Concentrations Of Phorbol 12-myristate 13-acetate Research Articles

Measurement of phagocytosis and oxidative burst of canine neutrophils: high variation in healthy dogs

Quantitative analysis of phagocytosis and oxidative burst in canine polymorphonuclear (PMN) cells was performed by flow cytometry techniques. Different concentrations of phorbol myristate acetate (PMA) were used to modulate PMN phagocytosis. A low concentration of PMA (3 nmol) resulted in increased phagocytic activity of canine PMN, which could not be enhanced by higher dosages. Experiments with a reference cell population showed high losses of PMN, most probably by adherence to plastic material. It was possible to avoid this loss by layering all ingredients on cushions of Histopaque®. However, Histopaque® had a negative influence on the phagocytic activity of canine PMN. The use of PMA led to a dosage-dependent increase in the oxidative burst measured by the production of reactive oxygen species (ROS). Cushions of Histopaque® were used to avoid cell loss. There was no negative influence of Histopaque® on ROS formation. Storage of canine PMN for 24 h at room temperature had no negative influence on phagocytosis or oxidative burst measurements. Variations in the ROS assays conducted by two different examiners could be eliminated by use of a Histopaque®-cushion.

혈관평활근세포에서 Phorbol 12-Myristate 13-Acetate의 전처리가 Interleukin-1β에 의한 Nitrite생성에 미치는 영향

Protein kinase C (PKC)가 interleukin-1 beta (IL-1<TEX>$\beta$</TEX>)에 의하여 산화질소(NO) 생성과정에 어떤 역할을 하는지를 검토하기 위하여, 혈관평활근세포에서 PKC 활성제인 phorbol 12-myristate 13-acetate (PMA)로 전처리한 후 IL-1<TEX>$\beta$</TEX>에 의하여 야기되는 NO생성을 nitrite (<TEX>$NO_2$</TEX>)로 정량하고, RT-PCR method를 이용하여 iNOS 발현에 미치는 영향을 검토하여 다음과 같은 결과를 얻었다. PMA (20, 200 nM)는 IL-1<TEX>$\beta$</TEX>에 의한<TEX>$NO_2$</TEX> 생성을 유의하게 증가시켰다. PMA 200 nM, phorbol 12,13-dibutyrate 500 nM로 전처리하여 8, 24시간 노출된 세포에서 IL-1<TEX>$\beta$</TEX>에 의한 NO2생성이 현저히 감소되었으나, PKC 비활성제인 4<TEX>$\alpha$</TEX>-phorbol-didecanoate 200 nM로 전처리한 경우는 영향을 받지 아니하였다. PMA 농도를 달리하여 24시간 전처리한 경우 IL-1<TEX>$\beta$</TEX>에 의한 <TEX>$NO_2$</TEX> 생성의 감소는 PMA의 농도가 20및 200 nM에서 현저하였다. RT-PCR method를 이용하여 iNOS 발현을 검토한바 IL-1<TEX>$\beta$</TEX> 100U/ml에 의한 iNOS발현이 PMA전처리 및 cycloheximide 또는 actinomycin D존재로서 현저히 억제 되었다. 이상의 결과로 미루어 혈관평활근세포에서 PMA 전처리로 야기되는 IL-1<TEX>$\beta$</TEX>에 의한 NO 생성의 감소는, PKC 조절저하작용에 의한 iNOS 발현의 억제로 야기되는 것 같다. To examine the role of protein kinase C (PKC) in regulation of interleukin-1 beta (IL-1<TEX>$\beta$</TEX>)-induced iNOS expression, IL-1<TEX>$\beta$</TEX>-induced nitrite production was observed in cultured vascular smooth muscle (VSM) cells pretreated with phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-butyrate (PDB) as PKC activator; 4<TEX>$\alpha$</TEX>-phorbol-didecanoate (PDD) as PKC non-activator. Nitrite production induced by IL-1<TEX>$\beta$</TEX> was increased by the presence of increasing concentration of PMA ranging from 2 to 200 nM. However, in VSM cells pretreated with PMA and PDB, IL-1<TEX>$\beta$</TEX>-induced <TEX>$NO_2$</TEX> production was decreased in proportion to the duration of pretreatment, and most significantly decreased in pretreatment time of 24 hours. Using RT-PCR method, the expression of iNOS mRNA induced by IL-1<TEX>$\beta$</TEX> was decreased in VSM cells pretreated with PMA 200 nM for 24 hours. These results suggest that decrease in IL-I<TEX>$\beta$</TEX>-induced nitrite production by the pretreatment of PMA result from inhibition of iNOS expression and the inhibition related to PMA-induced PKC down-regulation.

Inactive caspase 3 activates Akt in human leukemia cells susceptible or resistant to apoptosis induced by phorbol ester

Phorbol 12-myristate 13-acetate (PMA) is a protein kinase C (PKC) activator and tumor promoter that induces terminal differentiation in human myeloid leukemia cells. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest and apoptosis. In the present studies, we determined the effect of high intracellular levels of the anti-apoptosis Bcl-2 protein on caspase 3 activation and cyctochrome c release during phorbol ester 12-myristate 13-acetate (PMA)-induced apoptosis. For this, we used the U937 cells, Bcl-2 overexpressed U937 cells (U937/Bcl-2) and the PMA-resistant derivative cell line R-U937. The G1 arrest of U937 cells and U937/Bcl-2 cells induced by treatment with 20 nM PMA is associated with cyclin A down-regulation and accumulation of p21, cdks inhibitor. However, PMA had no effect on the levels of cyclin A expression and p21 expression under the same conditions of time and concentration of PMA in the R-U937 cells. Treatment with 20 nM PMA for 24 h produced morphological features of apoptosis and DNA fragmentation in U937 and U937/Bcl-2 cells, but not in R-U937 cells. This was associated with the caspase 3 activation and cyctochrome c release. R-U937 cells exhibited less cytochrome c release and sustained phosphorylation level of Akt during PMA-induced apoptosis. These findings indicate that R-U937 cells are resistant to PMA-induced apoptosis by a mechanism of the signaling defect in the activation of the caspase 3 that is involved in the execution of apoptosis.

Phorbol myristate acetate and Bryostatin 1 rescue IFN-gamma inducibility of MHC class II molecules in LS1034 colorectal carcinoma cell line

BackgroundThe expression of major histocompatibility complex class II (MHCII) antigens in both mouse and human tumors is rare, and these antigens are not easily inducible by IFN-gamma (IFNg). Since MHCII may play an important role in the development of host antitumor immune response, we explored the possibility of restoring MHCII inducibility in several IFNg-resistant tumor cell lines using protein kinase C (PKC) agonists phorbol myristate acetate (PMA) or Bryostatin.ResultsTumor cells were co-cultured with various concentrations of PMA and IFNg for 48 hr. The expression of MHCII antigens and receptors IFNgR1 and IFNgR2 was determined by flow cytometry. We showed that the presence of as little as 0.1 ng/ml of PMA in tissue culture restored the ability of weakly inducible LS1034 colon carcinoma cells to express MHCII in response to IFNg (100 – 10,000 IU/ml) in a dose-dependent manner. Likewise, Bryostatin 1, as low as 10 ng/ml produced a 5–6 fold upregulation of MHCII. The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM). Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA.ConclusionIn this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists. Bryostatin 1 may be considered for further investigation of IFNg-dependent MHCII induction in resistant tumors in vivo.

Involvement of protein kinase C in glutamate release from cultured microglia

Glutamate release from microglial cells may cause neuronal damage. To elucidate the mechanism of glutamate release, we examined the possible regulation by nitric oxide and protein kinase C. Cultured microglia prepared from the whole brains of newborn rats released glutamate by the stimulation with lipopolysaccharide (LPS) dose dependently. The time course study revealed that glutamate release showed a long lag time about 6 h after LPS stimulation, whereas about 3 h lag time was observed in LPS-induced NO production. An inhibitor for NO synthase, N G-nitro- l-arginine, could effectively inhibit the glutamate release. Glutamate release induced by LPS was enhanced by 1 nM phorbol myristate acetate (PMA). Furthermore, high concentrations of PMA (>10 nM) induced glutamate release even without LPS stimulation. Glutamate release stimulated either by 100 ng/ml LPS or 100 nM PMA was inhibited by staurosporine, and also by α-aminoadipate. These results provide insight into the pathways regulating microglial pathological activation by protein kinase C and may be a base for the protection against microglia-evoked neurotoxicity.

Protein kinase C stimulates release of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human decidual cells

Objective: Increased concentrations of amniotic fluid matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 have been observed in the context of premature rupture of membranes (PROM) and microbial invasion of the amniotic cavity. However, the source of the stimuli that contribute to the accumulation of these proteins in amniotic fluid remains to be identified. The present study was conducted to investigate MMP-2, MMP-9 and TIMP-1 secretion by decidual cells in response to activated protein kinase C (PKC). Methods: Decidual cells were isolated from term placentae, grown to confluence and incubated with control media or 10-11 to 10-8 mol/l concentrations of phorbol 12-myristate 13-acetate (PMA). Concentrations of MMP-2, MMP-9 and TIMP-1 in the culture supernatant were determined using sensitive and specific immunoassays. Substrate zymography was conducted to confirm MMP-9 assays. Results: PMA induced a concentration-dependent stimulation of release of MMP-9 (control vs. PMA 10-9 and 10-8 mol/l; p < 0.01) and TIMP-1 (control vs. PMA 10-9 and 10-8 mol/l; p < 0.001), but not MMP-2. A direct positive correlation was observed between MMP-9 and TIMP-1 release (r = 0.645; p < 0.001). Substrate zymography confirmed increased release of MMP-9 in response to PMA (control vs. PMA 10-8 and PMA 10-7 mol/l; p < 0.01). Conclusions: Activation of PKC within the decidua will result in enhanced MMP-9 release, which upon activation could contribute to degradation of matrices within fetal membranes leading to PROM.

The Histone Deacetylase Inhibitor Sodium Butyrate Interacts Synergistically with Phorbol Myristate Acetate (PMA) to Induce Mitochondrial Damage and Apoptosis in Human Myeloid Leukemia Cells through a Tumor Necrosis Factor-α-Mediated Process

Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (ΔΨ m) and cytochrome c release). Neutralization of endogenous TNFα by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G 2M populations, and dephosphorylation of p34 cdc2. These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFα-dependent pathway in which procaspase 8 and Bid activation play key roles.

Synergistic induction of apoptosis in human myeloid leukemia cells by phorbol 12-myristate 13-acetate and flavopiridol proceeds via activation of both the intrinsic and tumor necrosis factor-mediated extrinsic cell death pathways.

Previous studies have shown that coexposure to marginally toxic concentrations of phorbol 12-myristate 13-acetate (PMA; 10 nM) and the cyclin-dependent kinase inhibitor flavopiridol (FP; 100-200 nM) synergistically induces apoptosis in human myeloid leukemia cells U937 and HL-60 (i.e., >50% apoptotic at 24 h). Attempts have now been made to characterize the cell death pathway(s) involved in this phenomenon. In contrast to cytochrome c release and caspase-3 activation, which occur within 2.5 h of PMA/FP coexposure, caspase-8 activation and Bid cleavage appeared as later events. Such findings implicate the mitochondria-dependent pathway in the initial induction of apoptosis by PMA/FP. However, U937 cells ectopically expressing CrmA, dominant-negative caspase-8, or dominant-negative Fas-associated death domain that were highly resistant to tumor necrosis factor (TNF)/cycloheximide-induced lethality displayed significant, albeit incomplete, resistance to PMA/FP-induced apoptosis after 24 h. Furthermore, coadministration of TNF soluble receptor significantly attenuated PMA/FP-induced apoptosis in U937 (p < 0.02) and HL-60 (p < 0.03) cells at 24 h. PMA/FP coadministration also triggered substantial increases in TNFalpha mRNA and protein secretion compared with the effects of PMA administered alone. The protein kinase C (PKC) inhibitor bisindolylmaleimide (1 microM) completely blocked PMA/FP-induced TNFalpha secretion in U937 cells and attenuated apoptosis. Taken together, these results suggest that coadministration of PMA with FP in myeloid leukemia cells initially triggers mitochondrial damage, an event followed by the PKC-dependent induction and release of TNFalpha, supporting a model in which the synergistic induction of leukemic cell apoptosis by this drug combination proceeds via both mitochondrial- and TNF receptor-related apoptotic pathways.

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester.

To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.

Protein kinase C stimulates release of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human decidual cells

Objective: Increased concentrations of amniotic fluid matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 have been observed in the context of premature rupture ofmembranes (PROM) and microbial invasion of the amniotic cavity. However, the source of the stimuli that contribute to the accumulation of these proteins in amniotic fluid remains to be identified. The presentstudy was conducted to investigate MMP-2, MMP-9 and TIMP-1 secretion by decidual cells in response to activated protein kinase C (PKC).Methods: Decidual cells were isolated from term placentae,grown to confluence and incubated with control media or 10-11 to 10-8 mol/l concentrations of phorbol 12-myristate 13-acetate (PMA). Concentrations of MMP-2, MMP-9 and TIMP-1 in theculture supernatant were determined using sensitive and specific immunoassays. Substrate zymography was conducted to confirm MMP-9 assays.Results: PMA induced a concentration-dependent stimulationof release of MMP-9 (control vs. PMA 10-9 and 10-8 mol/l; p < 0.01) and TIMP-1 (control vs. PMA 10-9 and 10-8 mol/l; p < 0.001), but not MMP-2.A direct positive correlation was observed between MMP-9 and TIMP-1 release (r = 0.645; p < 0.001). Substrate zymography confirmed increased release of MMP-9 in responseto PMA (control vs. PMA 10-8 and PMA 10-7 mol/l; p < 0.01).Conclusions: Activation of PKC within the decidua will result in enhanced MMP-9 release, which uponactivation could contribute to degradation of matrices within fetal membranes leading to PROM.

Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

Adenosine triphosphate-evoked cytosolic calcium oscillations in human granulosa-luteal cells: role of protein kinase C.

ATP has been shown to modulate progesterone production in human granulosa-luteal cells (hGLCs) in vitro. After binding to a G protein-coupled P2 purinergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate activates protein kinase C (PKC) and intracellular calcium [Ca(2+)](i) mobilization, respectively. In the present study, we examined the potential cross-talk between the PKC and Ca(2+) pathway in ATP signal transduction. Specifically, the effect of PKC on regulating ATP-evoked [Ca(2+)](i) oscillations were examined in hGLCs. Using microspectrofluorimetry, [Ca(2+)](i) oscillations were detected in Fura-2 loaded hGLCs in primary culture. The amplitudes of the ATP-triggered [Ca(2+)](i) oscillations were reduced in a dose-dependent manner by pretreating the cells with various concentrations (1 nM to 10 microM) of the PKC activator, phorbol-12-myristate-13-acetate (PMA). A 10 microM concentration of PMA completely suppressed 10 microM ATP-induced oscillations. The inhibitory effect occurred even when PMA was given during the plateau phase of ATP evoked [Ca(2+)](i) oscillations, suggesting that extracellular calcium influx was inhibited. The role of PKC was further substantiated by the observation that, in the presence of a PKC inhibitor, bisindolylmaleimide I, ATP-induced [Ca(2+)](i) oscillations were not completely suppressed by PMA. Furthermore, homologous desensitization of ATP-induced calcium oscillations was partially reversed by bisindolylmaleimide I, suggesting that activated PKC may be involved in the mechanism of desensitization. These results demonstrate that PKC negatively regulates the ATP-evoked [Ca(2+)](i) mobilization from both intracellular stores and extracellular influx in hGLCs and further support a modulatory role of ATP and P2 purinoceptor in ovarian steroidogenesis.

Phorbol 12-myristate 13-acetate induces alteration in mucin gene expression and biological properties of colon cancer cells.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

The CD44 receptor of lymphoma cells: structure-function relationships and mechanism of activation.

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.

The effect of PKC activation on the survival of rat retinal ganglion cells in culture

Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C (PKC), an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept “in vitro” for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate (PMA), a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ng/ml produced the maximal effect (a two-fold increase on ganglion cells survival compared with 48 h control). This effect was totally abolished by 1.25 μM chelerythrine chloride (an inhibitor of PKC) and 30 μM genistein (an inhibitor of tyrosine kinase enzymes). Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 μM 5-fluoro-2′-deoxyuridine, an inhibitor of cell proliferation, or 25 μM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells “in vitro” may be mediated by a mechanism that involves PKC activation.

Differential Localization of Protein Kinase C δ by Phorbol Esters and Related Compounds Using a Fusion Protein with Green Fluorescent Protein

Enzyme localization often plays a controlling role in determining its activity and specificity. Protein kinase C (PKC) has long been known to translocate in response to physiological stimuli as well as to exogenous ligands such as the phorbol esters. We report here that different phorbol derivatives and related ligands, selected for differences in chemical structure and profile of biological activity, induce distinct patterns of redistribution of PKC delta. Localization of a PKC delta-green fluorescent protein (GFP) fusion construct was monitored in living Chinese hamster ovary cells as a function of ligand, concentration, and time using confocal laser scanning microscopy. delta-PKC-GFP was expressed predominantly in the cytoplasm, with some in the nucleus and perinuclear region. Phorbol 12-myristate 13-acetate (PMA) induced plasma membrane translocation followed by slower nuclear membrane translocation. As the concentration of PMA increased, the proportion of nuclear to plasma membrane localization increased markedly. In contrast to PMA, bryostatin 1, a unique activator of PKC that induces a subset of PMA-mediated responses while antagonizing others, at all doses induced almost exclusively nuclear membrane translocation. Like PMA, the complete tumor promoter 12-deoxyphorbol 13-tetradecanoate induced plasma membrane and slower nuclear membrane translocation, whereas the inhibitor of tumor promotion 12-deoxyphorbol 13-phenylacetate, which differs only in its side chain, induced a distinctive distribution of PKC delta-GFP. Finally, the novel constrained diacylglycerol derivative B8-DL-B8 induced a slow Golgi localization. We speculate that differential control of PKC delta localization may provide an interesting strategy for producing ligands with differential biological consequences.

Distinct protein kinase C isozymes signal mitogenesis and apoptosis in human colon cancer cells

Background & Aims: Protein kinase C (PKC) is a family of serine-threonine kinases that transmit signals from cell surface receptors. To determine if distinct PKC isozymes transmit proliferative and/or apoptotic signals in colon cancer cells, we examined the effects of 3 PKC agonists, phorbol 12-myristate 13 acetate (PMA), ingenol 3,20-dibenzoate (IDB), and bistratene A, and a selective PKC inhibitor, GF 109203X, on proliferation, apoptosis, and activation of individual PKC isozymes in 5 colon cancer cell lines. Methods: Effects were assayed by a formazan-based colorimetric assay, [ 3H]thymidine incorporation, fluorescent nuclear staining, annexin V binding, DNA fragmentation assay, and immunoblotting of cytoplasmic and membrane fractions for PKC isozymes. Results: Two cell lines, SNU-C1 and SNU-C4, showed proliferative responses to PMA (0.1-1 nmol/L) and IDB (10-1000 nmol/L) and marked apoptotic responses to PMA (>5 nmol/L) and bistratene A (>1 μmol/L). GF 109203X blocked proliferative and apoptotic effects of PMA with distinct IC 50s. Proliferative concentrations of PMA and IDB caused translocation of PKCϵ alone, whereas apoptotic concentrations of PMA and bistratene A induced translocation of PKCδ. Conclusions: Activation of PKCϵ and PKCδ triggers proliferative and apoptotic signals, respectively, in SNU-C4 colon cancer cells. These 2 isozymes may play important opposing roles in normal homeostasis and neoplastic transformation of the colorectal epithelium. GASTROENTEROLOGY 1999;117:848-857

Phorbol Ester Facilitates Apoptosis in Murine Fibroblasts Pretreated by Mild Ultraviolet Radiation

Although phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m(2)) alone exhibited a slight expression of Fas and Fas-L 36 to 48 h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72 h after exposure. The addition of PMA (0.8 x 10(-5) to 3.2 x 10(-5) M) to the medium 24 h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24 h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24 h with a high concentration of PMA (3.2 x 10(-5) M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P=0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P=0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.