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PubMed1981

The molecular structure and charge distribution of phorbol: parent compound of the tumor promoting phorbol diesters.

The geometry of phorbol, parent compound of the very highly active tumor promoting phorbol diesters, was totally optimized starting from the crystal structure of phorbol-5-bromofuroate-chloroform solvate (Petterson et al., J. Chem. Soc. B980, 1968). The full optimization of all atomic positions was done quantum chemically using the MINDO/3 potential energy surface and a modified Davidon-Fletcher-Powell search for local minima. The molecular structure and details of the electronic charge distribution are presented.

Chemical Phenomena↗
PubMed1983

The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate and stimulation of 3H-choline incorporation into endoplasmic reticulum membranes and other subcellular fractions of Krebs II ascites cells during in vitro incubation.

After transfer of Krebs II ascites cells from the mouse peritoneum to suspension culture addition of the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) causes an early stimulation of 3H-choline incorporation into phosphatidylcholine (PC). Choline transport into the treated cells, however, was unaffected. Within 30 min of TPA treatment 3H-choline incorporation was almost 300% above the control level. During a 5 hr period of suspension culture the overall patterns of 3H-choline incorporation were similar in TPA-treated and control cultures though the rate was greatly accentuated by the presence of the phorbol ester. Incubation of cells with cycloheximide prior to incubation with TPA did not result in an inhibition of the TPA-directed 3H-choline incorporation. After 3 hr incubation with TPA there were large increases in radioactivity in all subcellular fractions. At 20 hr, however, the values were not far from those of the control. During the first 3 hr of incubation with TPA the incorporation of 3H-choline into light rough (LR) and smooth (S) membranes was stimulated to levels of 400% and 320% respectively above control values. At later times the profiles of radioactivity in membrane subfractions in TPA-treated and control cultures were similar. The results illustrate an early effect of TPA on PC biosynthesis in Krebs II ascites cells while at later times of incubation the stimulatory effect was virtually abolished.

Animals↗
PubMed1992

The protein kinase C activators, phorbol 12-myristate,13-acetate and phorbol 12,13-dibutyrate, are convulsant in the pico-nanomolar range in mice.

Administration of phorbol 12-myristate,13-acetate (PMA, 10 fmol-10 nmol) or phorbol 12,13-dibutyrate (PDB, 0.2-495 nmol) (i.c.v.) to mice induced: hindlimb scratching, tremor, myoclonic jerks, hyperlocomotion, clonic seizure, followed by death or recovery. CD50 values for clonic seizures for PMA and PDB were 1.0 pmol and 1.2 nmol. 4-alpha-Phorbol (68-686 nmol) was inactive. The effects of PDB (24-247 nmol) were reduced by pretreatment with staurosporine (30 nmol, i.c.v.). Protein kinase C activators are potent convulsants in vivo.

Alkaloids↗
PubMed2002

4alpha-Phorbol negates the inhibitory effects of phorbol-12-myristate-13-acetate on human cilia and alters the phosphorylation of PKC.

In medium 199, ciliary beat frequency (CBF) in human nasal epithelium declines to 60% of baseline by 2 h and 1 nM phorbol-12-myristate-13-acetate (PMA) doubles the rate of decline by activating protein kinase C (PKC). We find that a reported negative control for PMA, 4alpha-phorbol (1 pM-1 nM)+/-1 nM PMA, not only maintains CBF at baseline, but arrests a pre-existing PMA-induced decline in CBF and alters the profile of multiple phosphorylated PKC species. Thus, 4alpha-phorbol not only potently prevents PMA from inhibiting CBF but also has potent effects on the phosphorylation of PKC.

Blotting, Western↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--IV. Lipophilic binding and molecular orientation of spin-labeled phorbol-12,13-diesters in human erythrocyte membrane.

In human erythrocyte membranes, membrane binding of spin-labeled TPA-analogous phorbol (doxyl)esters [(n,m)PA] was investigated during measurement of the kinetics of the decay of their electron paramagnetic resonance signal by ascorbate reduction. In membrane-bound (n,m)PA the reduction rate was dependent of the position of doxyl in the aliphatic chain of their 12-O-acyl moiety. To describe quantitatively the reaction kinetics observed, two hypotheses (models) were developed and used. Model 1 is based on the assumption that ascorbate reduction takes place in the extracellular space. In this case the experimental data could be fitted by the partition and permeability coefficients of (n,m)PA determining model 1 only, if non-realistic values of these parameters were used. The more refined model 2, corresponding to a bilayer membrane structure, assumes the reduction to take place in the hydrophilic region of the membrane. Assuming a finite probability of finding the doxyl group within the hydrophilic membrane region, model 2 describes quantitatively the dependence of the reduction rate on the position of the doxyl in the aliphatic chain of the (n,m)PA used. From the validity of this model it may be postulated that the molecular orientation of TPA-analogous (n,m)PA in the bilayer membrane is determined by an anchoring of their lipophilic ester moiety in the lipophilic region of the membrane bilayer, thus locating the hydrophilic phorbol moiety within the hydrophilic region of the membrane. With regard to the well-known categories of non-specific versus specific binding of bioactive phorbol esters to protein kinase C/membrane complexes it is deduced that anchoring of (n,m)PA (and hence TPA) in the hydrophobic interior of the membrane structure may be the molecular equivalent of their non-specific binding.

Ascorbic Acid↗
PubMed1983

Phorbol diester-induced H2O2 production by peritoneal macrophages. Different H2O2 production by macrophages from normal and BCG-infected mice despite comparable phorbol diester receptors.

Mouse peritoneal macrophages respond to environmental stimuli in different ways depending on their state of differentiation. Macrophages from mice with bacillus Calmette--Guerin (BCG) infection produced large amounts of H2O2 in response to phorbol diesters (PDEs), while those from noninfected mice produced little or no H2O2. The effects of PDEs on cells are mediated by specific cellular receptors for these ligands. The purpose of this study was to determine if the varying responses of macrophages from different groups of mice were caused by differences in their receptors for the PDE ligands. By all parameters studied, the binding of [20-3H]phorbol 12,13-dibutyrate ( [3H]PDBu) was similar in all macrophages irrespective of their ability to produce H2O2 in response to PDEs. Binding of [3H]PDBu was rapid at 23 degrees C reaching a maximum at 10-20 min with a subsequent decline to 50-60% of maximum by 30-60 min. Binding was slower at 0 degrees C reaching a maximum at 90-120 min. The binding was reversible, with dissociation kinetics paralleling association kinetics. The binding was saturable; the Kd's (45 to 91 nM) and number of binding sites (about 7-14 X 10(5)/cell or 11-12 pmol/mg protein) were essentially the same for the different classes of macrophages. The binding was specific, and analogs of PDBu inhibited [3H]PDBu binding to macrophages with potencies comparable to their potencies in causing in vivo tumor promotion and elicitation of other cellular responses in vitro. The ligands [3H]PDBu and [3H]PMA were degraded to comparable degrees by macrophages from normal or BCG-infected mice. Macrophages from C3H/HeJ and C3H/HeN mice, although known to differ in their abilities to respond to stimuli such as lymphokines and LPS, did not differ in their ability to produce H2O2 in response to PDEs or in their receptors for PDEs. Results of this study suggest that in vivo "activation" of macrophages in mice infected with BCG is not associated with a change in the cells' receptors for PDEs, but may be associated with "postreceptor" changes such as linkage of the PDE receptor with NAD(P)H oxidase, a change in NAD(P)H oxidase, or induction of synthesis of NAD(P)H oxidase.

Animals↗
PubMed1993

Insulin-mimetic actions of phorbol ester in cultured adult rat hepatocytes. Lack of phorbol-ester-elicited inhibition of the insulin signal.

The actions of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on glucose metabolism, amino acid transport and enzyme inductions were studied in primary cultures of adult-rat hepatocytes and compared with the effects of insulin. PMA and insulin stimulated glycolysis 5- and 7-fold respectively. The half-maximal effective dose of PMA was 60 nM. Stimulation of glycolysis was accompanied by an insulin- or PMA-dependent and okadaic acid-sensitive activation of 6-phosphofructo-2-kinase and pyruvate kinase, as well as by an increase in fructose 2,6-bisphosphate. Glucose production from glycogen was decreased to 50% by PMA and to 15% by insulin, whereas glycogen synthesis was stimulated 2- and 7-fold respectively. PMA also increased aminoisobutyrate uptake, induced ornithine decarboxylase and counteracted the glucagon-dependent induction of phosphoenolpyruvate carboxykinase. PMA strongly antagonized the hormonal activation of glycogen synthesis, but all other insulin actions assayed were not decreased by the phorbol ester. Whereas additive effects of PMA and insulin were not detected, PMA and a simultaneous increase in the glucose concentration had additive effects on glycolysis and glycogen metabolism. Cell exposure to insulin resulted in receptor autophosphorylation and a more than 10-fold activation of the receptor tyrosine kinase. PMA did not alter these effects, and also had no effect on the receptor phosphorylation status in the absence of insulin. Long-term (15 h) pretreatment of the cells with PMA abolished all PMA effects, but not the insulin effects. It is concluded that PMA does not generally antagonize the action of insulin in differentiated adult hepatocytes, and that insulin and PMA may use related signal-transduction pathways.

Aminoisobutyric Acids↗
PubMed1993

Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the 'de novo' pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake.

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the 'de novo' pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the 'de novo' pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).

Animals↗
PubMed1996

Transcriptional regulation of transferrin receptor expression during phorbol-ester-induced HL-60 cell differentiation. Evidence for a negative regulatory role of the phorbol-ester-responsive element-like sequence.

The mechanism involved in the regulation of transferrin receptor (TfR) expression during phorbol-ester-induced HL-60 cell differentiation was investigated. The mRNA of the TfR was constitutively expressed in proliferating HL-60 cells. Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h resulted in a gradual decrease in the expression of the TfR mRNA. Nuclear run-on assays revealed that the transcription of the TfR gene was inhibited by prior treatment of cells with PMA. The effect of PMA on the binding of nuclear proteins to the TfR gene promoter region was then investigated. Based on sequence similarity and previous footprinting data, the promoter region of the TfR gene seems to contain a sequence like that of the phorbol-ester-responsive element (TRE). Our results showed that the binding of nuclear extracts to the TfR gene promoter region containing the TRE-like sequence was increased in PMA-treated cells. This binding activity could be abolished by prior incubation of the nuclear extracts with a synthetic oligonucleotide containing the consensus TRE sequence. In vitro transcription assays revealed that prior incubation of the nuclear extracts of PMA-treated cells with excess consensus TRE oligonucleotide enhanced the gene transcription driven by the TfR gene promoter. These findings suggest that the TRE-like element may play a role in the inhibition of TfR gene transcription.

Binding Sites↗
PubMed1987

Basal and electrically stimulated release of [3H]noradrenaline and [3H]dopamine from rat amygdala slices in vitro: effects of 4 beta-phorbol 12,13-dibutyrate, 4 alpha-phorbol 12,13-didecanoate and polymyxin B.

The protein kinase C activator 4 beta-phorbol 12,13-dibutyrate (PDB) enhanced in a concentration-dependent manner the electrically stimulated release of [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) from rat amygdala slices in vitro. PDB enhanced the basal release of [3H]NA and [3H]DA as well. 4 alpha-Phorbol 12,13-didecanoate, which lacks the capacity to activate protein kinase C, was without effect on either basal or electrically stimulated release of [3H]NA and [3H]DA. Polymyxin B, which is a relatively selective protein kinase C inhibitor, decreased in a concentration-dependent manner the electrically stimulated release of both [3H]NA and [3H]DA from amygdala slices, whereas it enhanced the basal release of both neuromessengers. In the presence of 1.5 X 10(-7) M PDB, a concentration which when added to the superfusion medium alone doubled the electrically stimulated release of both [3H]NA and [3H]DA, polymyxin B again decreased in a concentration-dependent manner the release of both neuromessengers. At all polymyxin B concentrations used, the effect of the PKC inhibitor, expressed as percent inhibition, in the presence of PDB was approximately the same as that observed in the absence of PDB. This suggests that the antagonism between PDB and polymyxin B at the level of protein kinase C is not a competitive one. The effects of PDB and polymyxin B on basal release were additive. Taken together, these data suggest that in the amygdala presynaptically localized protein kinase C plays a role in signal transduction processes related to the exocytotic secretion of NA and DA from their nerve terminals.

Amygdala↗
PubMed1987

Calcium ionophore A23187 enhances human neutrophil superoxide release, stimulated by phorbol dibutyrate, by converting phorbol ester receptors from a low- to high-affinity state.

The calcium ionophore A23187 acted synergistically with phorbol dibutyrate (PDBu) to stimulate human neutrophil superoxide production. A23187 shortened the lag period and markedly increased the initial rate of neutrophil superoxide production induced by suboptimal concentrations of PDBu. 1 microM A23187 reduced the EC50 value for superoxide release from 56 to 8 nM PDBu. This effect of A23187 was correlated with enhanced binding of [3H]PDBu to its receptor and a reduction in the dissociation constant (Kd) from 27 to 10 nM, without altering the apparent total number of phorbol dibutyrate receptors. These actions of A23187 were abolished in the presence of EGTA or TMB-8, confirming a dependence on Ca2+.

Caenorhabditis elegans Proteins↗
PubMed1987

Differences between human and goose erythrocytes in response to phorbol esters and expression of phorbol ester receptors.

Phorbol esters inhibited the uptake of a fluorescent glucose analogue in goose but not in human erythrocytes. Specific phorbol-12,13-dibutyrate (PDB) binding sites were identified in both goose and human erythrocytes. In the absence of Ca2+ and phospholipid, PDB binding in whole cell lysates was similar to that in intact cells, but addition of Ca2+ (0.5 mM) and phosphatidyl serine (96 micrograms/ml) caused a 4-fold increase in the binding detected in lysates. Nonlinear least-squares analysis of the PDB binding isotherm revealed that the data for lysates from both goose and human cells were best fit by a two-site model, with goose erythrocytes having approximately 3 times as many sites per class of receptors. Subcellular fractionation of human lysates indicated that the high (Kd = 3.6 +/- 2.2 nM) and low (Kd = 20 +/- 5 nM) affinity sites could be accounted for by the contributions from cytosol and crude membrane, respectively. Separation of the high and low affinity sites was not achieved in goose lysates. PDB binding to intact goose erythrocytes exhibited the lower affinity (Kd approximately 30 nM) and was enhanced approximately 2-fold by incubation at 37 degrees C relative to incubation at 4 degrees C. This was due to an increased Bmax, with no change in Kd of the whole cell binding. Human erythrocytes did not demonstrate this temperature-enhanced binding of PDB to intact cells. These data are consistent with a temperature-induced translocation of PDB receptors from cytosol to membrane in goose erythrocytes. The failure of human erythrocytes to respond to PDB is not due to an absence of PDB receptors but may be related to the diminished number of receptors or to the lack of a temperature-induced increase in whole cell receptor number.

4-Chloro-7-nitrobenzofurazan↗
PubMedDate not supplied

Dissimilarities in superoxide anion production by human neutrophils stimulated by phorbol myristate acetate or phorbol dibutyrate.

Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBU) are known to translocate protein kinase C (PKC) and to induce superoxide anion (O2-.) production in human neutrophils. They are thus currently used to probe the role of PKC in O2-. production. We show here that under certain conditions, O2-. production induced by PMA is not associated with a decrease in cytosolic PKC activity, whereas these two events are associated after PDBU stimulation. (1) In the presence of extracellular calcium (1 mM), O2-. production was related to the concentration of PMA. PMA induced O2-. production at all the concentrations studied, but this was not associated with a decrease in cytosolic PKC levels up to 5 ng/ml PMA (50% maximum O2-. production). (2) Under PDBU stimulation, even at very low O2-. production levels, cytosolic PKC decreased and the decrease as well as the O2-. production were related to the concentration of PDBU. (3) For a given decrease in cytosolic PKC, O2-. production induced by PMA was much greater than that induced by PDBU. (4) In calcium-free medium, O2-. production induced by low concentrations of PMA (up to 5 ng/ml) was lower than that observed in the presence of 1 mM calcium, whereas modifications of cytosolic PKC activity were similar. (5) Cytochalasin B had no effect on PMA-induced O2-. production, regardless of the calcium content of the medium, and had no effect on the decrease in cytosolic PKC. On the contrary, following PDBU stimulation, cytochalasin B increased O2-. production, regardless of the medium, but induced a larger decrease in cytosolic PKC when Ca2+ was present. (6) Preincubation of PMN with 100 microM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before stimulation with PMA or PDBU led to similar inhibition of O2-. production whatever the degree of decrease in cytosolic PKC activity. These findings show that, in contrast to PDBU, O2-. production induced by PMA is not always related to cytosolic PKC activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

Effects of a phorbol ester and cyclosporin A on hippocampal synaptic plasticity in streptozotocin-induced-diabetic rats: reduced sensitivity to phorbol esters.

In streptozotocin-induced diabetic (STZ-diabetic) rats, an animal model of diabetes mellitus, a reduced expression of long-term potentiation (LTP) and enhanced long-term depression (LTD) are observed. This study examined the role of protein kinase C (PKC) and protein phosphatase 2B in hippocampal synaptic transmission in STZ-diabetic rats. The phorbol ester 4beta-phorbol-12,13-dibutyrate (PDB) induced a concentration-dependent potentiation of synaptic responses in area CA1 that could partially be inhibited by the PKC inhibitor chelerythrine. In slices from STZ-diabetic rats the effectivity of PDB to increase synaptic transmission was reduced compared to slices from control animals. In STZ-diabetic rats the protein phosphatase 2B (PP2B) inhibitor cyclosporin A inhibited LTD induction, but did not affect the induction of LTP. In conclusion, these data show a reduced response to PDB in STZ-diabetic rats, and indicate that the lack of LTP induction in these animals is not due to increased PP2B activity.

Alkaloids↗
PubMed1991

Prostratin, a nonpromoting phorbol ester, inhibits induction by phorbol 12-myristate 13-acetate of ornithine decarboxylase, edema, and hyperplasia in CD-1 mouse skin.

Pretreatment of CD-1 mouse skin with prostratin (12-deoxyphorbol 13-acetate) inhibited biological response to phorbol 12-myristate 13-acetate. The three responses examined were hyperplasia, induction of ornithine decarboxylase, and edema; the characteristics of inhibition depended on the specific response. Hyperplasia is the best short-term correlate of tumor promotion. Two or more pretreatments with 2.56 mumol (1 mg) prostratin, administered at intervals of 1-4 days, almost completely blocked the hyperplasia induced by phorbol 12-myristate 13-acetate applied 15 min to 6 h after the last pretreatment. Inducibility of hyperplasia was partially restored at 2 days and recovered by 4 days. Prostratin was more potent for inhibition of ornithine decarboxylase induction (50% inhibitory dose = 25.6 nmol) than it was for hyperplasia: the inhibition was largely attained by the first application, and the recovery from inhibition was slower (8 days). Edema was partially inhibited by prostratin (dose giving 50% of maximal inhibition = 512 nmol). We have previously demonstrated that prostratin is a protein kinase C activator. Our present results show that prostratin is a functional antagonist for a class of protein kinase C mediated responses. The findings emphasize the diversity of biological outcome for protein kinase C activators, presumably driven by the extensive heterogeneity in the protein kinase C pathway.

Animals↗
PubMed1994

Changes in gap-junction permeability, phosphorylation, and number mediated by phorbol ester and non-phorbol-ester tumor promoters in rat liver epithelial cells.

The effects of three tumor promoters on gap-junction permeability; connexin 43 and 26 mRNA levels, protein levels, and phosphorylation; and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the inhibition of gap-junction function. 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL), dieldrin (10 micrograms/mL), and heptachlor epoxide (10 micrograms/mL) inhibited gap-junctional intercellular communication (GJIC) assayed by fluorescent dye transfer by 80-90% after a 5-min exposure and by more than 90% within 1 h. Decreases in steady-state connexin 43 mRNA levels were detected by northern blot analysis within 1 h and paralleled changes in steady-state beta-actin mRNA, but these changes did not occur rapidly enough to account for the rapid loss of gap-junction function. A substantial loss in the number of connexin 43 immunostained gap-junctional membrane plaques was detected after a 15-min exposure to all three promoters, but little change had occurred at 5 min. Western blot analyses using connexin 43-specific antibodies showed changes in the degree of connexin 43 phosphorylation for all three tumor promoters. TPA induced the appearance of a fourth connexin 43-immunoreactive band (P3) and a concomitant decrease in the relative intensity of the unphosphorylated (P0) band within 5 min of treatment. P3, in addition to bands P1 and P2, disappeared after treatment with alkaline phosphatase. In contrast, dieldrin and heptachlor expoxide induced loss of P2 with a concomitant increase in the relative staining intensity of P0 within 1 h of exposure, but no changes were seen after 5 min. Connexin 43 phosphorylation levels recovered in parallel with the recovery of GJIC for all three tumor promoters. Connexin 26 mRNA levels showed little change after a 1-h exposure to three promoters, but reductions in connexin 26 immunofluorescent staining were observed. These results suggest that (i) TPA-induced hyperphosphorylation of connexin 43 occurred fast enough to account for inhibition of GJIC, (ii) dieldrin and heptachlor expoxide modulated connexin phosphorylation in a manner different from TPA by promoting hypophosphorylation of connexin 43, (iii) redistribution of plasma membrane gap-junctional plaques after treatment with phorbol ester and non-phorbol-ester tumor promoters occurred subsequent to changes in gap-junction permeability, and (iv) changes in connexin mRNA levels could not account for the losses in fluorescent dye coupling induced by these promoters.

Animals↗
PubMed1985

Bryostatin, a non-phorbol macrocyclic lactone, activates intact human polymorphonuclear leukocytes and binds to the phorbol ester receptor.

Bryostatin, is an antineoplastic agent with activity in both solid and liquid tumors. When added to tissue culture cells this agent shares a number of similarities with phorbol esters. In this report, we evaluate Bryostatin's effect on human polymorphonuclear leukocytes. Bryostatin stimulates the release of specific granules with a parallel dose response curve to phorbol 12-myristate 13-acetate (PMA), but induces release of superoxide at a significantly slower rate than PMA. Competition experiments demonstrate that Bryostatin, although sharing little structural similarity with PMA, can bind to the PMA receptor. In addition, both Bryostatin and PMA stimulate the phosphorylation of almost identical proteins in intact PMNs. These experiments suggest that Bryostatin may activate PMNs by binding to the PMA receptor, which is currently felt to be the calcium, phospholipid-dependent protein kinase.

Binding, Competitive↗
PubMed2005

Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2.

The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-alpha, or the PKC inhibitor Gö6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.

Anoikis↗