Inhibition of respiration by a phorbol ester tumor promoter in murine cultured cells

The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate is a potent inhibitor of mitochondrial respiration in both normal and methylcholanthrene-transformed C3H $\text{10T1}\text{2}$ mouse fibroblasts. This inhibition is seen at concentrations of tumor promoter in the range of 10^?8M, occurs within a few minutes after exposure of the intact cells, and is not seen with a biologically inactive analog. The effect appears to be exerted through inhibition of the function of an oligomycin-sensitive ATPase. It is possible, therefore, that alterations in mitochondrial function are associated with the process of tumor promotion.     

Modulation of type alpha transforming growth factor receptors by a phorbol ester tumor promoter

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.     

Calcium-dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter

The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two-phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase. Ionophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.     

Protein synthesis and degradation in cultured muscle is altered by a phorbol diester tumor promoter

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was observed to influence the relative rates of synthesis and degradation of several polypeptides in cultured chick embryo myotubes. The direction of influence partially correlated with whether the polypeptide was uniquely expressed in myotubes or also expressed in its proliferating precursors. The synthesis of all but one of the eight myotube-unique polypeptides examined was inhibited and the degradation of all but two was stimulated. The exceptions were intermediate filament subunits. In contrast, the metabolism of several non-myotube-unique polypeptides was either unaffected or influenced in the opposite direction. A method involving saturation of the intracellular leucine precursor pool with extracellular radioactive leucine suggested that the absolute rate of protein synthesis was only minimally, if at all, affected by the promoter. The effects on protein synthesis were at least partially reversible following removal of the promoter. The changes in synthesis and degradation did not reflect normal maturational changes in cultured myotubes and thus they appeared to be induced by TPA. The data suggest that under some circumstances the rates of synthesis and degradation of several proteins are inversely coupled in the myotube. They also provide a partial explanation for earlier ultrastructural observations that TPA caused a loss of myofilaments and an accumulation of intermediate filaments. Since myotubes occupy the G₁ (or G₀) stage of the cell cycle, and the DNA content of the cultures was not affected by TPA, TPA's effects were not mediated through cell cycle-dependent mechanisms.     

Induction of ornithine decarboxylase in rat liver by phorbol ester is mediated by prostanoids from Kupffer cells

Administration of phorbol 12-myristate 13-acetate (PMA) to rats in vivo resulted in the induction of ornithine decarboxylase activity in the liver which could be blocked by preinjection of indomethacin, a cyclooxygenase inhibitor. In vitro administration of PMA to primary cultures of rat parenchymal cells did not lead to an induction of ornithine decarboxylase activity. It was investigated to what extent non-parenchymal liver cells could play an intermediary role in the expression of the PMA effect on ornithine decarboxylase activity in parenchymal liver cells. Addition of conditioned medium from PMA-activated Kupffer cells to cultured parenchymal cells led to the induction of ornithine decarboxylase activity in parenchymal cells. This effect was not observed with conditioned medium from untreated Kupffer cells or from Kupffer cells treated with PMA plus indomethacin. Conditioned media from PMA-treated or untreated endothelial liver cells were ineffective in the induction of ornithine decarboxylase activity in parenchymal liver cells. Prostaglandin D2, the main eicosanoid produced by Kupffer cells, was able to stimulate the synthesis of ornithine decarboxylase in parenchymal liver cells (up to 40-fold) in a dose-dependent way. Prostaglandin (PG) D2 appeared to be a more potent inducer of ornithine decarboxylase activity in parenchymal cells than PGE1 and PGE2. It is concluded that intercellular communication inside the liver mediated by prostaglandins derived from activated Kupffer cells may form a mechanism to induce synthesis of specific proteins in parenchymal cells.     

The role of calcium in macrophage chemotaxis to the phorbol ester tumor promoter TPA

The significance of the macrophage in the inflammatory response that occurs concurrently with phorbol ester induced tumor promotion has not yet been determined. Biologically active phorbol ester tumor promoters modify several functional responses of macrophages including chemotaxis, cytotoxicity, secretion and prostaglandin synthesis and release. The present study examines calcium metabolism as a possible underlying biochemical mechanism through which 12-0-tetradecanoyl-phorbol-13-acetate (TPA) exerts its effects on macrophage chemotaxis. The chemotaxis of mouse resident peritoneal macrophages was evaluated in the presence of pharmacological agents known to alter cellular calcium metabolism. The calcium ionophore A23187 in microM concentrations enhanced macrophage chemotaxis to TPA by approximately 41%. This enhancement was dependent on the presence of extracellular calcium. TPA-induced chemotaxis was also enhanced by the histological dye ruthenium red (RR), an agent known to modify mitochondrial calcium fluxes and calcium-dependent neuronal transmission. Ruthenium red (0.1 and 1.0 microM) produced a maximal stimulation of macrophage chemotaxis to TPA of approximately 62%. An intracellular calcium antagonist, 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) inhibited macrophage chemotaxis to TPA in a dose related fashion (1.0 to 100 microM). Varying extracellular calcium concentrations (0-3.6 mM) had no effect on macrophage chemotaxis in response to TPA. In drug combination studies neither A23187 nor RR was able to overcome the inhibitory effects of TMB-8 on macrophage chemotaxis to TPA. These results indicate that intracellular calcium metabolism may be playing a significant role in modulating TPA's effect on macrophage chemotaxis, while extracellular calcium may be of little import. A possible mode of TPA's effect on the macrophage via mobilization of calcium from cellular storage sites is discussed.     

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.     

Changes in fibroblast contractility, morphology, and adhesion in response to a phorbol ester tumor promoter

We have studied the effects of the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the contractility, locomotion, morphology, and adhesion of two mammalian fibroblastic cell lines. Using the silicone rubber substratum technique, we have found that the first observable response to the tumor promoter is a rapid weakening of cell contractility (8-15 min). This is followed by gradual morphological changes, characterized by a hyperextension of the cells' leading lamellae, which stretch out to an unlimited degree, and occasionally even detach from the cell bodies. Treated cells also become able to crawl onto hydrophobic substrata which are insufficiently adhesive to support the spreading of untreated fibroblasts. We suggest that both the hyperextension and the ability to spread on nonadhesive surfaces can be explained as consequences of the reduced contractility, and that this reduced contractility may also help to explain the increased invasiveness and loss of anchorage dependence by transformed cells.     

The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and Bay K8644 and inhibited by 8-bromo-cyclic GMP

The role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release was studied using the isolated perfused rat heart. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the protein kinase C pathway, produced a dose-dependent increase in perfusate ANP immunoreactivity. Bay k8644, a putative calcium channel activator, and forskolin, which stimulates adenylate cyclase, induced a sustained increase in ANP secretory rate. TPA in combination with either Bay k8644 or forskolin induced higher ANP secretion than the calculated additive value for each agent. 8-bromo-cyclic GMP and sodium nitroprusside, when given alone, had no effect on ANP secretion, but delayed the TPA-stimulated increase in perfusate ANP. ANP secretion appears therefore to be mediated both by the phosphoinositide and the cAMP system, whereas the cGMP pathway may be inhibitory.     

Dynactin is essential for growth cone advance

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150^Glued, from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150^Glued, plays an essential role in growth cone advance.     

Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation at 5 microM. Carbachol increased the accumulation of [3H]inositol phosphates only in the presence of added guanine nucleotide. Calcium increased [3H]InsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal [3H]InsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous [3H]InsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited [3H] InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated [3H]InsP3 formation although calcium-stimulated [3H]InsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated [3H]InsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of [3H]InsP3 results from inhibition of the G protein interaction with phospholipase C.     

Nerve cell production in Hydra is deregulated by tumour-promoting phorbol ester

Summary The tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) interfers with nerve cell production in Hydra when applied to the animals' culture medium. Precursor cells exposed to 0.2 nM TPA during the first half of their S-phase are prevented from differentiating into nerve cells. Precursor cells which start their S-phase following a treatment with TPA give rise to nerve cells. The frequency is higher than in untreated control animals. Offprint requests to: S. Berking     

Regulation of neuronal growth cone filopodia by nitric oxide

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.     

Studies on immunomodulatory properties of isoniazid. IV. Effect of isoniazid on T lymphocyte activation by phorbol ester tumor promoter

Human peripheral blood T cells or continuous T cell lines were treated with phorbol myristate (PMA), a direct protein kinase C activator. The effect of isoniazid on PMA-treated cells was investigated. It was found that low doses of isoniazid augmented proliferation of T cells treated with PMA or stimulated with PMA and phytohemagglutinin. Similar results were shown in the Jurkat cell line. There was no influence of isoniazid upon proliferation of PMA-treated HT-2 cells, interleukin 2-dependent cell line. The obtained results suggest that, at least one target mechanism of isoniazid is located in T cell activation pathway after protein kinase C activation.     

Zymosan-stimulated tumor necrosis factor-alpha production by human monocytes. Down-modulation by phorbol ester.

In this study, we showed that human monocytes produced TNF-alpha in response to zymosan, a particulate agonist. Protein kinase C (PKC) seems to play a regulatory role in zymosan-induced TNF-alpha secretion. The pretreatment of monocytes with PMA induced a dose-dependent inhibition of zymosan-stimulated TNF production. This inhibition was likely due to an activation of PKC because it was prevented by inhibitors of PKC, sphingosine, and staurosporine. Moreover, PMA elicited a profound down-modulation of zymosan binding to monocytes. The inhibition of zymosan binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, PMA did not modify the expression of CD11b/CD18 receptor that is involved in zymosan recognition. In view of these findings, qualitative changes of CD11b/CD18 molecules might account for the inhibition of zymosan binding and TNF production. Thus, PMA specifically increased the association of CD11b/CD18 with the detergent-insoluble cytoskeleton. Cytochalasin B but not microtubule disrupters, nocodazole and colchicine, partially prevented the inhibition of zymosan binding. Hence, the inhibitory action of PMA on zymosan binding seems to be mediated by an increase in attachment of zymosan receptor to cytoskeleton and more likely to microfilaments. The regulatory activity of PKC might represent a first way of limiting cytokine over-production in response to pathogens which interact with monocytes via CD11/CD18 molecules.