Concentrations Of Phorbol 12-myristate 13-acetate Research Articles

Assessing the immunomodulatory potential of high-molecular-weight extracts from mushrooms; an assay based on THP-1 macrophages.

Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5-5) × 10(5) cells mL(-1). An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-α, IL-1β, IL-6 and IL-10.

Validación de la técnica de dihidrorodamina 123 para el diagnóstico de la enfermedad granulomatosa crónica en Colombia

IntroductionChronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in the respiratory burst of phagocytes. Affected patients often suffer from granulomas and recurrent infections, mainly due to encapsulated bacteria. AimTo standardize the dihydrorhodamine test (DHR) in Colombia used for the diagnosis of CGD by evaluating the respiratory burst in human blood neutrophils and monocytes after in vitro activation. MethodsPhagocyte respiratory burst in peripheral blood samples from 10 healthy controls was evaluated by flow cytometry after leukocyte activation with several concentrations of phorbol myristate acetate (PMA). The different oxidation patterns of DHR in X-linked or autosomal recessive CGD were also obtained. ResultsThe most suitable concentrations of PMA for the evaluation of the respiratory burst in peripheral blood were 0.2 to 5μg/ml. Reference values for this test in neutrophils from our population were established. It was shown that the oxidation patterns of DHR in monocytes were not always identical to those observed in neutrophils. ConclusionThe evaluation of DHR oxidation by flow cytometry is a screening method that easily identifies the different phenotypes of CGD, with good sensitivity and at a lower cost. However, it is crucial that every laboratory establishes its own normal range for this test, in order to achieve the accurate characterization of this condition. DHR oxidation patterns may be also evaluated in different blood cells, since cell type-specific defects have also been reported.

Short communication: Protein kinase C regulates glucose uptake and mRNA expression of glucose transporter (GLUT) 1 and GLUT8 in lactating bovine mammary epithelial cells

The aim of this study was to determine the role of protein kinase C (PKC) in regulating glucose uptake in lactating bovine mammary epithelial cells (BMEC). The BMEC were cultured and treated with different concentrations of phorbol 12-myristate 13-acetate (PMA;0, 10, 25, 50, 100, and 200ng/mL), the classic activator of PKC, for 48h. Compared with the cells with no PMA treatment, 50 and 100ng of PMA/mL significantly stimulated the glucose uptake of the BMEC, whereas the glucose uptake by the cells treated with the lowest and the highest amounts of PMA (25 and 200ng/mL, respectively) did not show a significant difference. Consistently, the mRNA expression of glucose transporter (GLUT) 1 and 8 showed a similar pattern of increase under the treatments of PMA. Furthermore, when the cells were pretreated with GF1090203X (0, 0.25, 0.5, 1, and 2μM), an inhibitor of PKC, for 30min before exposed to PMA (50ng/mL), the PMA-induced glucose uptake and GLUT1 and GLUT8 expression were decreased by GF1090203X in a dose-dependent manner. These results demonstrate that PKC is involved in the regulation of glucose uptake by BMEC, and this function may work, at least partly, through upregulating the expression of GLUT1 and GLUT8.

Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli

Macrophages are versatile cells of the immune system which react to various external stimuli through different polarization patterns which adjust the cells to the required function whether it is removal of pathogens or necrotic cells, tissue repair or propagation of inflammation. As much of macrophage behavior is determined by their polarization, appropriate models to study macrophage polarization are required. Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48h using 100ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48h of differentiation with 100ng/ml PMA. When we applied this protocol to study interleukin (IL) 10 dependent polarization, the cells were inert to the IL10 stimulus, as indicated by a failure to induce IL10 target genes such as SOCS3. Further investigation revealed that the cells were classically activated by the differentiation agent phorbol 12-myristate 13-acetate (PMA) as shown by induction of chemokine receptor CCR7. Subsequent reduction of PMA concentration during THP-1 macrophage differentiation significantly improved their response to IL10 as SOCS3 increased more than 40-fold. This increased responsiveness of the THP-1 macrophages was also confirmed for polarization with LPS and IFNγ. Up-regulation of classical activation markers CCL3, CCR7 and TNFα was enhanced from 18-, 21- and 70-fold, respectively, to 48-, 222- and 951-fold, respectively. Reduction of PMA concentration did not negatively affect macrophage differentiation or transfection efficiency. Expression of macrophage differentiation markers CD11b and CD68 as well as cell morphology remained unchanged. In addition transfection efficiency and rates of apoptosis and necrosis remained unaffected. Thus our revised protocol combines high transfection efficiency and cell vitality with a strong response to polarizing stimuli and therefore constitutes a suitable model for studying macrophage polarization in vitro.

AB0041 Possible role of cell apoptosis in the production and release of il-1β induced by pathogenic crystals

BackgroundDirect cell activation by crystals in vitro provokes the release of a number of cytokines and chemokines but not that of IL-1ß whose production and secretion needs the activation of...

Multianalyte Microphysiometry of Macrophage Responses to Phorbol Myristate Acetate, Lipopolysaccharide, and Lipoarabinomannan.

This study examined the hypothesis that mycobacterial antigens generate different metabolic responses in macrophages as compared to gram-negative effectors and macrophage activators. The metabolic activation of macrophages by PMA is a useful tool for studying virulent agents and can be compared to other effectors. While phorbol myristate acetate (PMA) is commonly used to study macrophage activation, the concentration used to create this physiological response varies. The response of RAW-264.7 macrophages is concentration-dependent, where the metabolic response to high concentrations of PMA decreases suggesting deactivation. The gram-negative effector, lipopolysaccharide (LPS), was seen to promote glucose and oxygen production which were used to produce a delayed onset of oxidative burst. Pre-incubation with interferon-γ (IFN-γ) increased the effect on cell metabolism, where the synergistic effects of IFN-γ and LPS immediately initiated oxidative burst. These studies exhibited a stark contrast with lipoarabinomannan (LAM), an antigenic glycolipid component associated with the bacterial genus Mycobacterium. The presence of LAM effectively inhibits any metabolic response preventing consumption of glucose and oxygen for the promotion of oxidative burst and to ensure pathogenic proliferation. This study demonstrates for the first time the immediate inhibitory metabolic effects LAM has on macrophages, suggesting implications for future intervention studies with Mycobacterium tuberculosis.

만성 골수성 백혈병 K562세포의 분화 내성 분획에서 백혈병 유지 세포의 동정

The present study investigated whether leukemia-maintaining cells reside in a differentiation-resistant fraction using a megakaryocytic differentiation model of K562 cells. Treatment with phorbol-12-myristate- 13-acetate (PMA) significantly inhibited the colony-forming efficiency of the K562 cells. At a PMA concentration of 1 nM or higher, colony was not formed, but approximately 40% of K562 cells still survived in soft agar. Approximately 70% of colony-forming cells that were isolated following the removal of PMA after exposure to the agent were differentiated after treatment with 10 nM PMA for 3 days. The differentiation rate of the colony-forming cells was gradually increased and reached about 90% 6 weeks after colony isolation, which was comparable to the level of a PMA-treated K562 control. Meanwhile, imatinib-resistant variants from the K562 cells, including K562/R1, K562/R2, and K562/R3 cells, did not show any colony-forming activity, and most imatinib-resistant variants were CD44 positive. After 4 months of culture in drug-free medium, the surface level of CD44 was decreased in comparison with primary imatinib-resistant variants, and a few colonies were formed from K562/R3 cells. In these cells, Bcr-Abl, which was lost in the imatinib-resistant variants, was re-expressed, and the original phenotypes of the K562 cells were partially recovered. These results suggest that leukemia-maintaining cells might reside in a differentiation-resistant population. Differentiation therapy to eliminate leukemia-maintaining cells could be a successful treatment for leukemia if the leukemia-maintaining cells were exposed to a differentiation inducer for a long time and at a high dose.

Peroxiredoxin 6 Enhances Phagocyte NADPH Oxidase (phox) Activity in Response to Agonists Acting Via Cell Surface Receptors and Intracellular Signaling Pathways.

Abstract 2139 IntroductionThe phagocyte NADPH oxidase (phox) is a multiprotein, transmembrane-enzyme-complex found in diverse professional phagocytes including neutrophils. Phox in its inactive state consists of the membrane-associated subunit cytochrome b558 (a dimer of gp91phox/Nox2 and p22phox) and cytoplasmic subunits which include p47phox, p67phox, p40phox and Rac1 or Rac2. Phosphorylation of phox proteins promotes tight interaction of the cytoplasmic phox components with cytochrome b558 and the resulting active complex catalyzes transmembrane electron transfer from cytoplasmic NADPH to molecular oxygen forming superoxide anion (O2−). O2− and other reactive oxygen species formed from it are microbicibal and crucial to pathogen killing by neutrophils.Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with a peroxiredoxin activity that reduces oxidized lipids and H2O2 and a PLA2 activity that cleaves the sn2 position of phospholipids forming the corresponding free fatty acid and lysophospholipid.Previous work in our laboratory has identified Prdx6 as a binding partner of p67phox and as an enhancer of O2− production by active phox; we have shown that this enhancement is due to products of the PLA2 activity of the peroxiredoxin but the precise mechanism by which these molecules act is unclear. Here we present evidence that Prdx6 causes maximal activation of phox when the complex is activated by physiological agonists (the formylated peptide fMLP and the particulate agonist serum opsonized zymosan (SOZ)) but not when it is activated by the non-physiological agonist phorbol 12-myristate 13-acetate (PMA). Because fMLF and SOZ interact with cell surface receptors but PMA enters the cell and activates protein kinase C (PKC) enzymes that directly phosphorylate phox proteins our data provides a clue as to the mode of action of Prdx6 in phox activation. MethodsPLB985 cells were used to model neutrophils. This myeloid cell line can be terminally differentiated to a neutrophil like state with phox activity by exposure to DMSO. Prdx6 was suppressed in these cells by stable expression of an shRNA resulting in an approximately 70% reduction of the protein. Following maturation of the cells they were stimulated with fMLP, SOZ or PMA and O2− production was measured by superoxide dismutase (SOD) inhibitable luminescence of Diogenes fluorophore. For fMLP, due to the transient nature of the O2- production, the entire amount of O2− production was measured. SOZ and PMA induced prolonged (>1hr) O2− production and O2− production over 1hr was thus measured. ResultsIn cells with Prdx6 suppressed, SOD inhibitable O2− production in response to fMLP was reduced by 53% ± 3% (SEM), n=34, which was significant (p<0.001). O2− produced in response to SOZ was reduced by 37% ± 9% (SEM), n = 4, which was also significant (p<0.05).In contrast, the response to PMA was not altered (p>0.5, n of at least 4) at a range of PMA concentrations from one causing slight activation of phox (10 ng/ml) to one causing maximal O2- production (1 μg/ml). ConclusionsfMLP binds to receptors on neutrophils and triggers intracellular signaling cascades that ultimately lead to phosphorylation and activation of phox. Similarly, opsonins of particulate stimuli bind cell surface complement receptors that trigger intracellular signaling cascades that activate phox. PMA on the other hand enters cells and activates PKC enzymes that directly phosphorylate phox proteins; it thus bypasses the physiological signaling pathways that mediate the fMLP and SOZ response.Since Prdx6 enhances the SOZ and fMLF mediated phox response but not the PMA mediated response we propose that in myeloid cells, products of the PLA2 activity of Prdx6 enhance the SOZ and PMA mediated signaling cascades. This may be via activation of signaling molecules in these pathways which would be consistent with the known ability of the fatty acid arachidonate to activate PKC enzymes, MAP kinases, PLA2 enzymes and a class Ia phosphatidylinositol 3-kinase and to mobilize Ca2+. Disclosures:No relevant conflicts of interest to declare.

Activation of the PKC Pathway Stimulates Ovarian Cancer cell Proliferation, Migration, and Expression of MMP7 and MMP10.

Postmenopausal women are at a higher risk of ovarian cancer due, in part, to increased levels of gonadotropins such as LH. Gonadotropins activate the PKA and PKC pathways which are altered in ovarian cancer. To determine the role of LH on ovarian cancer, we explored the effects of hCG, a LH mimic, and PMA (Phorbol-12-Myristate 13-Acetate), an activator of the PKC pathway, on ovarian cancer cell proliferation, apoptosis and cell cycle kinetics in Ovcar3 cells. Cells were serum starved for 24 hrs, treated with or without PMA (20 nM) or hCG (1 IU) for varying times, and then cells were collected. Our flow cytometry data shows that PMA increased the number of cells in the S phase of the cell cycle and decreased cells in the G0/G1 phase after 24 hrs. In order to determine whether the changes in cell cycle seen in Ovcar3 cells at 24 hr were translated into changes in cell proliferation, MTS and BrdU assays were performed. When Ovcar3 cells were treated with increasing concentrations of PMA (0, 0.2, 2 or 20 nM) or hCG (1 IU) for 24 hr, no changes in proliferation were observed. To explore the possibility that the changes in cell cycle at 24 hr were manifest in cell proliferation after 24 hr, cells were treated for 48 hrs. Administration of PMA (20 nM) for 48 hr resulted in an increase in Ovcar3 cell proliferation when analyzed by both the MTS and BrdU assays. To investigate the effects of PMA and hCG on ovarian cancer cell apoptosis, we utilized FACS analysis with an annexin V assay. There was an increase in apoptotic cells after 4 hr of treatment with PMA. However, after 8 hr, PMA treatment led to the presence of less apoptotic cells whereas no changes in apoptosis were observed after 12 hr. The PKC pathway is known to differentially regulate the expression of matrix metalloproteinases (MMPs) which have an active role in cancer metastasis. PMA treatment of Ovcar3 cells increased MMP7 and MMP10 mRNA levels after 8 hr of treatment and expression remained high after 12 hr before decreasing at 24 hr The expression of MMP2, MMP9, TIMP1 and TIMP3 was not altered. The mRNA expression of extracellular matrix metalloproteinase inducer (BSG), an activator of MMPs, was unaffected by PMA. Due to the role that MMPs play in migration, we investigated the effect of MMP induction on ovarian cancer cell migration using transwell assays. The use of the MMP inhibitor GM6001 blocked the increased migratory effects of PMA on ovarian cancer cells after 24 hrs. The MMP2/9 inhibitor in contrast, had no effect on ovarian cancer migration. Treatment of ovarian cancer cells with GM6001 did not cause a decrease in proliferation of ovarian cancer cells indicating that the inhibitory effect of GM6001 on migration is not due to a decrease in cell proliferation. Together, these studies show that activating the PKC pathway causes significant changes in cell cycle kinetics, proliferation and selective expression of MMPs that are involved in enhancing ovarian cancer cell proliferation and migration. Supported by NIH P20 RR15592, NIH HD057446.

Regulation of Protein Kinase C Inactivation by Fas-associated Protein with Death Domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.

Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal

To investigate the effect of sulfated cholecystokinin-8 (CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal (ICC) and its possible mechanisms. ICC were isolated from the gastric antrum of mice and cultured. Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC. The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca(2+) concentration ([Ca(2+)]i). A confocal laser scanning microscope was used to monitor [Ca(2+)]i changes. The selective CCK(1) receptor antagonist lorglumide, the intracellular Ca(2+)-ATPase inhibitor thapsigargin, the type III inositol 1,4,5-triphosphate (InsP(3)) receptor blocker xestospongin C and the L-type voltage-operated Ca(2+) channel inhibitor nifedipine were used to examine the mechanisms of [Ca(2+)]i elevation caused by CCK-8S. Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type III InsP(3) receptor (InsP(3)R3) in ICC. Protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca(2+)]i increment of ICC. ICC were successfully isolated from the gastric antrum of mice and cultured. Cultured ICC were identified by immunofluorescence staining. When given 80 nmol/L or more than 80 nmol/L CCK-8S, the [Ca(2+)]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca(2+)]i by 59.30% ± 4.85% (P < 0.01). Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca(2+)]i increment from 59.30% ± 4.85% to 14.97% ± 9.05% (P < 0.01), suggesting a CCK(1)R-mediated event. Emptying of intracellular calcium stores by thapsigargin (5 μmol/L) prevented CCK-8S (100 nmol/L) from inducing a [Ca(2+)]i increase. Moreover, pretreatment with xestospongin C (1 μmol/L) could also abolish the CCK-8S-induced effect, indicating that Ca(2+) release from InsP(3)R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC. On the other hand, by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine, a smaller but significant rise in the [Ca(2+)]i could be still elicited by CCK-8S. These data suggest that the [Ca(2+)]i release is not stimulated or activated by the influx of extracellular Ca(2+) in ICC, but the influx of extracellular Ca(2+) can facilitate the [Ca(2+)]i increase evoked by CCK-8S. CCK-8S increased the phosphorylation of InsP(3)R3, which could be prevented by chelerythrine. Pretreatment with lorglumide (5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP(3)R3. In the positive control group, treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP(3)R3. Pretreatment with various concentrations of PMA (10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of 100 nmol/L PMA was most obvious. Likewise, the effect of CCK-8S was augmented by the pretreatment with chelerythrine (10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect. CCK-8S increases [Ca(2+)]i in ICC via the CCK(1) receptor. This effect depends on the release of InsP(3)R-operated Ca(2+) stores, which is negatively regulated by PKC-mediated phosphorylation of InsP(3)R3.

Flow cytometry assays of respiratory burst in Atlantic salmon ( Salmo salar L.) and in Atlantic cod ( Gadus morhua L.) leucocytes

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.

Production of functional platelet-like particles by the megakaryoblastic DAMI cell line provides a model for platelet biogenesis

The aim of this study was to evaluate cell maturation and the platelet production capacity of the megakaryoblastic DAMI cell line, to characterize platelet-like particles produced and to investigate the mechanisms involved in their production. DAMI cell maturation was induced by phorbol myristate acetate (PMA) and thrombopoietin (TPO). Expression levels of GATA-1, Fli-1 and NF-E2 were evaluated using real-time PCR and western blot. Platelet-like particles were characterized by the presence of GPIb and GPIIb by flow cytometry, while the soluble fragment of GPIb, glycocalicin, was detected by enzyme immunoassay. Dense and alpha granules were evaluated by mepacrine staining and thrombospondin-1 detection, respectively, and by electron microscopy. Functional capacity of platelet-like particles was studied by measuring P-selectin membrane after thrombin stimulation by flow cytometry and actin polymerization using phalloidin-FITC by immunofluorescence. We found that stimulation of DAMI cells with high concentration of PMA and TPO induced the expression of transcription factors GATA-1 and Fli-1 followed by an increase in the isoform a of NF-E2. Mature DAMI cells give rise to extensions resembling proplatelets and later, produce platelet-like particles expressing GPIIb and GPIb on their surface and containing dense and alpha granules, which were confirmed by electron microscopy. Platelet functionality was demonstrated by the increase in P-selectin membrane expression after thrombin stimulation and by their ability to spread on fibrinogen matrices. DAMI cell line induced to differentiate into mature megakaryocytes is able to produce functional platelets providing a suitable model to study the mechanisms involved in platelet generation.

Green tea prevents down-regulation of gap junction intercellular communication in human keratinocytes treated with PMA

It has been suggested that connexin (Cx) gap junction proteins act as tumor suppressors and green tea has a potential to prevent tumor development, however, the studies on their association with human keratinocytes were rare. We evaluated the effects of a tumor promoter, phorbol-12-myristate-13-acetate (PMA), on the expression of Cxs and gap junction intercellular communication (GJIC) in human keratinocytes (HaCaT cells) and explored the preventive effects of green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG). We performed neutral red dye uptake assay to determine the optimal concentrations of PMA, EC, and EGCG for this study and confirmed the expression of Cx mRNAs using RT-PCR. We evaluated GJIC quantitatively using the 'scrape-loading dye transfer (SLDT)' technique after 24-h culture of HaCaT cells treated with agents. To analyze the expression change of Cxs, we also performed Western blot and immunocytochemistry. HaCaT cells were found to express Cx26, Cx30, Cx31, and Cx43, but not Cx29. In 'scrape-loading dye transfer' for functional study for GJIC, EC and EGCG significantly prevented PMA-induced down-regulation of GJIC. Western blot analyses revealed that EC and EGCG prevented down-regulation of Cx26 and Cx43 proteins in HaCaT cells treated with PMA. Immunocytochemistry showed decreased expression and abnormal location of Cx26 and Cx43 in HaCaT cells when treated with PMA, and EC and EGCG inhibited its effect. These results suggest an important role of GJIC played in carcinogenesis involving human keratinocytes and green tea as a useful anticancer diet.

Activation of p44/42 MAPK plays a role in the TBT-induced loss of human natural killer (NK) cell function.

Natural killer (NK) cells destroy (lyse) tumor cells, virally infected cells, and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen-activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen Toxicology 209:263-277, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function ((51)Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1-h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1-h exposure to 5 nM PMA caused a sixfold increase in phospho-p44/42 levels. Previous studies showed a fivefold increase in phospho-p44/42 in response to a 1-h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function.

Protein Kinase Cβ Modulates Ligand-induced Cell Surface Death Receptor Accumulation: A MECHANISTIC BASIS FOR ENZASTAURIN-DEATH LIGAND SYNERGY

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.

The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically applied. Protein kinase C (PKC) family members have been implicated in bone metabolism, which prompted us to use a pharmaceutical approach to manipulate PKC signalling in hMSCs. Inhibition of PKC resulted in a dose-dependent inhibition of dexamethasone-induced osteogenic differentiation. Surprisingly, PKC activation using phorbol 12-myristate 13-acetate (PMA) also resulted in inhibition of osteogenesis, although we observed that inhibition was more pronounced at low than at high concentrations of PMA. Furthermore, we observed that inhibition of PKCdelta blocked alkaline phosphatase (ALP, an early marker of osteogenic differentiation) expression, whereas inhibition of the conventional PKC subfamily and PKCmicro using Gö6976 resulted in an induction of ALP activity, collagen (I) expression and mineralization. In conclusion, inhibition of the conventional PKCs/PKCmicro and activation of PKCdelta could further benefit osteogenic differentiation of hMSCs in vitro and in vivo, which is currently under investigation.

Persistance et voie de l’inhibition de la production de superoxyde par les neutrophiles induite par isoflurane

Our previous work has demonstrated that treatment with isoflurane has a preconditioning-like inhibitory effect on superoxide production (SOP) by polymorphonuclear neutrophils. The current objectives were to determine persistency of this effect and to clarify where in the signalling pathway this inhibition of SOP occurred. The latter was accomplished using two receptor-dependent neutrophil agonists, platelet activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (fMLP), and two receptor-independent neutrophil stimuli, the protein-kinase C stimulator, phorbol myristate acetate (PMA), and the calcium ionophore, A23187. Arterial blood samples were obtained from eight dogs under baseline condition (conscious state), during isoflurane (1 MAC) administration, and 24 and 48 hr post-isoflurane (also in conscious state). Neutrophils were isolated and stimulated with 1 muM concentrations of PAF, fMLP, PMA, and A23187. SOP was measured spectrophotometrically. Isoflurane administration caused (1) an approximate 50% decrease in SOP during PAF or fMLP (P < 0.01 vs baseline), which remained evident from 24 to 48 hr following isoflurane; (2) an initial 29% decrease in SOP during PMA (P < 0.05 vs baseline), which returned to baseline by 24 hr following isoflurane; and (3) no change in SOP during A23187 (P > 0.05 vs baseline). Isoflurane administration caused prolonged (from 24 to 48 hr) decreases in agonist-induced SOP by neutrophils. This effect involved inhibition at site(s) in the signalling pathway upstream from protein kinase C. The current findings suggest that the intraoperative use of isoflurane may result in an extended impairment to the antibacterial host defense mechanism and that neutrophil inhibition may play a role in the delayed tissue protection afforded by treatment with volatile anesthetics.

SP146 Coordinating biological material collection in the context of multinational clinical trials

The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inflammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated significant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differentiation of THP-1 macrophages. Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, specifically in response to the pro-inflammatory stimulus, lipopolysaccharide (LPS). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied significantly, and was dependent upon the concentration of PMA used to stimulate the differentiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24 h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells.

The production of reactive oxygen species in peripheral blood neutrophils is modulated by airway mucous

Abstract Neutrophils are a major source of reactive oxygen species (ROS). The role of airway mucous on ROS production is unknown. The aim of our study was to investigate the direct influence of bronchoalveolar lavage fluid (BALF) and induced sputum (IS) alone or in combination with chemical/biological stimulus on ROS production in peripheral blood neutrophils during chronic obstructive pulmonary disease (COPD). Neutrophils were isolated from peripheral blood of 47 patients with moderate COPD and 14 healthy individuals (HI). BALF/RPMI (1:1) or IS/RPMI (1:1) from COPD patients were used to stimulate neutrophils alone or in combination with phorbolmyristate- acetate (PMA) (0.1–30 nM) or Staphylococcus aureus bacteria (0.7–500 bact/neutrophil). Relative generation of ROS was measured flow cytometrically. BALF/RPMI and in combination with relatively low PMA or all bacteria concentrations stimulated ROS; while, combination with relatively high PMA concentrations suppressed ROS in of COPD patients and HI. IS/RPMI and its combination with PMA inhibited ROS generation in both groups; whereas, IS stimulated or had a tendency to stimulate ROS production with relatively high bacteria concentrations. In conclusion, BALF and IS directly or in combination with chemical/biological factors modulated ROS production. This effect was stronger in neutrophils from COPD patients and depended on chemical/biological stimulus intensity.