fertility. In particular, it is apparent that without the most proximal region, the initial segment (IS), infertility results. Therefore, it is important to understand the development and regulation of this crucial epididymal region. We have previously shown that many functions of the IS are regulated by luminal fluid factors/lumicrine factors from the testis. This study provides evidence that lumicrine factors activated the ERK pathway only in epithelial cells of the IS from Postnatal Day (P) 14 to P19 and sustained this activation into adulthood. The activated ERK pathway promoted cell proliferation and differentiation in the developing IS, although in the adult, its role was switched to maintain cell survival. To understand further the regulation of cell proliferation in the IS, we examined the role of DUSP6, an MAPK1/3 (ERK1/2) preferred phosphatase that is also regulated by lumicrine factors in the IS. Utilizing Dusp6(-/-) mice, our studies, surprisingly, revealed that Dusp6 was a major regulator of cell proliferation in the caput and corpus regions, whereas components of the ERK pathway, together with PTEN and SRC, were the major regulators of cell proliferation in the IS. We hypothesize that region-specific regulation of cell proliferation is caused by differences in the balance of activities between pro- and antiproliferation signaling pathway components for each epididymal region. An understanding of the mechanisms of cell proliferation may provide clues as to why the epididymis rarely succumbs to cancer.
    Document Type:
    Reference
    Product Catalog Number:
    06-570
    Product Catalog Name:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker

  • Progesterone receptor enhances breast cancer cell motility and invasion via extranuclear activation of focal adhesion kinase. 20233709

    While progesterone plays multiple roles in the process of breast development and differentiation, its role in breast cancer is less understood. We have shown previously that progestins stimulate breast cancer cell migration and invasion because of the activation of rapid signaling cascades leading to modifications in the actin cytoskeleton and cell membrane that are required for cell movement. In this study, we have investigated the effects of progesterone on the formation of focal adhesion (FA) complexes, which provide anchoring sites for cell attachment to the extracellular matrix during cell movement and invasion. In T47-D breast cancer cells, progesterone rapidly enhances FA kinase (FAK) phosphorylation at Tyr(397) in a time- and concentration-dependent manner. As a result, exposure to progesterone leads to increased formation of FA complexes within specialized cell membrane protrusions. The cascade of events required for this phenomenon involves progesterone receptor interaction with the tyrosine kinase c-Src, which activates the phosphatidylinositol-3-kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase complex. In the presence of progesterone, T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices, which is reversed by small interfering RNAs abrogating FAK. In conclusion, progesterone promotes breast cancer cell movement and invasion by facilitating the formation of FA complexes via the rapid regulation of FAK. These results provide novel mechanistic views on the effects of progesterone on breast cancer progression, and may in the future be helpful to develop new strategies for the treatment of endocrine-sensitive breast cancers.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • CG0006, a novel histone deacetylase inhibitor, induces breast cancer cell death via histone-acetylation and chaperone-disrupting pathways independent of ER status. 21184271

    We previously reported that CG0006, a novel hydroxamate-based pan-histone deacetylase inhibitor (HDACI), suppresses the growth of human cancer cells. Here, we tested the ability of CG0006 to inhibit breast cancer cell proliferation in relation to estrogen receptor (ER) status, and examined changes in the expression of cell-cycle regulatory proteins. CG0006 effects on the proliferation of multiple human cancer cell lines were tested using MTT and MTS assays. Changes in estrogen-signaling proteins and cell-cycle regulatory proteins were examined by western blotting and quantitative RT-PCR, and cell-cycle effects were tested using flow cytometry. CG0006 increased histone H3 and H4 acetylation, up-regulated p21 protein, and promoted cell-cycle arrest, inducing G(2)/M-phase accumulation in ER-positive MCF7 cells, and G(1)- and G(2)/M-phase accumulation in ER-negative MDA-MB-231 cells. In both cell types, CG0006 treatment (1 μM) reduced the levels of the estrogen-signaling proteins ERα and cyclin D1, and promoted massive degradation of cell-cycle regulatory proteins. CG0006 down-regulated the histone deacetylase HDAC6 at the protein level in association with a subsequent increase in Hsp90 and α-tubulin acetylation. HDAC6 depletion using small interfering RNA produced a protein-degradation phenotype similar to that of CG0006 treatment. These findings suggest that CG0006 inhibits breast cancer cell growth by two different pathways: a histone acetylation-dependent pathway, and a non-epigenetic pathway that disrupts chaperone function.
    Document Type:
    Reference
    Product Catalog Number:
    06-748

  • The interferon-gamma-induced murine guanylate-binding protein-2 inhibits rac activation during cell spreading on fibronectin and after platelet-derived growth factor trea ... 20505078

    Exposure of cells to certain cytokines can alter how these same cells respond to later cues from other agents, such as extracellular matrix or growth factors. Interferon (IFN)-gamma pre-exposure inhibits the spreading of fibroblasts on fibronectin. Expression of the IFN-gamma-induced GTPase murine guanylate-binding protein-2 (mGBP-2) can phenocopy this inhibition and small interfering RNA knockdown of mGBP-2 prevents IFN-gamma-mediated inhibition of cell spreading. Either IFN-gamma treatment or mGBP-2 expression inhibits Rac activation during cell spreading. Rac is required for cell spreading. mGBP-2 also inhibits the activation of Akt during cell spreading on fibronectin. mGBP-2 is incorporated into a protein complex containing the catalytic subunit of phosphatidylinositol 3-kinase (PI3-K), p110. The association of mGBP-2 with p110 seems important for the inhibition of cell spreading because S52N mGBP-2, which does not incorporate into the protein complex with p110, is unable to inhibit cell spreading. PI3-K activation during cell spreading on fibronectin was inhibited in the presence of mGBP-2. Both IFN-gamma and mGBP-2 also inhibit cell spreading initiated by platelet-derived growth factor treatment, which is also accompanied by inhibition of Rac activation by mGBP-2. This is the first report of a novel mechanism by which IFN-gamma can alter how cells respond to subsequent extracellular signals, by the induction of mGBP-2.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • R-Ras is required for murine dendritic cell maturation and CD4+ T-cell priming. 22174156

    R-Ras is a member of the RAS superfamily of small GTP-binding proteins. The physiologic function of R-Ras has not been fully elucidated. We found that R-Ras is expressed by lymphoid and nonlymphoid tissues and drastically up-regulated when bone marrow progenitors are induced to differentiate into dendritic cells (DCs). To address the role of R-Ras in DC functions, we generated a R-Ras-deficient mouse strain. We found that tumors induced in Rras(-/-) mice formed with shorter latency and attained greater tumor volumes. This finding has prompted the investigation of a role for R-Ras in the immune system. Indeed, Rras(-/-) mice were impaired in their ability to prime allogeneic and antigen-specific T-cell responses. Rras(-/-) DCs expressed lower levels of surface MHC class II and CD86 in response to lipopolysaccharide compared with wild-type DCs. This was correlated with a reduced phosphorylation of p38 and Akt. Consistently, R-Ras-GTP level was increased within 10 minutes of lipopolysaccharide stimulation. Furthermore, Rras(-/-) DCs have attenuated capacity to spread on fibronectin and form stable immunologic synapses with T cells. Altogether, these findings provide the first demonstration of a role for R-Ras in cell-mediated immunity and further expand on the complexity of small G-protein signaling in DCs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • MT1-MMP- and Cdc42-dependent signaling co-regulate cell invasion and tunnel formation in 3D collagen matrices. 19934222

    Complex signaling events control tumor invasion in three-dimensional (3D) extracellular matrices. Recent evidence suggests that cells utilize both matrix metalloproteinase (MMP)-dependent and MMP-independent means to traverse 3D matrices. Herein, we demonstrate that lysophosphatidic-acid-induced HT1080 cell invasion requires membrane-type-1 (MT1)-MMP-mediated collagenolysis to generate matrix conduits the width of a cellular nucleus. We define these spaces as single-cell invasion tunnels (SCITs). Once established, cells can migrate within SCITs in an MMP-independent manner. Endothelial cells, smooth muscle cells and fibroblasts also generate SCITs during invasive events, suggesting that SCIT formation represents a fundamental mechanism of cellular motility within 3D matrices. Coordinated cellular signaling events are required during SCIT formation. MT1-MMP, Cdc42 and its associated downstream effectors such as MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) and Pak4 (p21 protein-activated kinase 4), protein kinase Calpha and the Rho-associated coiled-coil-containing protein kinases (ROCK-1 and ROCK-2) coordinate signaling necessary for SCIT formation. Finally, we show that MT1-MMP and Cdc42 are fundamental components of a co-associated invasion-signaling complex that controls directed single-cell invasion of 3D collagen matrices.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3211

  • Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors. 18948384

    This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2-hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4-green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2-hybrid and glutathione-S-transferase pulldown analyses show Robo4 binding to a Wiskott-Aldrich syndrome protein (WASP), neural Wiskott-Aldrich syndrome protein, and WASP-interacting protein actin-nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4-mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation-promoting factors to promote endothelial cell migration.
    Document Type:
    Reference
    Product Catalog Number:
    07-066

  • Receptor-interacting protein shuttles between cell death and survival signaling pathways. 19955213

    Cross-talk between apoptosis and survival signaling pathways is crucial for regulating tissue processes and mitigating disease. We report that anoikis-apoptosis triggered by loss of extracellular matrix contacts-activates a CD95/Fas-mediated signaling pathway regulated by receptor-interacting protein (RIP), a kinase that shuttles between CD95/Fas-mediated cell death and integrin/focal adhesion kinase (FAK)-mediated survival pathways. RIP's death domain was critical for RIP and Fas association to mediate anoikis. Fas or RIP attenuation reduced this association and suppressed anoikis, whereas their overexpression had the reverse effect. Overexpressing FAK restored RIP and FAK association and inhibited anoikis. Thus, RIP shuttles between CD95/Fas death and FAK survival signaling to mediate anoikis.
    Document Type:
    Reference
    Product Catalog Number:
    05-182
    Product Catalog Name:
    Anti-FAK Antibody, clone 2A7