Specific binding of phorbol ester tumor promoters to mouse skin

The tumor promoter 20-³H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K_D = 2.4 × 10^?8 M). The potencies of phorbol esters for inhibiting binding of ³H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K_I = 0.74 nM; phorbol 12,13-didecanoate, K_I = 16 nM; phorbol 12,13-dibenzoate, K_I = 82 nM; mezerein, K_I = 98 nM; phorbol 12,13-diacetate, K_I = 3 μM; phorbol 12,13,20-triacetate, K_I = 5.6 μM; phorbol 13-acetate, K_I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, ³H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the ³H-PDBu binding activity mediates phorbol ester tumor promotion. ³H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE₂, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Induction of phorbol ester responsiveness in conventional B cells after activation via surface Ig.

The signals required to induce S phase entry in murine splenic B cells were found to be altered by prolonged treatment with low doses of anti-Ig antibody. Whereas fresh splenic B cells are stimulated by the combination of a phorbol ester protein kinase C agonist plus a calcium ionophore, anti-Ig-treated splenic B cells were stimulated by phorbol ester alone, in the absence of a comitogen. The majority of these phorbol ester responsive B cells expressed CD5. The phorbol ester responses of anti-Ig-treated splenic B cells paralleled those previously reported for untreated peritoneal CD5+ B cells in a number of respects: responses were not idiosyncratic to phorbol esters but occurred with nonphorbol protein kinase C agonists; phorbol ester responses were enhanced by IL-4; and, phorbol ester responses occurred rapidly and were greater at 24 than at 48 h. However, the effect of agents that act to raise intracellular levels of cAMP distinguished between anti-Ig-treated splenic B cells and untreated peritoneal B cells in that the phorbol ester responses of the former were enhanced whereas the responses of the latter were inhibited. The present results add a functional dimension to the phenotypic similarity between splenic B cells treated with anti-Ig and resident peritoneal B cells that constitutively express CD5; however, some differences in behavior were noted.     

Regulation of chromaffin cell secretion and protein kinase C activity by chronic phorbol ester treatment

Bovine adrenal chromaffin cells were exposed to phorbol esters to determine the effects of reduced levels of protein kinase C on secretion of hormones. Treatment with active phorbol esters such as 4 beta-phorbol 12, 13-didecanoate (PDD) reduced levels of protein kinase C activity with a maximal 80-90% reduction in activity after 16-24 h treatment (greater than or equal to 500 nM PDD). Treatment with PDD also inhibited catecholamine secretion from chromaffin cells evoked by nicotine, barium, and scorpion venom (50-70%, t1/2 approximately 6 h) and by veratridine (80%, t1/2 less than 15 min). Secretion induced by these agents in phorbol ester-treated cells returned to that of untreated cells by 3-4 days despite no recovery of protein kinase C activity. Potassium-evoked secretion was not inhibited by phorbol ester treatment. Catecholamine secretion from digitonin-permeabilized cells was more sensitive to calcium between 1 and 24 h, but not greater than or equal to 48 h, after addition of phorbol ester. The results suggest that phorbol esters inhibit secretion by activation of protein kinase C resulting in inhibition of ion channels or receptors but not of the secretory machinery itself; hence, protein kinase C may usually machinery itself; hence, protein kinase C may usually attenuate secretory responses in the adrenal chromaffin cell.     

Efficacy of karanjin and phorbol ester fraction against termites (Odontotermes obesus)

The non-edible oil seeds of Jatropha curcas (physic nut) and Pongamia pinnata (karanja) contain some toxic components (phorbol esters in J. curcas and karanjin in P. pinnata), which may be used as biopesticides. In this study, the active components of J. curcas and P. pinnata oil were extracted and their efficacy against the termites Odontotermes obesus (Rambur), was tested. The phorbol ester fraction of J. curcas and karanjin of P. pinnata oil were found to be effective against termites. A mortality rate of 100% was achieved in 6 h with karanjin and in 12 h with phorbol ester fraction. The LC₅₀ levels of karanjin and phorbol esters fractions were 0.038 and 0.071 g ml⁻¹, respectively, after 24 h at a 95% (0.05) confidence limit.     

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.     

Calcium ionophore plus phorbol ester can substitute for antigen in the induction of cytolytic T lymphocytes from specifically primed precursors

Previous studies indicated that Ca++ ionophores and phorbol esters in synergy could substitute for the initial activation step of normal T lymphocytes or T cell clones leading to increased expression of receptors for the growth factor interleukin 2 (IL 2) and secretion of interleukins, with the mitogenic signal for T cell proliferation being dependent on the presence of IL 2. In this study, the question was addressed as to whether T lymphocytes activated through the Ca++ ionophore ionomycin and the phorbol ester 12-o-tetradecanoyl phorbol 3-acetate (TPA) also acquired the competence to kill relevant target cells. The results indicate that T lymphocytes from primed mice proliferate and lyse the relevant allogeneic target cells after in vitro stimulation with ionomycin plus TPA, and that T lymphocyte preparations enriched for a subpopulation bearing the Lyt-2 marker are dependent on exogeneous sources of IL 2 to proliferate and become competent killer cells, whereas preparations enriched for subpopulations bearing the L3T4 marker grow independently of exogenous IL 2.     

Effects of diacylglycerols on LLC-PK1 renal epithelia: Similarity to phorbol ester tumor promoters

As previously shown using phorbol ester tumor promoters (see Mullin and O'Brien: Am. J. Physiol., 251:C597–C602, 1986), diacylglycerols induce leakiness in LLC-PK₁ renal epithelial tight junctions. The similarity between phorbol ester and diacylglycerol action includes effects on (1) cell morphology, (2) dome formation, (3) transepithelial resistance and potential difference, (4) transepithelial flux of D-mannitol, and (5) mitogenesis. Four diacylglycerols have been tested: 1,2-dioctanoylglycerol; 1,2-dicaprylglycerol; 1,2-dioleoylglycerol; and 1-oleoyl-2-acetoyl-sn-3-glycerol. Their relative effectiveness depended upon the phenomenon being observed. Unlike phorbol esters, diacylglycerol effects were reversible within hours at 37°C in the continued presence of diacylglycerol, and effects were more pronounced when cell sheets were exposed to diacylglycerols from the basolateral cell surface. Overall, these findings indicate that previous results with phorbol esters may be attributed to the protein kinase C signal transduction system, and this system may therefore exert a role in transepithelial permeability.     

Activity correlations in the phorbol ester series

Activity correlations in the phorbol ester series. The production of inflammation by phorbol esters on mammalian skin correlates on a structural basis with in vitro measurements of lymphocyte mitogenesis and mobilization of prostaglandins. All of the pro-inflammatory phorbol esters tested in our laboratory have been shown to activate the enzyme protein kinase C, and such an interaction could in large part explain the induction of an inflammatory response in vivo. Certain of these compounds additionally induce aggregation of human and rabbit blood platelets. This activity does not structurally correlate with the induction of inflammation but may correlate with the known tumour-promoting actions of phorbol derivatives. Compounds which induce platelet aggregation stimulate the secretion of a biologically active substance which we have termed 'Factor W'. The production of Factor W occurs into human plasma following platelet stimulation by phorbol tumour-promoting agents. It is an unstable substance, distinct in its aggregating properties from phorbol esters, ADP, 5-hydroxytryptamine, thrombin, platelet aggregating factor and the products of arachidonate oxidative metabolism.     

Actions of a tumor-promoting phorbol ester in a cloned rat pituitary cell

Tumor promoters, such as phorbol myristate acetate (PMA), facilitate carcinogenesis by mechanisms that may involve changes in intracellular Ca²⁺ metabolism and distribution of Ca²⁺, as well as activation of a Ca²⁺-and phospholipid-dependent protein kinase, referred to as protein kinase C. We compared the actions of PMA on GH₃ cloned pituitary cells with those of thyrotropin releasing hormone (TRH), an established Ca²⁺-mobilizing agent. The TRH treatment produced a⁴⁵Ca efflux, inhibited⁴⁵Ca uptake, diminished chlortetracycline fluorescence, and stimulated cAMP accumulation and protein synthesis in a Ca²⁺-dependent manner. Like TRH, PMA produced an efflux of⁴⁵Ca and inhibited⁴⁵Ca uptake; however, the phorbol ester stimulated cAMP accumulation and protein synthesis in the absence of external calcium and failed to alter chlortetracycline fluorescence. The TMB-8, a putative inhibitor of the mobilization of membrane-associated Ca²⁺, did not alter PMA-induced stimulation of protein synthesis. The results suggest that PMA-induced changes in Ca²⁺ metabolism are not caused by the mobilization of membrane-associated calcium. Alternative proposals are that PMA (1) inhibits Ca²⁺ influx and/or (2) mobilizes calcium from nonmembranous storage sites. Further study is needed to characterize the mechanism through which tumor-promoting phorbol esters influence Ca²⁺ metabolism and to ascertain the significance of changes in Ca²⁺ metabolism to cellular processes affected by these substances.     

Enhancement of phorbol ester induced cell aggregation after alterations in asparagine-linked oligosaccharides

Summary— Human erythroleukemia (K-562) cells grown in the presence of phorbol 12,13-dibutyrate formed aggregates of cells not seen in untreated control cultures. Furthermore, the proportion of cells in aggregates and the size of the aggregates both increased dramatically in cultures treated with both phorbol ester and kifunensine, an inhibitor of asparagine-linked oligosaccharide processing. Relative to control cells, phorbol ester treated cells exhibited a greater proportion of N-linked oligosaccharides of the complex-type. Kifunensine prevented this change and caused an accumulation of Man₉GlcNAc₂. The enhanced aggregation of cells treated with phorbol ester plus kifunensine depended on phorbol ester concentration and was blocked by inhibitors of protein kinase C (H7, sphinganine and sangivamycin). In flow cytometry analysis, phorbol ester treated K-562 cells showed an increase in CD44, a glycoprotein involved in cell adhesion. Moreover, monoclonal antibody to CD44 augmented reaggregation of phorbol ester treated cells. The results implicate phorbol ester induction of CD44 in aggregation of K-562 cells and demonstrate that the presence of high mannose-type asparagine-linked oligosaccharides on cell glycoproteins correlates with increased aggregation of phorbol ester treated cells.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

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.     

Differential effects of phorbol ester on the beta-adrenergic response of normal and ras-transformed NIH3T3 cells

The basal and isoproterenol stimulated levels of cyclic AMP in NIH3T3 and H-ras transformed NIH3T3 cells were equivalent. In exponentially growing cells, the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) inhibited the beta-adrenergic response of NIH3T3 cells, but not of the ras-transformed line. Another line of NIH3T3 cells transformed by a non-ras gene exhibited the normal loss of beta-adrenergic response by the tumor promoter. These results are consistent with a role for p21 in signal transduction related to the effects of TPA.     

Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology.

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.     

The effects of phorbol ester on alloantigen presentation

B cells and Ia+ thyroid cells fail to stimulate alloreactive T cells in a primary mixed leukocyte reaction (MLR) and fail to activate some allo-class II (I-A) reactive T cell hybridomas. We now demonstrate that B cells can specifically stimulate a primary MLR in combination with the phorbol ester, PMA, but not with interleukin 1 (IL 1) or calcium ionophore. The primary MLR induced with B cells plus PMA can be blocked by either monoclonal anti-I-A or anti-L3T4 antibodies. In contrast, thyroid cells that can be induced to express Ia antigens after incubation with interferon-gamma fail to stimulate a primary MLR even in the presence of PMA or IL 1. We confirmed these observations by using the alloreactive T cell hybridoma, HTB-9.3, which does not react to stimulator B cells. In the presence of PMA, however, this I-Ab-specific hybridoma line was able to respond to relevant but not to control stimulator B cells. Furthermore, the response of HTB-9.3 to B cells plus PMA was also blocked by anti-I-A or anti-L3T4 antibody. In contrast to B cells, Ia+ thyroid cells could not activate HTB-9.3 even in the presence of PMA or IL 1. The data indicate that for primary class II restricted allo-responses, B cells provide signals that can be complemented with the phorbol ester PMA, whereas Ia+ thyroid cells do not, suggesting the existence of additional requirements for T cell activation.     

Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester-treated Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane-bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.     

Differential actions of phorbol ester and diacylglycerol on inhibition of granulosa cell maturation

Hormonal induction of granulosa cell maturation is inhibited by phorbol esters and permeant synthetic diacylglycerols, but these activators of protein kinase C differ in their effects on cAMP production and actions. Both agents prevented the induction of luteinizing hormone receptors and progesterone biosynthesis by follicle-stimulating hormone, choleragen, and forskolin, but only diacylglycerol abolished the cAMP responses to these stimuli. Granulosa cell aggregation and aromatase activity were inhibited by phorbol ester but not completely by diacylglycerol. In intact granulosa cells, cytosolic C kinase activity was rapidly decreased by phorbol ester but unaffected by diacylglycerol. Although diacylglycerol has a marked inhibitory action on cAMP production, the more prominent suppression of granulosa cell differentiation by phorbol ester may be related to its rapid and prolonged action on kinase C.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

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.