scholarly journals Enhancement of enteric adenovirus cultivation in a novel Ras-overexpressing cell line

2014 ◽  
Vol 95 (1) ◽  
pp. 171-178 ◽  
Author(s):  
Jiyeon Si ◽  
Misoon Kim ◽  
Mi Young Lim ◽  
GwangPyo Ko
Keyword(s):  
Cell Line ◽  
Mitogen Activated Protein Kinase ◽  
Stable Cell Line ◽  
Drinking Water Contamination ◽  
Ras Protein ◽  
Efficient Detection ◽  
Mitogen Activated Protein ◽  
Enteric Adenovirus ◽  
Mammalian Cell Cultivation ◽  
Extracellular Regulated Kinase

Enteric human adenoviruses (HAdVs; serotypes 40 and 41) have been identified as an emerging cause of drinking water contamination. Due to their fastidious characteristics, HAdVs are difficult to cultivate and therefore not detected easily by standard mammalian cell cultivation methods. Here we found that human embryonic kidney 293 cells, transformed transiently with Ras, enhanced HAdV replication by more than threefold. We also constructed a stable cell line overexpressing the Ras protein, 293-Ras, in which the replication of three HAdV strains of serotypes 40 and 41 was increased markedly. However, only HAdV replication was enhanced; infection of 293 and 293-Ras cells with human rhinivorus-6 showed no significant differences in replication rate. Infected 293-Ras cells exhibited an increased level and phosphorylation of extracellular regulated kinase (ERK). In addition, the Ras-mediated increase in HAdV replication was impaired by the mitogen-activated protein kinase/ERK kinase (MEK1) inhibitor U0126, suggesting direct involvement of the MEK1/ERK pathway in enhanced HAdV replication. Based on these results, we suggest that the 293-Ras cell line be used for the efficient detection and cultivation of HAdV strains in both clinical and environmental specimens.

scholarly journals Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway

2004 ◽  
pp. 233-240 ◽  
Author(s):  
AM Nanzer ◽  
S Khalaf ◽  
AM Mozid ◽  
RC Fowkes ◽  
MV Patel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Thymidine Incorporation ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Gh3 Cells ◽  
Rat Pituitary ◽  
Mitogen Activated Protein

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.

scholarly journals Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes.

1996 ◽  
Vol 184 (3) ◽  
pp. 1027-1035 ◽  
Author(s):  
R Trotta ◽  
P Kanakaraj ◽  
B Perussia
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitogen Activated Protein Kinase ◽  
Monocytic Cell ◽  
Fast Kinetics ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.

Synergistic Activation of Mitogen-Activated Protein Kinase by Cyclic AMP and Myeloid Growth Factors Opposes Cyclic AMP’s Growth-Inhibitory Effects

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 537-553 ◽  
Author(s):  
Angel Wai-mun Lee
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cell Line ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein ◽  
Erk Activity ◽  
Myeloid Progenitors

Abstract Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90rsk. cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1–mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.

scholarly journals Glial Cell Line-derived Neurotrophic Factor Signals through the RET Receptor and Activates Mitogen-activated Protein Kinase

1996 ◽  
Vol 271 (39) ◽  
pp. 23619-23622 ◽  
Author(s):  
Carolyn A. Worby ◽  
Quinn C. Vega ◽  
Yi Zhao ◽  
Hanna H.-J. Chao ◽  
Audrey F. Seasholtz ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Line ◽  
Glial Cell ◽  
Neurotrophic Factor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein

scholarly journals N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

1994 ◽  
Vol 14 (7) ◽  
pp. 4722-4730 ◽  
Author(s):  
K A Cadwallader ◽  
H Paterson ◽  
S G Macdonald ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Membrane Targeting ◽  
Ras Proteins ◽  
Kinase Activation ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Protein Kinase Activation ◽  
Mitogen Activated Protein

Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.

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