Protein Kinase C-Activating Tumor Promoters Enhance the Differentiation of Astrocytes in Aggregating Fetal Brain Cell Cultures

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte-specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4 alpha-phorbol 12,13-didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C-kinase, it is suggested that C-kinase plays a regulatory role in the growth and differentiation of normal astrocytes.     

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF

Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca(2+) increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.     

Inducible gene expression from multiple promoters by the tumor-promoting agent, PMA.

Phorbol ester tumor promoters affect a broad scope of changes in mammalian cells. This report describes the activation of expression of an introduced chloramphenicol acetyltransferase (CAT) reporter gene by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in a variety of fibroblast and hematopoietic cell lines. PMA-mediated activation appears to be promoter region specific, yet widespread. Enhanced gene expression is observed for four out of five promoter systems tested, and, in some cases, is dependent on the cellular environment. Further experiments indicate that PMA mediates elevated gene expression by rapidly increasing steady state levels of CAT mRNA. The broad range of promoters affected by PMA may help explain the high potency of this agent in tumor production.     

Effect of phorbol and bryostatin I on chondrogenic expression of chick limb bud, in vitro

The ability of phorbol esters to promote tumor formation and alter cell differentiation has been attributed to its action on a number of critical cellular functions, in particular, on protein phosphorylation, through the activation of protein C kinase. The present paper describes the effects of PMA (phorbol 12-myristate 13 acetate) on in vitro chondrogenesis in non-passaged, embryonic limb bud cells, relative to the effects of Bryostatin I. This compound also activates C kinase and binds competitively to the phorbol ester receptor, yet does not affect cell differentiation. Levels of PMA as low as 10(-7) M markedly reduced cartilage formation in 4-day cultures, as indicated by nodule count and Alcian blue staining for chondroitin sulfate. Coadministration of Bryostatin I at equimolar concentration prevented the PMA inhibitory effect on chondrocytic expression. This confirms other findings that phorbol activation of C kinase cannot exclusively account for the activity of phorbol on cell expression, i.e., that other pathway(s) must also be involved. Altering the time of PMA exposure demonstrated that PMA inhibited chondrocyte phenotypic expression, rather than cell commitment: early (0-48 h) exposure to PMA (during chondrocytic commitment in vitro) had little inhibitory effect on the staining index, whereas, exposure from 49-96 h (presumably post-commitment) and 0-96 h had moderate and strong inhibitory effects, respectively, on cartilage synthesis. Further research on the phorbol/Bryostatin I interaction should add to our knowledge of the control processes involved in tumor promotion and cell differentiation.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.     

Phorbol 12-myristate 13-acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10^?6 M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10^?7 and 10^?6 M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10^?6 M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-α PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen. © 1994 Wiley-Liss, Inc.     

Protein kinase C in erythroid and megakaryocytic differentiation: possible role in lineage determination

Erythroid differentiation of normal human hematopoietic progenitor cells was drastically inhibited by phorbol ester, 12-myristate 13-acetate (PMA), an agent known to activate the class of serine-threonine kinases, protein kinase C (PKC). This inhibition was accompanied by augmented megakaryocytic differentiation as demonstrated by expression of megakaryocyte-specific mRNAs and proteins. These effects of PMA were reversed by two specific antagonists of PKC. Analysis of single colonies transferred from cultures not containing PMA to PMA-containing cultures indicated that, in this system, PMA exerts megakaryocytic differentiating activity directly on cells which may have already initiated a progression toward the erythroid pathway of differentiation. These results suggest that modulation of PKC activity plays a role in erythroid and megakaryocytic differentiation, and may constitute an important selective signal between these pathways during normal blood cell development.     

Stimulation of progesterone production by phorbol-12-myristate-13-acetate in MA-10 Leydig tumor cells

The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (alpha-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Upregulation of recepteur d'origine nantais tyrosine kinase and cell invasiveness via early growth response-1 in gastric cancer cells

Abnormal accumulation and activation of the recepteur d'origine nantais (RON) has been implicated in carcinogenesis of epithelial tumors. RON expression was induced by the tumor promoter, phorbol 12-myristate 13-acetate (PMA), in gastric adenocarcinoma AGS cells. Studies with deleted and site-directed mutagenesis of Egr-1 promoter and with expression vectors encoding Egr-1 confirmed that Egr-1 is essential for RON expression. In addition, AGS cells pretreated with PMA showed remarkably enhanced invasiveness, which was partially abrogated by siRNA-targeted RON and Egr-1. These results suggest that tumor promoter induces RON expression via Egr-1, which, in turn, stimulates cell invasiveness in AGS cells.     

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.     

Suppression of cyclic AMP-dependent protein kinase activity in murine melanoma cells by 12-0-tetradecanoylphorbol-13-acetate

The effect of the potent tumor promoter 12-0-tetradeconylphorbol-13-acetate (TPA) on the cyclic AMP metabolism of B16 mouse melanoma cells was examined. TPA (10^?7M) slightly increased the growth rate and inhibited melanin production by these cells. Although TPA had little effect on basal or hormone stimulated cyclic AMP levels, it did significantly suppress cyclic AMP-dependent protein kinase activity from treated cells in a dose-dependent fashion. Other phorbol ester and non-phorbol ester tumor promoters also suppressed cyclic AMP-dependent protein kinase activity while the non-promoter, phorbol, did not alter cyclic AMP-dependent protein kinase activity.     

Functional role of extracellular signal-regulated kinase activation and c-Jun induction in phorbol ester-induced promoter activation of human 12(S)-lipoxygenase gene

The functional role of mitogen-activated protein kinase (MAPK) signaling and c-Jun induction in phorbol 12-myristate 13-acetate (PMA)-induced human 12(S)-lipoxygenase gene expression was studied in human epidermoid carcinoma A431 cells. Among the family of MAPK, PMA only increased the activity of extracellular signal-regulated kinase (ERK). Treatment of cells with PD98059, which is an inhibitor of mitogen-activated protein kinase kinase (MEK), decreased the PMA-induced expression of 12(S)-lipoxygenase. Transfection of cells with Ras, Raf and ERK2 dominant negative mutants inhibited the PMA-induced promoter activation of the 12(S)-lipoxygenase gene in all cases. PMA-induced expression of c-Jun was inhibited by pretreatment with PD98059. Following treatment with PMA, the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in cells increased with time. Enhancement of binding between the c-Jun-Sp1 complex and the Sp1 oligonucleotide was observed in cells treated with PMA, suggesting the possible interaction of c-Jun-Sp1 with GC-rich binding sites in the gene promoter. These results indicate that PMA treatment induced ERK activation mainly through the Raf-MEK-ERK signaling pathway following induction of c-Jun expression, and the formation of the c-Jun-Sp1 complex. Finally, PMA activated the promoter activity of the 12(S)-lipoxygenase gene in cells overexpressing protein kinase C (PKC)delta but not PKCalpha, indicating that PKCdelta played the functional role in mediating the gene activation of 12(S)-lipoxygenase induced by PMA.     

Bryostatins mimic the effects of phorbol esters in intact human platelets

Bryostatin-7 induces aggregation of human platelets and the phosphorylation of specific platelet proteins. Both the rate and extent of aggregation are similar to that induced by the tumor promoter phorbol ester 12-myristate 13-acetate (PMA); however, the rate of response is markedly reduced compared to that induced by thrombin. The addition of bryostatin-7 to 32P-labeled platelets results in a time-dependent incorporation of 32P into proteins of 20, 47 and 250 kDa; proteins of similar molecular mass are phosphorylated in response to the addition of thrombin or PMA. The time courses and dose responses of the phosphorylations induced by bryostatin-7 are similar to those found with PMA. In addition, bryostatin-7 increases the level of 32P incorporation into platelet polyphosphoinositides, which also occurs in response to PMA. These results suggest that, in intact human platelets, bryostatin-7 mimics the phorbol ester tumor promoter by directly activating protein kinase C.     

The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.