Studies on the adhesive interaction between purified human eosinophils and cultured vascular endothelial cells

Many recent studies have established the eosinophil as a primary effector cell in the pathology of allergic diseases. However, relatively little is known about the mechanisms by which eosinophils accumulate and are activated at local sites of tissue inflammation in allergic or other eosinophil-dependent pathologic states. Because the adherence of leukocytes to vascular endothelial cells (VEC) is a critical initial event in eosinophil infiltration, we have studied the interaction of purified human eosinophils with cultured human umbilical vein endothelial cells. Treatment of VEC with stimuli known to activate endothelial cells, including purified human IL-1, rTNF-alpha, bacterial endotoxin LPS, and the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate resulted in time- and dose-dependent increases (from two- to fourfold) in adhesiveness for eosinophils. Adherence induced by optimal concentrations of IL-1 (2 U/ml), TNF (1 micrograms/ml), and LPS (1 microgram/ml) is dependent upon the CD18 leukocyte cell surface adherence glycoproteins, because a mAb (60.3) directed against the common beta-subunit of the complex inhibits adherence induced by these stimuli. Several agents directly activated eosinophils to display increased adhesiveness to both VEC and gelatinized plates. The bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine (10(-8) to 10(-6) M), TNF (1 to 1000 ng/ml), and 12-O-tetradecanoyl-phorbol-13-acetate (0.3 to 3 ng/ml) all increased eosinophil binding to VEC by two to fivefold. Platelet-activating factor (PAF; 10(-8) to 10(-6) M), but not lyso-PAF, caused approximately a twofold increase in eosinophil binding to both VEC and gelatinized tissue culture plates, suggesting that activation of eosinophils may be responsible for the known ability of PAF to induce eosinophilic responses. These results suggest that the initiation of an eosinophilic infiltrate in vivo can result from activation of endothelial cells, activation of eosinophils, or activation of both cell types.     

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.     

The effect of tumor-promoting phorbol diesters on terminal differentiation of cells in culture

Summary Phorbol diesters with tumor-promoting activity, in particular, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), can induce or inhibit terminal differentiation in a variety of cell systems, with specificity for particular cell lineages. The phorbols are excellent tools to investigate the expression and control of differentiation in some cells and the mechanism by which oncogenic agents interfere with the process of terminal differentiation. The mechanism of action of the phorbols on different target cells is not understood at the present time. It is felt that the status of the cell is of major importance as, in some cases, opposite effects can be achieved by the same concentration of the phorbol diester used. Changes in membranes, receptors, in secretion of prostaglandins and in the level of cyclic AMP have all been reported. However, the relationship of these changes with the alterations in the genetic program involved in the differentiation process is not clear, and the recent report of a possible cell receptor for phorbol diesters should elucidate their mechanism of action. The findings on the effect of phorbol diesters on differentiation have suggested the testable hypothesis that promotion could be mediated through inhibition of cellular differentiation. It has also been suggested that changes in differentiating systems could be of future use in screening for unknown tumor promoters, however, this possibility seems quite remote. Finally, phorbol diesters with tumor-promoting activity appear to exert a specific effect on differentiation of leukemic cells of both mouse and human origin, and therefore, the application of this particular phenomenon in experimental therapy should be the subject of future investigations.     

Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes

Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4 alpha-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. The catecholamine isoproterenol induced a 51% desensitization. Incubation of 32Pi labeled erythrocytes with TPA promoted a 3- to 4-fold increase in phosphorylation of the beta-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating beta-adrenergic receptor function.     

Tumor promotion by depleting cells of protein kinase C delta.

Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function.     

Phorbol ester-stimulated T lymphocytes show enhanced adhesion to human endothelial cell monolayers

Phorbol esters have been used to study changes in the adhesiveness of T cells to endothelial cells (EC) after activation. The phorbol esters 12-O-tetra-decanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (P(Bu)2), but not the biologically inert 4-alpha-phorbol-12,13-didecanoate, strongly increased the binding of 51Cr-labeled T cells to human umbilical vein EC monolayers in microtiter wells. Binding to fibroblasts and gelatin-coated plastic was also increased, but to a lesser extent. Increased binding was observed at 0.3 ng/ml, with maximal enhancement at 33 to 100 ng/ml. Enhancement occurred within 1 min, with maximal increase after 15 min. Preincubation studies with P(Bu)2 showed that binding enhancement was entirely attributable to an effect on T cells, with no action on EC. Additive binding enhancement was seen when phorbol esters and agents that alter adhesion by acting on EC (LPS, IL 1, or IFN-gamma) were used together. The increase in T cell adhesion to EC after T cell activation may contribute to the selective emigration of activated T cells from the blood into developing inflammatory lesions. The rapid increase in binding suggests that this may be an important mechanism for immediate localization of circulating T cells, particularly sensitized T cells, in the cellular immune response, perhaps involving the activation of these cells at the endothelial blood-tissue interface.     

Role of phorbol ester receptors in the 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-induced down-regulation of colony-stimulating factor (CSF-1) binding to murine peritoneal exudate macrophages

Treatment of murine peritoneal exudate macrophages (PEM) by tumor-promoting phorbol esters (TPA) results in a rapid loss of binding activity to radioactive-labeled colony-stimulating factor ([125I]-CSF-1) on the cell surface. The inhibitory effect of TPA on PEM is transient; treated cells recover full [125I]-CSF-1 binding activity in less than 6 hr at 37 degrees C either in the presence or after the removal of added TPA. The role of phorbol ester receptors in the induction of [125I]-CSF-1 binding inhibition was studied. The biologically active ligand [3H]-phorbol 12,13-dibutyrate ([3H]-PDBu) bound specifically to cultured murine PEM. At 0 degree C, stable and equilibrium binding occurred after 2-3 hr. Scatchard analysis revealed linear plots with a dissociation constant and receptor number per cell of 20.9 nM and 3.9 X 10(5)/cell, respectively. Treatment of PEM with biologically active phorbol esters at 37 degrees C rapidly inhibited the binding activity of [3H]-PDBu on cell surface (down-regulation) and rendered these cells refractory to the TPA-induced [125I]-CSF-1 binding inhibition by the subsequent TPA treatment. The inhibition of phorbol ester binding activity on TPA-treated PEM is caused by a reduction in the total number of available phorbol ester receptors rather than by a decrease in receptor affinity as judged by Scatchard analysis. The disappearance of [3H]-PDBu binding activity is reversible and transient. However, unlike CSF-1 receptors the restoration of phorbol ester receptors on TPA-treated PEM is a very slow process; a prolonged incubation of up to 72 hr after the removal of TPA was required for PEM to regain fully its [3H]-PDBu binding activity. Furthermore, the degree of TPA-induced CSF-1-receptor down-regulation is closely associated with the number of available phorbol ester receptors present on PEM at the time of treatment. Thus, the refractoriness to TPA diminished as the phorbol ester receptors on PEM recovered. A 72-hr incubation time at 37 degrees C was needed for PEM to lose their refractoriness and again become fully sensitive to TPA-induced CSF-1-receptor down-regulation. This study provides evidence that the loss of CSF-1-receptors induced by TPA treatment requires the presence of phorbol ester receptors and proceeds presumably via a co-internalization of both CSF-1 and phorbol ester receptors; the refractoriness to TPA is thereby induced by a transient loss of available phorbol ester receptors.     

Metabolism of Catecholamine Esters by Cultured Bovine Brain Microvessel Endothelial Cells

The metabolism of epinine (N-methyldopamine) and epinine diesters (acetyl, benzoyl, pivaloyl, and isobutyryl) by brain endothelium was investigated using primary cultures of bovine brain microvessel endothelial cells. 3,4-Dihydroxyphenylacetic acid (DOPAC), the product of monoamine oxidase (MAO)-mediated degradation of epinine, was the only metabolite detected by HPLC with electrochemical detection following incubation of the cell monolayers with epinine or its esters. This metabolism could be inhibited by the MAO inhibitors pargyline, clorgyline, and deprenyl, with the system being most sensitive to inhibition by clorgyline. Compared with epinine, incubation of cell monolayers with the diester prodrugs led to increased drug (epinine plus epinine diesters) tissue levels. With the exception of the diacetyl ester, lower levels of DOPAC were observed with the diester prodrugs than with the parent compound. Hydrolysis by serine-dependent esterases appears to be necessary for the subsequent oxidation by MAO. The permeabilities of epinine and the diester prodrugs through endothelial cell monolayers grown on porous supports were related to their lipophilicity and molecular weight.     

A correlation between phorbol diester-induced protein phosphorylation and superoxide anion generation in HL-60 cells during granulocytic maturation

As HL-60 cells matured along the granulocytic pathway, phorbol diester-induced superoxide anion production was compared to phorbol diester-induced protein phosphorylation using an in vitro phosphorylation technique. Maturation was induced by 0, 2, 4, or 6 days incubation with dimethyl sulfoxide (Me2SO). In 0 day Me2SO HL-60 cells, phorbol 12-myristate 13-acetate induced phosphorylation of protein pp29 (Mr = 28,600) and to a lesser extent protein pp76 (Mr = 76,300). With increased time of Me2SO incubation, phorbol 12-myristate 13-acetate induced phosphorylation of pp212 (Mr = 211,800), pp134 (Mr = 134,200), and pp76, whereas the phosphorylation of pp29 did not change appreciably. In close agreement with this increase in protein phosphorylation was the observed increase in phorbol diester-induced superoxide anion formation. Morphological characterization of cells during Me2SO-induced differentiation reveals that these increases in phorbol diester responses are probably attributable to the proportional rise in metamyelocytes, band, and segmented neutrophils. A variety of phorbol diesters increased superoxide anion generation in HL-60 cells differentiated into granulocyte-like cells by 6-day incubation with Me2SO. The structure-activity relationship of these phorbol diester derivatives for protein phosphorylation was strongly correlated to their ability to increase superoxide anion generation. Thus, we propose that phorbol diester-induced phosphorylation of pp212, pp134, and pp76, but not pp29 may play a role in mediating the functional response of phorbol diester-induced superoxide anion generation in HL-60 cells differentiated into mature granulocyte-like cells.     

Sensitization of frog erythrocyte adenylate cyclase system by tumor-promoting phorbol diesters

Preincubation of frog erythrocyte lysates with tumor-promoting phorbol diesters leads to an increase in adenylate cyclase activity. This stimulatory effect of phorbol diesters was specific. Incubation with 12-O-tetradecanoylphorbol 13-acetate led to increases in basal (38%) and isoproterenol- (40%), fluoride- (25%), and Mn-stimulated (68%) adenylate cyclase activities compared with control. The inactive phorbol diesters (4 alpha-phorbol 12,13-didecanoate and beta-phorbol) were ineffective in promoting increases in adenylate cyclase activity. The effect of active phorbol diesters was also observed on isolated frog erythrocyte membranes in the absence of cell supernatant, although to a much lesser extent than in the whole lysates. Addition of the cell supernatant or of purified protein kinase C to the membranes maximized the sensitization by the phorbol diesters. These data are consistent with the notion that some component(s) of the adenylate cyclase system is (are) phosphorylated by protein kinase C, resulting in an enhancement of enzyme activity.     

Modulation of survival and proliferation of BSC-1 cells through changes in spreading behavior caused by the tumor-promoting phorbol ester TPA

The effect of a tumor-promoting phorbol ester on spreading behavior was investigated to clarify the involvement of the interactions between cells and substratum in the maintenance of cell viability and the control of cell proliferation. BSC-1 cells did not spread and lost cell viability after a 24-h incubation in the absence of calf serum. Addition of calf serum initially induced radial spreading and then polarized spreading, with the formation on stress fibers and focal contact-like structure, and enhanced survival. Vitronectin also induced both radial spreading and polarized spreading, and enhanced cell survival. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced radial spreading with actin ribbons in the absence of serum. It improved the survival of cells attached to the substratum, but not in suspension. TPA suppressed polarized spreading, formation of stress fibers and of focal contact-like structure, and cell proliferation, in the presence of serum. Phorbol did not have any effect. These results suggest that enhancement of radial spreading and inhibition of polarized spreading of BSC-1 cells by TPA are closely related to the enhancement of cell survival and inhibition of cell growth.     

Human T lymphocyte activation by tumor promoters: role of protein kinase C

Protein kinase C (PKC) has a major role in a ligand-receptor-mediated signal transduction system in a variety of cell types including T lymphocytes. One of the early phenotypic changes associated with T cell activation is the expression of cell surface receptors for interleukin 2 (IL 2). To test the role of PKC in regulation of IL 2 receptor (IL 2-R) expression and T cell activation in general, we used tumor promoters (TP) as modulators of PKC and compared their effects on intact human T cells and on the enzymatic activity of T cell-derived PKC in a cellfree system. In T cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced IL 2-R expression and proliferation associated with cytosol-to-membrane PKC translocation. A dose of TPA (1 to 4 ng/ml) that induced about 50% of the maximal activation of PKC in the enzymatic assay also induced half-maximal effects on cell proliferation, IL 2-R expression, and PKC redistribution in intact T cells. Structure-function studies with several phorbol ester analogs and non-phorbol ester TP directly correlated tumor promotion activity with the ability to activate PKC and induce IL 2-R. An inhibitor of PKC, chlorpromazine, was found to suppress TPA-mediated proliferation and IL 2-R expression, and inhibited T cell-derived PKC by competing with the phospholipid. Ca2+ ionophore, which synergizes with TPA in induction of T cell proliferation, facilitated the TPA-induced PKC translocation to the membrane. The results thus demonstrate a direct correlation between the effects of various chemicals on: subcellular redistribution of PKC in T cells; induction of T cell proliferation and IL 2-R expression; and activation of T cell-derived PKC in vitro. These data provide further support for the role of PKC in transduction of activation signals in T cells and in regulation of IL 2-R expression.     

Interaction of IL 1 and TPA in modulation of eosinophil function

The tumor co-promotor TPA is believed to enhance a wide variety of cellular processes by interacting with protein kinase C. Interleukin (IL 1) is a family of highly active molecules which augments the host response to infection. We have explored the interactions of these activators of cell function on the modulation of selected eosinophil functions. The effects of purified monocyte-derived IL 1 on the eosinophil functions of oxidative metabolism (as measured by superoxide anion production) and degranulation (as measured by release of the granular enzymes arylsulfatase and beta-glucuronidase) have been examined. Superoxide anion production by eosinophils stimulated with standard doses of the stimulant phorbol myristic acetate (TPA) (1 microgram/ml) was augmented approximately 20% by preincubation with IL 1. However, IL 1 alone had no effect on superoxide anion production. At suboptimal doses of TPA, there was a dose-dependent inhibition of superoxide anion production in the presence of IL 1. Calcium ionophore (2 X 10(-7) M) markedly enhanced superoxide anion production elicited by 0.1 ng/ml of TPA, but had only modest effects in the absence of TPA. When IL 1 was added to eosinophils stimulated by TPA in the presence of calcium ionophore, there was a dose-dependent increase in superoxide anion production. In contrast to other cell types, degranulation as measured by the release of arylsulfatase and beta-glucuronidase was not elicited by the addition of TPA (1 microgram/ml). Although calcium ionophore (2 X 10(-6) M) caused enzyme release (24.2% release of beta-glucuronidase, 29.4% release of arylsulfatase), this release was inhibited by the addition of TPA. The addition of IL 1 alone caused an approximate twofold increase in enzyme release, but pretreatment with IL 1 (1 U) reduced ionophore-mediated degranulation (p less than or equal to 0.05). Studies employing purified monocyte IL 1 were confirmed by recombinant IL 1-beta. These studies demonstrate for the first time that eosinophil function is modulated by IL 1. IL 1 may also modify the response of eosinophils to other stimuli such as ionophore and TPA. Because TPA is known to act by direct binding to protein kinase C, these studies also demonstrate that, in eosinophils, activation of protein kinase C by phorbol esters may augment one cellular function (oxidative metabolism) while inhibiting another cellular function (degranulation). Similarly, phorbol esters may act synergistically with calcium ionophore in regulation of one function (oxidative metabolism) and act antagonistically with another function (degranulation). The concept that IL 1 uniformly enhances cell function may need to be re-evaluated.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

Phorbol ester inhibits luteinizing hormone-, forskolin-, and dibutyryl cyclic AMP-induced progesterone production in chicken granulosa cells

The possible role of protein kinase C in avian granulosa cell steroidogenesis was studied in vitro by examining the effect of tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on progesterone synthesis in chicken granulosa cells in short-term (3h) incubations. TPA (1-100 nM) caused a marginal but nonsignificant increase in progesterone production in granulosa cells isolated from the largest preovulatory follicle. When incubated in combination with luteinizing hormone (5-100 ng/mL), TPA suppressed the stimulatory effects of submaximally and maximally effective doses of the gonadotropin in a concentration-related manner. Similarly, the phorbol ester inhibited the steroidogenic responses to forskolin and dibutyryl cyclic AMP. By comparison, TPA had no appreciable effect on the metabolism of exogenous pregnenolone substrate to progesterone. Our data indicate that the tumor-promoting phorbol ester influences steroidogenic steps distal to cyclic AMP generation but at or before pregnenolone formation, and that protein kinase C may be a negative regulator of steroid biosynthesis in chicken granulosa cells.     

Structural requirements for diacylglycerols to mimic tumor-promoting phobol diester action on the epidermal growth factor receptor

The structural requirements for diacylglycerols to mimic the action of tumor-promoting phorbol diesters on the epidermal growth factor (EGF) receptor of A431 human epidermoid carcinoma cells were investigated. Five biological effects were considered: inhibition of high affinity 125I-EGF binding, change in the phosphorylation state of the EGF receptor, inhibition of the EGF-dependent tyrosine phosphorylation of the EGF receptor, inhibition of [3H]phorbol 12 beta, 13 alpha-dibutyrate binding, and stimulation of calcium- and phospholipid-dependent protein kinase (C-kinase) in vitro. A marked effect of the acyl chain length, 3-10 carbons, of symmetric sn-1,2-diacylglycerols was observed on their ability to mimic the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). sn-1,2-Dipropanoylglycerol did not mimic the effects of PMA, but sn-1,2-didecanoylglycerol potently mimicked PMA action. A correlation was found between the ability of these diacylglycerols to stimulate the activity of C-kinase in vitro and to mimic the effects of PMA on the EGF receptor in intact cells. Analogues of sn-1,2-dioctanoylglycerol in which the 3' hydroxyl group was substituted with hydrogen, thio or chloro moieties were inactive when assayed for their ability to stimulate C-kinase in vitro and mimic PMA action in intact cells. We conclude that the hydroxyl group of a diacylglycerol is vital for the interaction with the phorbol diester receptor. The stringent correlation between the potency of the 11 diacylglycerol analogues tested to modulate C-kinase in vitro and to mimic PMA action in vivo provides strong evidence for the hypothesis that C-kinase plays a central role in the regulation of A431 cell EGF receptors by tumor-promoting phorbol diesters.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Novel tumor-promoting property of tamoxifen.

The tumor-promoting phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) cooperates with c-Src overexpression to transform rat fibroblasts. TPA transforms c-Src-overexpressing cells by depleting the delta isoform of protein kinase C (PKCdelta). Tamoxifen, which has both estrogen-mimetic and estrogen-antagonist properties, has been widely used to improve the prognosis of breast cancer patients. However, with extended use, there is an increased risk for endometrial and other cancers that can be observed within 10 years of treatment. We report here that tamoxifen, similar to TPA, cooperates with c-Src overexpression to transform 3Y1 rat fibroblasts. Tamoxifen induced both DNA synthesis and anchorage-independent cell proliferation in c-Src-overexpressing, but not in parental, 3Y1 rat fibroblasts. Tamoxifen also induced an association between c-Src and PKCdelta that resulted in the tyrosine phosphorylation and down-regulation of PKCdelta. These phenotypes were not induced by estrogen, indicating that the effect of tamoxifen was in addition to any estrogen-mimetic effects. Thus, in addition to the hyperplasia-inducing capability of an estrogen-mimetic, tamoxifen has an additional tumor-promoting capability similar to that of TPA. The dual tumor-promoting capability of both estrogen- and TPA-mimetic properties for tamoxifen may contribute to the increased incidence of endometrial cancers observed in the relatively short exposure period of <10 years.     

Collagenase is a major gene product of induced rabbit synovial fibroblasts

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.