Inhibition of mitogen-activated kinase signaling sensitizes HeLa cells to Fas receptor-mediated apoptosis.
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The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation. (+info)
Activating signal cointegrator 1, a novel transcription coactivator of nuclear receptors, and its cytosolic localization under conditions of serum deprivation.
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Activating signal cointegrator 1 (ASC-1) harbors an autonomous transactivation domain that contains a putative zinc finger motif which provides binding sites for basal transcription factors TBP and TFIIA, transcription integrators steroid receptor coactivator 1 (SRC-1) and CBP-p300, and nuclear receptors, as demonstrated by the glutathione S-transferase pull-down assays and the yeast two-hybrid tests. The ASC-1 binding sites involve the hinge domain but not the C-terminal AF2 core domain of nuclear receptors. Nonetheless, ASC-1 appears to require the AF2-dependent factors to function (i.e., CBP-p300 and SRC-1), as suggested by the ability of ASC-1 to coactivate nuclear receptors, either alone or in cooperation with SRC-1 and p300, as well as its inability to coactivate a mutant receptor lacking the AF2 core domain. By using indirect immunofluorescence, we further show that ASC-1, a nuclear protein, is localized to the cytoplasm under conditions of serum deprivation but is retained in the nucleus when it is serum starved in the presence of ligand or coexpressed CBP or SRC-1. These results suggest that ASC-1 is a novel coactivator molecule of nuclear receptors which functions in conjunction with CBP-p300 and SRC-1 and may play an important role in establishing distinct coactivator complexes under different cellular conditions. (+info)
Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system.
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Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni(2+)-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level. (+info)
Oocytectomy does not influence synthesis of hyaluronic acid by pig cumulus cells: retention of hyaluronic acid after insulin-like growth factor-I treatment in serum-free medium.
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Mouse oocytes secrete a factor that enables cumulus cells to undergo expansion in response to FSH (1 microg/ml), whereas expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. The aim of this study was to assess FSH-induced synthesis of hyaluronic acid (HA) by porcine cumulus cells before and after oocytectomy. In addition, we studied the effect of insulin-like growth factor-I (IGF-I) on the ability of cumulus cells to synthesize and retain HA in response to FSH in serum-free medium. Porcine oocyte-cumulus complexes and complexes from which the oocytes had been removed by oocytectomy were cultured for 24 h in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride, fetal calf serum (FCS, 5%), and FSH. After 24 h, incorporation of [(3)H]glucosamine into HA was measured either in complexes alone (retained HA) or in medium plus complexes (total HA). Specificity of incorporation of radioactivity into HA was confirmed by the sensitivity to highly specific Streptomyces hyaluronidase. Our results suggest that 1) the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis; 2) oocytes do not influence retention of HA within the complex; 3) FSH-induced synthesis of HA by cumulus cells is decreased in medium with polyvinylpyrrolidone (PVP)-supplemented (total and retained HA) compared to FCS-supplemented medium; 4) IGF-I enabled cumulus cells to synthesize HA in response to FSH in PVP-supplemented medium in a manner similar to that observed when serum is present in the medium. (+info)
Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.
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BACKGROUND AND OBJECTIVE: The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. DESIGN AND METHODS: Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. RESULTS: An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. INTERPRETATION AND CONCLUSIONS: The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC. (+info)
Enhancement of Borna disease virus transcription in persistently infected cells by serum starvation.
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Transcription of Borna disease virus (BDV) in persistently infected MDCK (MDCK/BDV) cells increased in the fetal bovine serum free media as detected by Northern blot analysis. Especially, the amount of 1.9-kb RNA without cap formation at the 5' end and polyadenylation at the 3' end, increased as compared to other mRNA molecules of BDV. Growth arrest of MDCK/BDV cells observed in the condition of serum starvation might be important for increasing viral transcription. Since N-cadherin is the responsible factor for cell-to-cell contact, MDCK/BDV cells were cultured in calcium free medium which inhibits the interaction of N-cadherin. However, inhibition of cell-to-cell contact by N-cadherin is not effective on up regulation of viral transcription. Our finding in this study indicates that enhancement of BDV transcription by serum starvation is a useful technique for further investigation in understanding of mechanisms of BDV transcription. (+info)
c-Myc does not prevent glucocorticoid-induced apoptosis of human leukemic lymphoblasts.
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Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. The signal transduction pathways leading to this clinically-relevant form of apoptosis have, however, not been sufficiently elucidated. GC bind to their specific receptor, a ligand-activated transcription factor of the Zn-finger type, that activates or represses transcription of GC-responsive genes. Previous studies in leukemia cells suggested that transcriptional repression of c-myc expression might be the crucial event in GC-induced apoptosis, although in other systems, c-Myc apparently increased the sensitivity to cell-death inducers. To address this controversy, we stably transfected the GC-sensitive human T-ALL cell line CEM-C7H2 with constructs allowing tetracycline-regulated expression of c-Myc. Subsequent analyses of these cell lines showed that overexpression of c-Myc per se had little, if any, effect on cell viability, although it rendered the cells more sensitive to apoptosis induced by low serum, confirming the functionality of the expressed transgene. More importantly, however, when the cells were treated with GC in the presence of exogenous c-Myc, they underwent apoptosis exceeding that in cells treated in the absence of transgenic c-Myc. The data indicate that c-myc downregulation is not critical for induction of cell-death by GC in this system, and support the notion that c-Myc sensitizes cells to apoptosis-inducing agents. (+info)
Anti-apoptotic activity of p53 maps to the COOH-terminal domain and is retained in a highly oncogenic natural mutant.
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The tumour suppressor p53 plays a complex role in the regulation of apoptosis. High levels of wild type p53 potentiate the apoptotic response, while physiological range, low levels of the protein have an anti-apoptotic activity in serum starved immortalized fibroblasts. Here we report that primary fibroblast-like cells that show normal growth control are also efficiently protected from apoptosis by the endogenous p53 activity. The capacity to inhibit apoptosis is not restricted to the wild type protein: the R-->H175 p53 mutant fully retains the anti-apoptotic activity of the wild type p53, providing a possible explanation for its high oncogenicity. Using a series of point and deletion mutants of p53 under the control of tetracycline-regulated promoter we show that certain mutants, like the wild type, protect cells at low levels but lead to apoptosis when overexpressed. This latter effect is lost upon deletion of a proline-rich domain in the NH2 part of the protein. The anti-apoptotic activity can be mapped to the extreme carboxy-terminal part of the protein and is therefore independent of other well characterized p53 activities. Our results add a new level of complexity to the network of interactions mediated by p53 in normal physiology and pathology. (+info)