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At least 19 recordsLinked to original sources
PubMed1987

A phorbol ester, phorbol 12-myristate 13-acetate, and a calcium ionophore, A23187, can mimic the luteolytic effect of prostaglandin F2 alpha in isolated rat luteal cells.

To explore the possible role of protein kinase C and calcium in the luteolytic process, we treated luteal cells with a protein kinase C activator, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and with the calcium ionophore, A23187. Lower concentrations of PMA could clearly mimic the inhibitory, luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) on LH-induced cAMP and progesterone production. A nontumor promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no inhibitory effect, indicating a specific PMA effect. The calcium ionophore, A23187, also gave a marked inhibition of LH-induced cAMP and progesterone production. Hormone-stimulated adenylate cyclase activity was markedly impaired after preincubation of the cells with PGF2 alpha, PMA, or A23187, but no effect was seen when the substances were added to isolated membranes. In addition, the stimulation of progesterone production in the luteal cells with the cAMP analogs, 8-bromo-cAMP and (Bu)2cAMP, was almost totally abolished when PMA or A23187 was present. We conclude that PMA and A23187 in many ways mimic the effect of PGF2 alpha in luteal cells. The inhibition of steroidogenesis is partly dependent on depressed activity of the hormone-sensitive adenylate cyclase, but also obtained by inhibiting steps distal to cAMP formation. Both points of action seem to be calcium and/or protein kinase C dependent. In contrast, higher concentrations of PMA markedly stimulated steroidogenesis without affecting the cAMP level, a stimulation not seen after incubation with 4 alpha-phorbol 12,13-didecanoate, suggesting again a specific PMA effect. The stimulation of steroidogenesis by higher concentrations of PMA seems to be specific, but the interpretation of this finding is unclear at present. In conclusion, PMA and A23187 mimic some of the luteolytic properties of PGF2 alpha, not only inhibiting the luteal cAMP system, but also by inducing lesions in the steroidogenic steps beyond the cAMP system.

8-Bromo Cyclic Adenosine Monophosphate↗
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↗
PubMed1991

Effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on islet-cell responsiveness.

Collagenase-isolated rat islets were labelled for 2 h in myo-[2-3H]inositol solution supplemented with 2.75 mM-glucose. The phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.1 or 1 microM) was also present in some experiments. After labelling, islets were washed and then perifused in 2.75 mM-glucose to establish basal [3H]inositol-efflux and insulin-secretory rates. Subsequently, the responses of these islets to stimulation with various agonists were assessed. Inositol phosphate accumulation was measured at the termination of the perifusion. In separate experiments, the cellular location of protein kinase C (PKC) after PMA pretreatment was measured by quantitative immunoblotting of membrane and cytosolic fractions. The following observations were made. (1) Labelling in 0.1-1 microM-PMA had no deleterious effect on total [3H]inositol incorporation during the 2 h labelling period. However, islets labelled for 2 h in 1 microM-PMA were unable to respond, in terms of increases in insulin release, to a 1 microM-PMA stimulus during the subsequent perifusion. (2) As compared with the responses of control islets labelled in 2.75 mM-glucose alone, islets labelled in the additional presence of 1 microM-PMA displayed a significant impairment in phosphoinositide (PI) hydrolysis, but an enhancement of both first-and second-phase insulin secretion, in response to subsequent 20 mM-glucose stimulation. (3) Decreasing extracellular Ca2+ level to 0.1 mM and including the Ca(2+)-channel antagonist nitrendipine (0.5 microM) along with 1 microM-PMA during the [3H]inositol-labelling period did not alter the response of the islets to the subsequent addition of 20 mM-glucose. Glucose-induced PI hydrolysis was still inhibited and 20 mM-glucose-induced insulin release was still enhanced. (4) A markedly amplified and sustained insulin-secretory response to 200 microM-tolbutamide in the presence of 2.75 mM-glucose was also obtained from 1 microM-PMA-pretreated islets. This contrasts sharply with the small and transient response to tolbutamide noted in control islets. (5) When present only during the perifusion phase of the experiments, nitrendipine (0.5 microM) abolished the amplified insulin-secretory responses to both 20 mM-glucose and 200 microM-tolbutamide noted in PMA-pretreated islets. (6) Prior labelling in 1 microM-PMA dramatically amplified the insulinotropic effect of 25 mM-K+ or 5 microM-A23187 stimulation. The amplified insulin-secretory response to K+, but not to A23187, was abolished by inclusion of nitrendipine during the perifusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

The phorbol ester phorbol myristate acetate inhibits human immunodeficiency virus type 1 envelope-mediated fusion by modulating an accessory component(s) in CD4-expressing cells.

The phorbol ester phorbol myristate acetate (PMA) strongly inhibits human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation; it has been suggested that this inhibitory effect is due to the transient downmodulation of the surface-associated CD4 receptors by PMA (I. H. Chowdhury, Y. Koyanagi, S. Kobayashi, Y. Hamamoto, H. Yoshiyama, T. Yoshida, and N. Yamamoto, Virology 176:126-132, 1990). Surprisingly, PMA treatment of cells expressing truncated (A2.01.CD4.401) and hybrid (A2.01.CD4.CD8) CD4 molecules, which are not downmodulated (P. Bedinger, A. Moriarty, R. C. von Borstel II, N. J. Donovan, K. S. Steimer, and D. R. Littman, Nature [London] 334:162-165, 1988), inhibited their fusion with CD4- (12E1) cells expressing vaccinia virus-encoded HIV-1 envelope glycoprotein (gp120-gp41) and with chronically HIV-1-infected H9 (MN, IIIB, or RF) cells. PMA pretreatment of T (12E1) and non-T (HeLa, U937.3, and Epstein-Barr virus-transformed B) cell lines expressing vaccinia virus-encoded CD4 also blocked fusion with 12E1 cells expressing vaccinia virus-encoded gp120-gp41. Interestingly, pretreatment of the gp120-gp41-expressing 12E1 cells with PMA did not alter their fusion with untreated CD4-expressing cells. Although the inhibitory effect of PMA was rapid and treatment for 1.5 h with 5 ng of PMA per ml was sufficient to reduce fusion by more than 50%, the recovery after treatment was slow and more than 40 h was needed before the cells regained half of their fusion potential. The inhibitory effect of PMA was blocked by staurosporine in a dose-dependent fashion, suggesting that it is mediated by protein kinase C. PMA treatment of A2.01.CD4.401 cells reduced the number of infected cells 6.7-fold, as estimated by a quantitative analysis of the HIV-1 MN infection kinetics, probably by affecting the stage of virus entry into cells. CD26 surface expression was not significantly changed by PMA treatment. We conclude that PMA inhibits the CD4-gp120-gp41-mediated fusion by modulating an accessory component(s), different from CD26, in the target CD4-expressing cells. These findings suggest a novel approach for identification of accessory molecules involved in fusion and may have implications for the development of antiviral agents.

Alkaloids↗
PubMed1988

Inhibition of the chemotactic peptide-induced elevation of intracellular calcium in differentiated human leukemic (HL-60) cells by the phorbol ester phorbol 12-myristate 13-acetate.

The chemotactic peptide formylmethionylleucylphenylalanine rapidly elevated the intracellular calcium concentration, in a concentration-dependent manner, of human leukemic (HL-60) cells which had been differentiated to polymorphonuclear leukocyte-like cells by pretreatment with dimethyl sulfoxide (1.3%). Preincubation of the cells with phorbol 12-myristate 13-acetate, a protein kinase C-activating phorbol ester, inhibited the formylmethionylleucylphenylalanine-induced rise in intracellular calcium in a time- and concentration-dependent manner. 4 alpha-Phorbol 12,13-didecanoate, which does not activate protein kinase C, was inactive in this capacity. The use of calcium-free medium suggested that the elevation of intracellular calcium by formylmethionylleucylphenylalanine consisted of rapid intracellular release followed by calcium influx. Phorbol 12-myristate 13-acetate (10(-7) M) inhibited both components of the elevation of intracellular calcium, whereas 10(-8) M phorbol 12-myristate 13-acetate inhibited only the calcium influx. The influx of calcium was not prevented by verapamil, which blocks the voltage-dependent calcium channels. These data suggest that, in differentiated HL-60 cells, 12-O-tetradecanoylphorbol-13-acetate rapidly inhibits both the intracellular release of calcium and calcium influx through non-voltage-dependent calcium channels in response to formylmethionylleucylphenylalanine.

Calcium↗
PubMed1989

Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate.

A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.

Cell Count↗
PubMed1989

Immunodeficiency after allogeneic bone marrow transplantation in man. Effect of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187) in vitro.

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.

Adolescent↗
PubMed1988

Expression of tartrate-resistant acid phosphatase in B-CLL treated with phorbol ester or phorbol ester plus calcium ionophore.

Cells from 3 patients with B-chronic lymphocytic leukaemia (B-CLL) and 1 with B-prolymphocytic leukaemia (B-PLL) were treated in vitro with the phorbol ester 12-0-tetradecanoylphorbol (TPA), the calcium ionophore A23187, or a combination of TPA and A23187. TPA induced the cells to adhere to the culture flask or to clump in dense clusters; single cells became enlarged, often with cytoplasmic elongations. Cells treated with TPA plus A23187 acquired a plasmacytoid morphology and formed regular aggregates in culture. Only TPA alone induced the expression of tartrate-resistant acid phosphatase (TRAP) as documented by isoelectric focusing on horizontal thin-layer gels. The TRAP isoenzyme was first detected after 24 h of TPA treatment; its intensity increased during further TPA exposure, being maximally expressed at 72/96 h. The results suggest that, while TPA triggers B-CLL cells to convert to hairy cell leukaemia (HCL)-type cells, the double stimulus which more closely imitates physiological activation initiates a 'normal' differentiation programme which leads to plasma cells.

Acid Phosphatase↗
PubMed1986

Transient modulation and internalization of T4 antigen induced by phorbol esters.

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.

Adult↗
PubMed1984

Inhibition of transferring binding and iron uptake of hematopoietic cell lines by phorbol esters.

Phorbol esters inhibit cell growth and the binding of transferrin to receptors on K 562, HL 60 and U 937 human leukemic cell lines. Exposure of these cells to 12-0-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C results in a 40% reduction of the specific binding of 125I-transferrin, which is apparent within 15 min. Half-maximal inhibition occurs at about 1 nM. Other tumor promoting phorbol esters also inhibit 125I-transferrin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA reduces the number of transferrin receptors, and does not alter the degradation or the internalization of transferrin. In addition, TPA inhibits iron uptake by these cell lines. These effects are specific, since phorbol esters do not affect either cell growth or the binding of transferrin to Friend erythroleukemia cells and Raji cell line. On the basis of these findings it is suggested that the inhibition of transferrin binding may represent one of the mechanisms by which phorbol esters affect the growth and the differentiation of hematopoietic cell lines.

Biological Transport↗
PubMed1982

Inhibition of insulin receptor binding by phorbol esters.

Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 37 degrees C results in a 50% reduction of the specific binding of 125I-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit 125I-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 degrees C than at 37 degrees C. It is reversible within 2 h at 37 degrees C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors.

Animals↗
PubMed1992

Modulation of interferon signaling in human fibroblasts by phorbol esters.

Phorbol esters activate the expression of a variety of early-response genes through protein kinase C-dependent pathways. In addition, phorbol esters may promote cell growth by the inhibition of expression of cellular gene products regulated by antiproliferative agents such as interferons (IFN)s. In human diploid fibroblasts, phorbol 12-myristate 13-acetate (PMA) selectively inhibits the IFN-alpha-induced cellular gene ISG54. Using transient transfection assays, we have delineated two elements in the promoter of this gene that are necessary for the inhibitory actions of PMA. These elements include (i) the IFN-stimulated response element (ISRE) which is necessary for IFN-alpha-induced cellular gene expression, and (ii) an element located near the site of transcription initiation. IFN-alpha treatment resulted in the rapid induction of ISGF3, a multisubunit transcription factor which binds to the ISRE. PMA caused a substantial reduction in IFN alpha-induced ISGF3 in both nuclear and cytoplasmic extracts, as determined by electrophoretic mobility shift assays with the ISRE as a probe. In vitro reconstitution experiments revealed that IFN-alpha activation of the ISGF3 alpha component of ISGF3 was not affected by PMA. Further experiments were consistent with the possibility that PMA regulated the activity of a cellular factor which competed with ISGF3 gamma for binding of the activated ISGF3 alpha polypeptides. Electrophoretic mobility shift assays using the cap site of ISG54 as a probe demonstrated the formation of a specific complex whose DNA binding activity was not affected by treatment of cells with PMA or IFN-alpha. Competitive inhibition studies were consistent with the DNA-protein complex at the cap site of ISG54 containing proteins with DNA binding sites in common with those which also interact with the ISRE. These data suggest a unique regulatory mechanism by which phorbol esters can modulate IFN signaling.

Base Sequence↗
PubMed1984

Functional studies on B-cell hybridomas with B-cell surface antigens. III. Differentiative response to phorbol esters.

Phorbol esters have been shown to induce differentiation of human lymphoid cells into the mature stage. Murine lymphocytes, however, have not been found to be induced the terminal differentiation by these products. In this study, TH2.52, a subclone of B-cell hybridomas between M12.4.1 B lymphoma of BALB/c mice and normal B cells of C57BL/6 (B6) mice was treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and the differentiative effect of TPA was examined. TPA treatment inhibited the spontaneous proliferation of TH2.52 and induced significant IgM secretion by the hybrid. In contrast, M12.4.1 did not develop any IgM secretion when treated with TPA. The differentiative effect of phorbol esters on TH2.52 closely correlated with their tumor-promoting activity. In addition, the differentiative response of TH2.52 to TPA was completely blocked by retinoic acid (RA). Moreover, TH2.52 cells treated with TPA were demonstrated to decrease the expression of Iab, Iad molecules as well as IgM molecules on the cell membrane by analyses of flow microfluorometry (FMF) and quantitative absorption tests. On the other hand, Iad expression of M12.4.1 did not change under the same conditions. The result clearly demonstrates that TH2.52 cells can be induced to differentiate into IgM-secreting cells after treatment with TPA, followed by the decrease in the expression of B-cell surface antigens on the cell membrane.

Animals↗
PubMed1993

Post-transcriptional regulation of apolipoprotein E expression in mouse macrophages by phorbol ester.

Phorbol ester-mediated differentiation of THP-1 cells (a human monocytic cell line) into mature macrophages is associated with a transcriptional induction of apolipoprotein E (apoE) expression [Auwerx, Deeb, Brunzell, Peng and Chait (1988) Biochemistry 27, 2651-2655]. Endotoxin, on the other hand, which may also act through activation of protein kinase C, is a potent inhibitor of apoE expression in mouse macrophages [Werb and Chin (1983) J. Biol. Chem. 258, 10642-10648]. The present experiments examine the effect of phorbol ester, an activator of protein kinase C, on the apoE expression in mouse thioglycollate-elicited peritoneal macrophages. Phorbol ester inhibits apoE expression in a specific, time- and dose-dependent manner. A 75% inhibition in the rate of apoE secretion, but not that of total protein, was observed following a 4.5 h incubation with 160 nM phorbol ester, although nearly full inhibition was obtained with 40 nM. The changes in apoE secretion were paralleled by similar changes in apoE synthesis, indicating synthesis as the primary site of action. The decreased rates of apoE synthesis are shown not to be due to increased apoE degradation. The profound inhibition of apoE synthesis was not accompanied by significant changes in apoE mRNA levels at any concentration of phorbol ester (up to 16 microM), or length of treatment (up to 24 h), suggesting a post-transcriptional locus of regulation of apoE expression. Although the early changes in apoE synthesis correlate with increased microsomal protein kinase C activity, the suppression of apoE expression persists even during conditions of nearly complete (> 95%) loss of protein kinase C activity, suggesting that the direct or indirect effect of protein kinase C on apoE expression is mediated by a stable phosphorylated protein, or that the observed effects are mediated through a protein kinase C species that is not readily downregulated by phorbol esters. The presented studies clearly demonstrate the potential importance of the translational regulation of apoE expression through the protein kinase C signal transduction pathway.

Animals↗
PubMed1986

Regulation of magnesium but not calcium transport by phorbol ester.

Phorbol esters in the presence of Ca2+ apparently mimic diacylglycerol in activating protein kinase C. Resulting phosphorylations alter multiple cellular processes including inhibition of the action of Ca2+-mobilizing agonists. In contrast to this inhibition of Ca2+ mobilization, addition of 4 beta-phorbol 12-myristate 13-acetate (PMA) to murine S49 lymphoma cells stimulated Mg2+ influx severalfold without any detectable alteration of Mg2+ efflux or of Ca2+ influx or efflux. Stimulation of Mg2+ influx did not require extracellular Ca2+, was half-maximal at 10 nM PMA, and was characterized by a marked increase in the Vmax of Mg2+ influx without change in the Ka for Mg2+. Stimulation of Mg2+ influx was not mimicked by 4 alpha-phorbol didecanoate, which does not activate protein kinase C and was not the result of Na+/H+ exchange activity. The effect of PMA on Mg2+ influx was inhibited by the beta-adrenergic agonist (-)-isoproterenol, which we have previously shown to inhibit Mg2+ influx by a non-cyclic AMP-dependent mechanism (Maguire, M. E., and Erdos, J. J. (1980) J. Biol. Chem. 255, 1030-1035). Forskolin, a direct activator of adenylate cyclase, also inhibited PMA stimulation of Mg2+ influx, suggesting the presence of both cyclic AMP-dependent and -independent influences on Mg2+ influx. We have also previously demonstrated that Mg2+ influx occurs solely into a small subcytoplasmic pool (Grubbs, R. D., Collins, S. D., and Maguire, M. E. (1984) J. Biol. Chem. 259, 12184-12192). PMA did not alter this compartmentation; rather, it almost doubled the content of this cytosolic Mg2+ pool. These data indicate that, in addition to phorbol ester modulation of intracellular Ca2+ mobilization, substantial changes in Mg2+ flux and content occur. They further demonstrate that Mg2+ influx is regulated by a variety of stimuli. It seems likely that such alterations in Mg2+ flux and content would have physiological consequences.

Animals↗
PubMed1982

Responses of amphibian embryos and blastomeres to a tumor-promoting phorbol ester.

Phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) alter the morphology and differentiated states of numerous cell types in vitro. It is likely that these molecules are teratogenic for vertebrate embryos in which cytodifferentiation, cell-tissue interactions, and cellular motility are prominent. By examining embryonic effects, I thought that insights into mechanisms might be gained that would not be apparent in adult tissues. The purpose of this study was to characterize the responses of frog (Bombina orientalis) embryos and embryonic cells to TPA. Effects of 10 to 100 ng TPA per ml on whole embryos included epithelial dissociation, inhibition of muscle segment histogenesis and adhesive organ differentiation, and acceleration of cell sheet movements during gastrulation. Possibly correlated with accelerated gastrulation, TPA also induced circumferential rotations of lobopodial blebs in blastula cells cultured on plastic. This capability is normally not seen until later developmental stages and is most prominent in areas undergoing morphogenetic movements. For blastula cells cultured on agar, TPA (1.0 to 10 ng/ml) inhibited cytokinesis but not karyokinesis, leading to the formation of abnormal multipolar spindles. A similar uncoupling of cytokinesis and karyokinesis in adult tissues could lead to mitotic aneuploidy, possibly an important step in tumor promotion. The amphibian embryo should be a useful organism for further studies on phorbol ester tumor promoters.

Animals↗
PubMed1992

Protein kinase C isoenzymes display differential affinity for phorbol esters. Analysis of phorbol ester receptors in B cell differentiation.

Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.

Animals↗