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The Journal of Biological Chemistry Sep 1985In this study we have used (phorbol-12-O-tetradecanoylphorbol 13-acetate) and its biologically inactive analogue, 4 alpha-phorbol 12,13-didecanoate), to investigate...
In this study we have used (phorbol-12-O-tetradecanoylphorbol 13-acetate) and its biologically inactive analogue, 4 alpha-phorbol 12,13-didecanoate), to investigate platelet protein phosphorylation with special emphasis on the properties of a membrane protein-cytoskeleton (transmembrane) complex during platelet activation. Our data indicate that phorbol-12-O-tetradecanoylphorbol 13-acetate (but not 4 alpha-phorbol 12,13-didecanoate) induces both a specific platelet shape change and the preferential phosphorylation of a 180-kDa protein (presumably due to the activation of protein kinase C on the cytoplasmic side of the membrane). Further analysis reveals that the 180-kDa protein can be iodinated by lactoperoxidase and is sensitive to trypsin treatment, indicating exposure of this protein on the outer cell surface. The 180-kDa protein has also been found to contain wheat germ agglutinin-binding sites. All evidence indicates that the 180-kDa polypeptide is a transmembrane glycoprotein and, most importantly, that this protein is found to be preferentially accumulated into a specific membrane-cytoskeleton complex during activation via phorbol-12-O-tetradecanoylphorbol 13-acetate treatment. We believe that the observed phosphorylation of this protein may be closely related to the formation of a complex between several membrane proteins and the cytoskeleton during the initial stages of platelet activation.
Topics: Blood Platelets; Cytoskeletal Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Phorbol Esters; Phosphoproteins; Phosphorylation; Tetradecanoylphorbol Acetate
PubMed: 4044578
DOI: No ID Found -
Journal of Cellular Physiology Jul 1992In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial...
In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC-PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp-like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane-associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two-stage carcinogenesis are discussed.
Topics: Animals; Cell Membrane Permeability; Cells, Cultured; Enzyme Activation; Epithelial Cells; Epithelium; Immunoblotting; Intercellular Junctions; Kidney Tubules, Proximal; Microscopy, Electron; Phorbol Esters; Protein Kinase C; Time Factors
PubMed: 1618921
DOI: 10.1002/jcp.1041520106 -
Journal of Cellular Physiology Nov 1988To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting...
To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.
Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Cell Division; Cell Survival; Colony-Stimulating Factors; Enzyme Activation; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Interleukin-3; Isoquinolines; Phorbol 12,13-Dibutyrate; Phorbol Esters; Piperazines; Protein Kinase C; Tetradecanoylphorbol Acetate
PubMed: 3142884
DOI: 10.1002/jcp.1041370219 -
Proceedings of the National Academy of... Mar 1986Phorbol esters bind to and activate a calcium phospholipid-dependent protein kinase (C kinase). Some researchers believe that activation of C kinase is necessary for the...
Phorbol esters bind to and activate a calcium phospholipid-dependent protein kinase (C kinase). Some researchers believe that activation of C kinase is necessary for the induction of phorbol ester biologic effects. Our research indicates that bryostatin, a macrocyclic lactone that binds to the phorbol ester receptor in human polymorphonuclear leukocytes, also binds to this receptor in the human promyelocytic leukemia cell line, HL-60. Bryostatin activates partially purified C kinase from HL-60 cells in vitro, and when applied to HL-60 cells in vivo, it decreases measurable cytoplasmic C kinase activity. Unlike the phorbol esters, bryostatin is unable to induce a macrophage-like differentiation of HL-60 cells; however, bryostatin, in a dose-dependent fashion, blocks phorbol ester-induced differentiation of HL-60 cells and, if applied within 48 hr of phorbol esters, halts further differentiation. These results suggest that activation of the C kinase by some agents is not sufficient for induction of HL-60 cell differentiation and imply that some of the biologic effects of phorbol esters may occur through a more complex mechanism than previously thought.
Topics: Binding, Competitive; Bryostatins; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phorbol Esters; Protein Kinase C; Receptors, Drug; Receptors, Immunologic
PubMed: 3456591
DOI: 10.1073/pnas.83.5.1334 -
Journal of Natural Products May 1995The novel phorbol ester 12-deoxyphorbol 13-(3E,5E-decadienoate) [1] was isolated as the anti-HIV principle of Excoecaria agallocha leaves and stems collected in...
The novel phorbol ester 12-deoxyphorbol 13-(3E,5E-decadienoate) [1] was isolated as the anti-HIV principle of Excoecaria agallocha leaves and stems collected in northwest Australia. The structure was determined by spectral means. Compound 1 was also a potent displacer of [3H]-phorbol dibutyrate from rat brain membranes.
Topics: Animals; Antiviral Agents; Chromatography, Liquid; HIV-1; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phorbol Esters; Plants, Medicinal; Rats; Virus Replication
PubMed: 7623051
DOI: 10.1021/np50119a020 -
Proceedings of the National Academy of... Jul 1983In the presence of phosphatidylserine, [20-3H]-phorbol 12,13-dibutyrate [( 3H]PBt2) bound specifically to a single class of binding sites in mouse brain cytosol...
In the presence of phosphatidylserine, [20-3H]-phorbol 12,13-dibutyrate [( 3H]PBt2) bound specifically to a single class of binding sites in mouse brain cytosol (supernatant at 100,000 X g). The dissociation constant for binding was 3.1 X 10(-9) M, and at saturation 23.2 pmol of [3H]PBt2 was bound per mg of cytosolic protein. Less than 1 pmol of [3H]PBt2 per mg bound in the absence of phospholipids. Phosphatidic acid, sphingomyelin, and phosphatidylinositol also were able to reconstitute binding activity, whereas phosphatidylcholine and phosphatidylethanolamine were relatively ineffective. [3H]PBt2 binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 4.4 X 10(-11) M), phorbol 12,13-didecanoate (Ki = 7.7 X 10(-9) M), phorbol 12,13-diacetate (Ki = 4.4 X 10(-7) M) and 4-O-methylphorbol 12-myristate 13-acetate (Ki = 5.1 X 10(-7) M). The apparent Ki values of the phorbol-related diterpenes for inhibiting binding agreed reasonably closely with the values previously determined for mouse brain membrane binding. The biologically inactive derivatives phorbol (30 microM) and 4 alpha-phorbol 12,13-didecanoate (30 microM) did not inhibit binding. The aporeceptor was eluted in one peak during Ultrogel 44 column chromatography, corresponding to a molecular weight of approximately equal to 77,000. Calcium phospholipid-dependent protein kinase C activity was eluted with a profile similar to that of the cytosolic aporeceptor-binding activity.
Topics: Animals; Apoproteins; Binding, Competitive; Brain; Caenorhabditis elegans Proteins; Carcinogens; Carrier Proteins; Cytosol; Female; Kinetics; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug
PubMed: 6308606
DOI: 10.1073/pnas.80.14.4208 -
Cancer Research Nov 1984Phorbol esters induce, in the chemotactically responsive Walker 256 carcinosarcoma cells, functional responses that are similar to those induced by peptide chemotactic...
Phorbol esters induce, in the chemotactically responsive Walker 256 carcinosarcoma cells, functional responses that are similar to those induced by peptide chemotactic factors. These responses are presumed to result from ligand binding to cellular receptors, although this has not been formally demonstrated. In this study, it was shown that tritium-labeled phorbol dibutyrate [( 3H]PDB) bound to the Walker cells in a time- and concentration-dependent manner. Binding was inhibited by excess unlabeled PDB and was reversible. Half-maximal binding was achieved with a 31 nM concentration of [3H]PDB and occurred within 15 min after addition of the ligand. This is in accord with biological activity (i.e., cell-to-substrate adherence). Half-maximal cell adherence was observed with 25 nM PDB. Increased adhesiveness was detected as early as 15 min after exposure of the cells to the ligand. The response peaked at 30 to 45 min after exposure and fell off rapidly thereafter. A number of phorbol esters successfully competed with [3H]PDB binding to the cells. There was a direct relationship between the ability of these agents to compete for binding and their ability to induce biological activity. Using cell-to-substrate adherence as an indicator of biological activity allowed separation of responding and nonresponding populations. The biologically responsive cells and the nonresponsive cells both bound high levels of [3H]PDB. This suggests that receptor occupancy is, by itself, not sufficient for biological activity and that, in Walker cells, one or more points of control exist subsequent to ligand binding.
Topics: Animals; Binding, Competitive; Caenorhabditis elegans Proteins; Carcinogens; Carcinoma 256, Walker; Carrier Proteins; Chemotaxis; Kinetics; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Drug; Receptors, Immunologic
PubMed: 6593115
DOI: No ID Found -
Science (New York, N.Y.) Dec 1983Autoradiography with 3H-labeled phorbol dibutyrate was used for the light microscopic detection of phorbol ester receptors in rat fetuses. In 15- and 18-day fetuses, as... (Comparative Study)
Comparative Study
Autoradiography with 3H-labeled phorbol dibutyrate was used for the light microscopic detection of phorbol ester receptors in rat fetuses. In 15- and 18-day fetuses, as well as in adult rats, receptors were found to be concentrated in the central nervous system. The localization of receptors in the ventral marginal zone of the fetal neural tube, the lens of the eye, and other sites suggests a role for phorbol ester receptors in cellular process extension and cell-cell interaction.
Topics: Animals; Autoradiography; Brain; Brain Chemistry; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Communication; Cell Division; Central Nervous System; Eye; Fetus; Intestines; Lens, Crystalline; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Drug
PubMed: 6316499
DOI: 10.1126/science.6316499 -
Cancer Research May 1982Two lines of EL4 mouse thymoma cells, one which responds to phorbol ester tumor promoters with production of T-cell growth factor and inhibition of proliferation and one... (Comparative Study)
Comparative Study
Two lines of EL4 mouse thymoma cells, one which responds to phorbol ester tumor promoters with production of T-cell growth factor and inhibition of proliferation and one which does not respond, have been examined for the presence of specific phorbol ester-binding components. Both lines displayed saturable specific binding as determined by using [20-3H]phorbol 12,13-dibutyrate in a whole-cell-binding assay. Specific binding in each line was maximal within 10 min at 37 degrees but rapidly decreased to about 30% within 30 min. Maximal binding at 4 degrees was reached after 50 min and was stable for at least 8 hr; however, this level was only about 30% of the maximum obtained at 37 degrees. Saturation of the specific binding after 3 hr at 4 degrees obtained at a concentration (30 to 50 nM) which is consistent with that yielding maximal T-cell growth factor production in the responding line but greater than that at which inhibition of proliferation was detected in those cells. Scatchard analysis of these data was consistent with the existence of a single class of binding sites with a Kd of 11 +/- 0.6 (S.D.) nM for the T-cell growth factor-producing cells and 18 +/- 1.9 nM for the nonproducing line. The numbers of sites per cell measured in the responding and nonresponding lines were 5.6 +/- 1.3 x 10(4) and 7.1 +/- 0.2 x 10(4), respectively. Competition by a series of phorbol esters for [20-3H]phorbol 12,13-dibutyrate binding in the responding line showed the same order of potency as production of T-cell growth factor and inhibition of proliferation by the analogs. These data support identification of the phorbol ester-binding component in the responding cells as the receptor mediating T-cell growth factor production. The presence of binding components in unresponsive cells in numbers and with characteristics indistinguishable from those of responsive cells suggests that the failure to respond is due to modification of a step which succeeds the initial binding event.
Topics: Animals; Binding, Competitive; Cell Line; Interleukin-2; Kinetics; Lymphokines; Mice; Neoplasms, Experimental; Phorbol Esters; Phorbols; Receptors, Mitogen; T-Lymphocytes; Thymoma; Thymus Neoplasms
PubMed: 6978177
DOI: No ID Found -
Biochemical and Biophysical Research... Nov 1985Bryostatins (2 ng/ml), when combined with insulin in serum-free culture medium, are strongly mitogenic for Swiss 3T3 cells that have been arrested in the G1/G0 phase of...
Bryostatins (2 ng/ml), when combined with insulin in serum-free culture medium, are strongly mitogenic for Swiss 3T3 cells that have been arrested in the G1/G0 phase of the cell cycle. The mitogenic effect of the bryostatins is similar to that of 12-O-decanoylphorbol-13-acetate (TPA). A prior treatment of the cultures with TPA eliminated the mitogenic response to bryostatin and to a second addition of TPA. Conversely, a prior treatment of the cultures with bryostatin eliminated the mitogenic response to TPA. Bryostatin potently inhibited the binding of [3H]phorbol dibutyrate to a high affinity receptor in the cells. The findings suggest that the bryostatins and TPA act via the same receptor, possibly protein kinase C.
Topics: Animals; Bryostatins; Caenorhabditis elegans Proteins; Carcinogens; Carrier Proteins; Cells, Cultured; Insulin; Lactones; Macrolides; Mice; Mitogens; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Drug; Receptors, Immunologic; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Thymidine
PubMed: 3907633
DOI: 10.1016/0006-291x(85)91898-4