phorbol-12-13-didecanoate and phorbol-12-13-dibenzoate

The pathogenesis of neuronal dysfunction in the gangliosidoses is poorly understood. Studies of the feline gangliosidoses and in vitro experiments implicate ganglioside inhibition of protein kinase C (PKC) in the pathogenesis of these neurological diseases. Therefore, in the present study, the binding of [3H]phorbol-12, 13 dibutyrate was measured to determine the levels of PKC in cerebral cortex of cats with GM1 gangliosidosis (mutant) and age matched normal siblings. This binding of ([3H]PDB) to cerebral cortex homogenates in both normal and mutant cats was highly specific. The specificity of receptors was ascertained also from displacement studies using nonradioactive phorbol ester analogues to displace [3H]PDB bound to its receptors. In both mutant and normal cat brain, phorbol 12,13-dibutyrate (PDB), 4-beta-phorbol 12,13-didecanoate (beta-PDD) and 4-beta-phorbol 12,13-dibenzoate (beta-PDBz) were highly potent (approximately to same degree) and effective in displacing [3H]PDB. On the other hand, 4-beta phorbol 12,13-diacetate (beta-PDA) was a weak displacer and 4-alpha-phorbol did not displace the bound [3H]PDB in either normal or mutant brain. Scatchard analysis of the binding data indicated a homogenous single class of binding sites in normal and mutant brain (Normal: Kd = 1.42 x 10(-7) M, Bmax = 8.40 pmoles/mg protein. Mutant: Kd = 1.60 x 10(-7) M, Bmax = 10.00 pmoles/mg protein). Sphingosine inhibited the binding to approximately the same extent in normal and mutant cortex. These studies demonstrate the presence of highly specific, homogenous, single type phorbol ester receptors in cerebral cortex of cats with GM1 gangliosidosis which are qualitatively and quantitatively similar to normal cat brain.

Topics: Animals; Binding, Competitive; Caenorhabditis elegans Proteins; Carrier Proteins; Cats; Cerebral Cortex; G(M1) Ganglioside; Gangliosidoses; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Drug; Sphingosine

1. The stimulation of (3H)-thymidine incorporation by lymphocytes in response to phorbol and seven other phorbol related compounds was investigated. 2. Lymphocytes from each of a small group of individuals were treated with the test compounds over wide concentration ranges. 3. All the tested compounds, including the most active, 12-0-tetradecanoylphorbol-13-acetate, were far less effective lymphocyte mitogens than the plant lectin phytohaemagglutinin. 4. Inter-individual differences were detected in the maximum response to the phorbols but not in their stimulating potency, as estimated by the concentration producing a half maximal response. 5. The rank order of the lymphocyte stimulating potency of the tested compounds was similar to the rank order of both tumour promoting activity and irritant potency in mouse skin. 6. Lymphocyte stimulation was paralleled equally well by these two mouse skin responses.

Topics: Adolescent; Adult; Animals; Humans; Irritants; Lymphocyte Activation; Male; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Phytohemagglutinins; Skin Tests; Tetradecanoylphorbol Acetate

We confirm here previous studies that have shown synergy between anti-CD3 and phorbol esters in the induction of T-cell proliferation. However, for this study we have used tonsillar rather than peripheral blood T cells, and have compared the role of different phorbol esters rather than different anti-CD3 antibodies in the activation process. Three phorbol esters (phorbol myristate acetate, -dibenzoate and -didecanoate) showed a synergistic relationship. However, the concentration of the dibenzoate and the didecanoate forms required was higher than the concentration of myristate acetate. A second group of phorbol esters (alpha-phorbol didecanoate, a beta phorbol with no side chain, and a monomyristate) did not activate T cells. This difference in activation efficiency between the phorbol derivatives correlates with the pattern of neutrophil activation that has been described previously using the same compounds. In contrast to both the neutrophil and the proliferation studies, if the T cells were preincubated with the different phorbol esters and then subsequently cultured with anti-CD3, the pattern of response was different. Only phorbol myristate acetate induced a proliferative response; all the other compounds were inactive. Taken in conjunction with the known structural differences between the different phorbol derivatives, these results suggest that the relative lipophilic properties of the different molecules which activate T cells may be an important determinant in the induction of a response. Thus changes in the relative lipophilic properties of an antigen could be one way to alter its relative immunogenicity.

Topics: Antibodies, Monoclonal; Cells, Cultured; Humans; Lymphocyte Activation; Phorbol Esters; T-Lymphocytes; Tetradecanoylphorbol Acetate

The effects of TPA, PDD, PDB, PDA, or MEZ on epithelial and mesenchymal skin tumors induced by a s.c. injection of MCA were studied histologically. Group-I mice received only MCA. At 6 weeks after MCA injection, mice in groups II to VII received acetone, 1.8 nmol TPA, PDD, PDB, PDA, or 6.1 nmol MEZ respectively in 0.1 ml acetone twice weekly until tumor development. Alterations in skin tumor induction patterns were also studied in animals that had been exposed to TPA or acetone for 10 weeks prior to s.c. injection of MCA. Exposure of mouse skin to TPA before or after carcinogen administration increased 2- to 3.5-fold, the incidence of carcinoma and mixed tumors of epithelial and mesenchymal histogenesis. The average time of tumor induction decreased in mice treated with MCA + TPA and 100% of the test animals in the TPA + MCA group developed tumors. In contrast, TPA-related phorbol esters inhibited skin tumor development, particularly trichoepithelioma and fibrosarcoma and increased the average time of tumor induction.

Topics: Animals; Carcinoma; Diterpenes; Female; Mesenchymoma; Methylcholanthrene; Mice; Mice, Inbred Strains; Phorbol Esters; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate

The effect of tumor promoter phorbol esters on cell proliferation was investigated in human breast cancer cell line MCF-7. During a 4-day culture period, the various phorbol ester derivatives TPA, PDD, PDBu, PDBz and PDA inhibited the proliferation of MCF-7 cells in a dose-dependent manner, with respective IC50 of 0.06, 0.75, 2.4, 3.6 and 15 X 10(-9) M. The 4-O-met-TPA, alpha PDD and alph PHR were ineffective at 2 X 10(-7) M, the highest concentration tested. Using a 3H-PDBu probe, we demonstrated the presence of specific, high affinity binding sites in intact cultured cells, with a Kd of about 9 X 10(-9) M. Unlabelled TPA, PDD, PDBU and PDBz competed with 3H-PDBu with respective IC50 of 35, 12.5, 150 and 220 X 10(-9) M. High concentrations of PDA, 4-O-met-TPA and alpha PDD slightly inhibited the 3H PDBu binding, whereas alpha PHR did not until 10(-5) M. The correlation that we observed between the relative potencies of the various phorbol derivatives for inhibiting both PDBu binding and cell proliferation, suggests that tumor promoter phorbol esters may induce growth arrest in MCF-7 cells by the mediation of protein kinase C.

Topics: Breast Neoplasms; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Humans; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Tetradecanoylphorbol Acetate

The tumor promoters mezerein and phorbol myristate acetate, and the phorbol diesters phorbol diacetate, phorbol dibenzoate, and phorbol didecanoate synergistically enhanced the production of lymphotoxin (LT) by phytohemagglutinin-stimulated human peripheral blood or tonsil and adenoid lymphocytes. LT production was elevated 2-20-fold, depending on such parameters as the nature of the promoter and dose, the lectin dose, and the lymphocyte source. The increased LT levels were primarily due to enhanced production of the alpha-light (alpha L) class of LT. The alpha L-class obtained from supernatants from promoter-synergized, lectin-stimulated lymphocyte cultures was compared with the alpha L from lectin-stimulated cultures. They were indistinguishable by molecular sieving on Ultrogel AcA44, were both composed primarily of the alpha 2-subclass as determined by ion-exchange chromatography on DEAE-Sepharose, and were immunologically cross-reactive. Lectin-affinity chromatography on concanavalin A-Sepharose and on lentil-lectin--Sepharose revealed that both alpha L preparations were dominated by components with affinity for these matrices. Affinity chromatography on alkyl sorbents also indicated very similar hydrophobicities. Chromatofocusing of the alpha L preparations demonstrated a comparable pattern of isoelectric points. Thus, the use of these drugs in lectin-stimulated human lymphocyte cultures provides an effective means for significantly increasing the yield of alpha L-LT suitable for biochemical purification and analysis, and biological testing in vitro.

Topics: Carcinogens; Chromatography, Affinity; Chromatography, Ion Exchange; Diterpenes; Humans; In Vitro Techniques; Lymphocytes; Lymphotoxin-alpha; Phorbol Esters; Phorbols; Phytohemagglutinins; Terpenes; Tetradecanoylphorbol Acetate

The effect of phorbol diesters on histone phosphorylation in BALB/c mouse lymphocytes, cells which do not respond to these agents with cell division, but with other biochemical and biological changes, was investigated. A technique for fractionating the proteins was used which was more powerful than those used previously in similar studies of phorbol diester effects on the metabolism of these proteins. Exposure of lymphocytes to tumor-promoting phorbol esters resulted in a rapid and specific increase in phosphorylation of the nuclear histone proteins H2B and H4. Within 2 hr, the phosphorylation of these two proteins rose to levels 6- to 8- and 2- to 4-fold greater, respectively, than those in control cells, when lymphocytes were exposed to 800 nM 12-O-tetradecanoylphorbol-13-acetate. Lower levels were observed with other phorbol analogues commensurate with their relative tumor-promoting abilities. Lymphocyte mitogens did not increase phosphorylation under the conditions used. The potential ability of the cell system used for defining early in vivo and in vitro phorbol diester effects, and those which are independent of cell division, is discussed.

Topics: Animals; Cells, Cultured; Histones; Lymphocytes; Mice; Mice, Inbred BALB C; Phorbol Esters; Phorbols; Phosphorylation; Tetradecanoylphorbol Acetate

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.

Topics: Animals; Ascitic Fluid; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Kinetics; Macrophages; Mice; Mice, Inbred C3H; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Receptors, Cell Surface; Receptors, Colony-Stimulating Factor; Temperature; Tetradecanoylphorbol Acetate

Topics: Animals; Antibody Formation; B-Lymphocytes; Cytotoxicity, Immunologic; Female; Macrophages; Mice; Neoplasms, Experimental; Phorbol Esters; Phorbols; Structure-Activity Relationship; Tetradecanoylphorbol Acetate