Embryo Fibroblasts (embryo + fibroblast)

Distribution by Scientific Domains

Kinds of Embryo Fibroblasts

  • mouse embryo fibroblast

  • Selected Abstracts

    PPAR,1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion

    Young C. Cho
    Abstract Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor ,1 (PPAR,1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPAR,1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l -mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPAR,1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPAR,1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPAR, was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPAR,1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPAR, synthesis and subsequent adipocyte differentiation. © 2003 Wiley-Liss, Inc. [source]

    Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers

    Michael W. Wolff
    Abstract Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300,500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE®, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor-intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host-cell DNA levels for human vaccines. This study compares different ion exchange and pseudo-affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell-derived MVA-BN® after cell homogenization and clarification. In parallel, the overall performance of classical bead-based resin chromatography (Cellufine® sulfate and Toyopearl® AF-Heparin) was investigated. The two tested pseudo-affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead-based column chromatography methods. Overall virus recovery was ,60% for both pseudo-affinity MA and the Cellufine® sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo-affinity chromatography materials. Cellufine® sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo-affinity with anion exchange MA a further reduction of host-cell DNA was achieved. Biotechnol. Bioeng. 2010. 105: 761,769. © 2009 Wiley Periodicals, Inc. [source]

    A FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assembly

    CYTOSKELETON, Issue 1 2008
    Priscila M. F. Siesser
    Abstract Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK ,/, mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK ,/, cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source]

    Influence of glutathione S-transferase pi and p53 expression on tumor frequency and spectrum in mice

    Laurent Gate
    Abstract The role of glutathione S-transferase , (GST,) in tumor development has been previously suggested; however the exact function of this enzyme in carcinogenesis remains unclear. GST, has been identified as a modulator of cell signaling by interacting with and inhibiting c-Jun N-terminal kinase (JNK). This kinase has been in turn described as a regulator of p53 stability and transcriptional activity. To study the possible interaction between GST, and p53, we crossed GST,-deficient animals with p53,/, mice. Double knock out animals were viable but developed tumors within 6 months of age; the life span of these animals was however similar to that of GST,+/,/p53,/, and GST,+/+/p53,/,. Mice heterozygous for p53 lived significantly longer than the p53,/, animals and developed tumors much later, and the expression of GST, did not significantly modify the life span of the animals. In contrast, in a wild-type p53 background, GST,,/, mice developed tumors with a significantly higher frequency than heterozygous and wild-type animals with a median tumor free life span 20 weeks shorter. In addition, in p53+/+ background, one third of the GST,,/, animals developed lung adenomas, while less than 10% of GST,+/, and GST,+/+ presented such tumors. GST, expression did not alter the expression of tumorigenesis markers such as COX-2 or ornithine decarboxylase in response to phorbol ester. Furthermore, GST,-deficient mouse embryo fibroblasts were more sensitive to H2O2 -induced apoptosis. P53,/, cells, independent of GST, status, were more sensitive to UV and other DNA damaging agents than their wild-type counterparts. These results suggest that GST, may play a protective role in the development of spontaneous tumors. [source]

    Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture

    AGING CELL, Issue 6 2003
    Rita A. Busuttil
    Summary Oxidative damage is a causal factor in aging and cancer, but it is still not clear how DNA damage, the cellular responses to such damage and its conversion to mutations by misrepair or misreplication contribute to these processes. Using transgenic mice carrying a lacZ mutation reporter, we have previously shown that mutations increase with age in most organs and tissues in vivo. It has also been previously shown that mouse cells respond to oxidative stress, typical of standard culture conditions, by undergoing cellular senescence. To understand better the consequences of oxidative stress, we cultured mouse embryo fibroblasts (MEFs) from lacZ mice under physiological oxygen tension (3%) or the high oxygen tension (20%) associated with standard culture, and determined the frequency and spectrum of mutations. Upon primary culture, the mutation frequency was found to increase approximately three-fold relative to the embryo. The majority of mutations were genome rearrangements. Subsequent culture in 20% oxygen resulted in senescence, followed by spontaneous immortalization. Immortalization was accompanied by an additional three-fold increase in mutations, most of which were G:C to T:A transversions, a signature mutation of oxidative DNA damage. In 3% oxygen, by contrast, MEFs did not senesce and the mutation frequency and spectrum remained similar to primary cultures. These findings demonstrate for the first time the impact of oxidative stress on the genomic integrity of murine cells during senescence and immortalization. [source]