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.     

The protein kinase C activator phorbol-12-myristate-13-acetate enhances cyclic AMP accumulation in pheochromocytoma cells

The protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), augments the cyclic AMP accumulation induced by forskolin in pheochromocytoma (PC 12) cells with an EC50 value of 14 nM, while having no effect on basal values. At a concentration of 100 nM PMA markedly augmented the magnitude of the forskolin response and, in addition, caused a slight increase in the potency of forskolin. PMA also enhanced the maximal cyclic AMP accumulation produced by 2-chloroadenosine, and caused a slight increase in potency of the adenosine analog. Since PMA mimics the effect of diacylglycerols that form during the turnover of the membrane lipid, phosphatidylinositol, the results suggest an interrelationship between the systems involved in phosphatidylinositol turnover and cyclic AMP generation in PC 12 cells.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Forskolin and 12-0-tetradecanoylphorbol-13-acetate mimic thyrotropin-stimulated protein iodination in mouse thyroid

In the present study, the capacity of the diterpene, forskolin, and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), to stimulate protein iodination in freshly dispersed mouse thyroid open follicles was assessed. Although both agents stimulated 125I incorporation into TCA precipitable material, dose response curves (0.1 - 25 microM) showed that maximal concentrations of either agonist alone failed to reproduce the stimulatory effect of a maximal concentration of thyrotropin (TSH; 50 mU/ml). When a maximal concentration of forskolin (20 microM) and TPA (10 microM) were added in combination, the stimulatory effect was additive and mimicked the effect of TSH. TPA had no significant effect on either basal or forskolin-stimulated cyclic-AMP production. We conclude that the regulation of protein iodination by TSH may involve both the adenylate cyclase-cyclic-AMP-dependent protein kinase system and the diacylglycerol-activated calcium/phospholipid-dependent protein kinase C pathway.     

Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells.

The relative potency with which phorbol esters inhibited histamine-stimulated aminopyrine accumulation (an index of acid secretion) paralleled that which has been established for the activation of purified protein kinase C. The inhibitory effect of 1-oleoyl-2-acetylglycerol on aminopyrine accumulation stimulated by various secretagogues was similar to that of 12-O-tetradecanoylphorbol 13-acetate. Protein kinase C activity was present in a parietal-cell-enriched fraction. In conclusion, protein kinase C could be involved in mechanisms regulating gastric acid secretion.     

Two protein kinase C activators, bryostatin-1 and phorbol-12-myristate-13-acetate, have different effects on haemopoietic cell proliferation and differentiation.

Primary B lymphocytes can be induced to proliferate and certain haemopoietic cell lines such as HL60 and U937 can be induced to differentiate by the addition of phorbol esters, which have been shown to activate protein kinase C. Several non-phorbol esters, such as the bryostatins, have also been shown to bind to and activate protein kinase C. Although bryostatin-1 and 12-O-tetradecanoylphorbol-13-acetate (TPA) compete for and activate protein kinase C to the same degree and with similar kinetics and also induce similar levels of expression of the CD23 cell-surface antigen, bryostatin-1 is a weak mitogen for B lymphocytes and fails to induce the differentiation of both HL60 and U937 cells. Such an outcome suggests that these two activators have different binding properties for the enzyme that have a physiological consequence which may be useful for analysing the role that protein kinase C plays in both differentiation and proliferation. Analysis of competition assays between bryostatin-1 and TPA leads us to put forward a model where protein kinase C is required to be constantly reactivated and recycled during proliferation and differentiation which can be accomplished by TPA but not by bryostatin, although we cannot exclude the differential activation of some of the sub-species of the kinase by the two agonists.     

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)     

Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.     

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35%) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.     

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of 125I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two types of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

Effects of the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate on the wasp,Bracon hebetor

A single oral dose of the tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), caused a rapid necrosis of the ovarioles, aberrations in the developmental sequence of oocytes, and a concomitant dose-dependent decline in egg production in the wasp, Bracon hebetor. TPA and its metabolities were found to have a biological half life of 26.7 h, with a peak concentration in the ovarioles in 3 h. Damage to ovariole tissue was persistent despite the relatively short half life. Other tissues in the wasp were largely unaffected, although TPA induced lethargy that persisted until death. There was no shortening of life span. Inhibition of intercellular transport and metabolic cooperation may account for decreased fecundity and fertility, but interaction with a phorbol ester receptor is more likely to account for developmental changes and central nervous system poisoning.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of ¹²⁵I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two type of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

An activator of protein kinase C (phorbol-12-myristate-13-acetate) augments 2-chloroadenosine-elicited accumulation of cyclic AMP in guinea pig cerebral cortical particulate preparations

Norepinephrine and histamine markedly augment accumulations of cyclic AMP elicited by 2-chloroadenosine in a guinea pig cerebral cortical vesicular preparation. In addition, these biogenic amines stimulate phosphatidylinositol turnover. Phosphatidylinositol turnover is associated with mobilization of internal calcium and with stimulation of protein kinase C. Phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, has no effect on cyclic AMP levels alone, but in a concentration-dependent manner enhances accumulations of cyclic AMP elicited by 2-chloroadenosine. PMA, like norepinephrine, also enhances accumulations of cyclic AMP elicited by histamine. PMA has no effect on the synergistic accumulations of cyclic AMP elicited by combinations of amines and 2-chloroadenosine. PMA also augments accumulations of cyclic AMP elicited by forskolin. The results suggest that activation of phosphatidylinositol turnover by biogenic amines may lead via stimulation of protein kinase C to enhanced responsiveness of cyclic AMP-generating systems.     

12-0-tetradecanoylphorbol-13-acetate and concanavalin A enhance glucose uptake in thymocytes by different mechanisms

The effects of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) and the plant lectin concanavalin A (Con A) on glucose uptake in murine thymocytes were studied. TPA induces a rapid dose-dependent increase in the uptake of 2-deoxyglucose and in the transport of 3-0-methylglucose. Con A also elicits a time- and dose-dependent enhancement of 2-deoxyglucose uptake. The effect of Con A, however, is less pronounced. The effect of combined treatment of thymocytes with Con A and TPA is not additive. Cytochalasin B completely inhibits the basal, as well as TPA- and Con A-enhanced, 2-deoxyglucose uptake. Dexamethasone markedly inhibits basal 2-deoxyglucose uptake, but is less inhibitory to enhanced 2-deoxyglucose uptake induced by TPA and Con A. The effect of TPA on 2-deoxyglucose uptake and 3-0-methylglucose transport is refractory to inhibition by isobutyl methyl xanthine, dibutyryl cyclic AMP, and ethyleneglycol tetraacetic acid. These agents markedly inhibit the enhancement of 2-deoxyglucose (2-DOG) uptake by Con A. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, also selectively inhibits Con A enhancement of 2-DOG uptake. Taken together, the results suggest that Con A and TPA exert their stimulatory effect on glucose uptake by different activating mechanisms, but they may share a final common transport pathway.     

Opposing effects of phorbol-12-myristate-13-acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The ‘cross‐talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP‐dependent signaling by structurally related human D1‐like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, followed by analysis of dopamine‐mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist‐evoked D1 receptor signaling, whereas constitutive and dopamine‐mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester‐regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ?, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca²⁺‐independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross‐talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1‐like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.