Tumor-promoting phorbol esters induce angiogenesis in vitro

A crucial event during angiogenesis is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. To investigate the possible role of proteases in endothelial cell invasiveness in vitro, bovine microvascular endothelial cells (BMEC) grown on collagen gels were treated with phorbol myristate acetate (PMA), a tumor promoter that markedly increases their production of collagenase and plasminogen activator. Whereas control BMEC were confined to the surface of the gels, PMA-treated BMEC invaded the underlying collagen matrix, where they formed an extensive network of capillary-like tubular structures. This phenomenon, which mimics some of the events occurring during angiogenesis in vivo, required protein synthesis and intercellular contact, was accompanied by collagen degradation, and was prevented by the metalloprotease inhibitor 1,10-phenanthroline.     

Tumor-promoting phorbol esters induce metamorphosis and multiple head formation in the hydroid Hydractinia

Abstract. A pulse-type application of tumor-promoting phorbol esters (e.g., 12-tetra-decanoyl-phorbol-13-acetate, TPA) initiates the metamorphosis of planula larvae into polyps. Phorbol esters replace or bypass a lipophilic inducer which, in the natural habitat, is produced by environmental bacteria of the genus Alteromonos . In regeneration assays, isolated gastric regions of adult polyps were induced by TPA to form heads at both ends and often in the middle, too. TPA increases the "positional value," as does an endogenous, extractable factor X. The morphogenetic effectiveness of various phorbol esters is correlated with their tumor-promoting potency. These findings show that signaltransducing systems responding to phorbol esters are present even in basic metazoan animals. However, protein kinase-C activity could not be detected, and 1 -oleoyl-2-acetyl-glycerol could not replace TPA. On the other hand, the biological activity of phorbol esters is promoted by reducing the Mg²⁺ and increasing the K⁺ concentration of the external medium, and is strongly amplified by lithium or cesium. Thus, the existence of a more elementary, presumably cation-mediated transducing system is suggested.     

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.     

Tumor-promoting phorbol esters and vanadate alter the sensitivity of HeLa S3 cells to poliovirus type 1

Treatment of HeLa S3 cells with tumor-promoting phorbol esters and vanadate increased their sensitivity to type 1 poliovirus. Since the sensitization could not be accounted for by increased virus binding or virus production, it appears that virus entry was facilitated by the treatments. When HeLa S3 cells were incubated with TPA for prolonged periods of time, they became resistant to poliovirus due to reduced ability to bind the virus.     

Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor

4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.     

Tumor-promoting phorbol esters interfere with the vasoactive intestinal peptide receptor/effector system in rat prostatic epithelial cells

Pretreatment of rat prostatic epithelial cells with the tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate resulted in a decrease of both the potency of vasoactive intestinal peptide (VIP) upon the stimulation of cyclic AMP accumulation and the affinity of the receptors of this peptide. These effects were dose-dependent and could be reproduced by other stimulators of protein kinase C (PKC). Thus, it is conceivably that phosphorylation of VIP receptors by PKC regulates VIP receptor function in the prostate gland.     

Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.     

Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

Tumor-promoting phorbol esters inhibit procollagen synthesis at a pretranslational level in JB-6 mouse epidermal cells.

Collagen synthesis was inhibited in JB-6 mouse epidermal cells after exposure to 12-O-tetradecanoylphorbol-13-acetate under conditions leading to irreversible neoplastic transformation. In vitro translation and hybridization studies demonstrated a dramatic decrease in collagen mRNA in 12-O-tetradecanoylphorbol-13-acetate-treated cells, suggesting that the inhibition of collagen synthesis in response to 12-O-tetradecanoylphorbol-13-acetate is due to regulation at a pretranslational level.     

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.     

Tumor-promoting phorbol esters stimulate the phosphorylation of ribosomal protein S6 in quiescent Reuber H35 hepatoma cells

The addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) to serum-starved quiescent Reuber H35 hepatoma cells results in a rapid 5- to 11-fold increase in the incorporation of 32Pi into a Mr = 32,000 ribosomal protein. The Mr = 32,000 protein was the major phosphorylated protein extracted from isolated 80 S ribosomes and was identified as the 40 S ribosomal protein S6 based upon its migration in two-dimensional gels. Insulin, which has been demonstrated to increase the phosphorylation of S6 in a number of cell lines, caused a 10- to 20-fold increase in the incorporation of 32Pi into this Mr = 32,000 ribosomal protein. S6 phosphorylation was dose- and time-dependent being detected as early as 5 min following the addition of 1.6 microM TPA. Maximal phosphorylation of ribosomal protein S6 was achieved by 60 min and remained elevated for at least 90 min in the presence of TPA. The 50% effective dose for TPA was estimated to be 0.14 microM. Based upon the altered migration of S6 in pH 8.5 urea-polyacrylamide gels, it was demonstrated that the increased 32Pi labeling of S6 by TPA was due to a net increase in the incorporation of phosphates into the S6 molecule. Non-tumor-promoting phorbol esters were ineffective in increasing the phosphorylation of S6. In whole cells, exogenously added 1 mM 8-bromoadenosine 3':5'-monophosphate failed to substantially increase phosphorylation of S6 suggesting that the TPA-induced phosphorylation of S6 occurs via a cyclic AMP-independent mechanism. The S6 amino acid residue phosphorylated in response to TPA was phosphoserine. A possible role for protein kinase C in the phosphorylation of ribosomal protein S6 is discussed.     

Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

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.     

Inhibition of insulin receptor binding by phorbol esters

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

Diacylglycerols, unlike phorbol esters, do not induce homologous desensitization or down-regulation of protein kinase C in Swiss 3T3 cells

Addition of 1-oleoyl-2-acetyl-glycerol (OAG), 1,2-dioctanoyl-glycerol (diC8) or phorbol-12, 13-dibutyrate (PDBu) to cultures of Swiss 3T3 cells rapidly increases the phosphorylation of the Mr 80,000 protein kinase C (PKC) substrate, inhibits EGF binding and stimulates DNA synthesis. Prolonged incubation (40 h) with PDBu completely blocked these responses to all agents and down-regulated PKC. In contrast, a similar treatment with OAG or diC8, at mitogenic concentrations, neither induced homologous cellular desensitization nor decreased the immunoreactive level or activity of PKC. The results show that PKC down-regulation can be dissociated from PKC-mediated mitogenesis in Swiss 3T3 cells.     

Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.     

Calphostin-C stimulates epidermal growth factor receptor phosphorylation and internalization via light-dependent mechanism

Calphostin-C with perylenequinone structure is known to bind the regulatory domain of protein kinase C (PKC) and to inhibit kinase activity in vitro in a light-dependent fashion. We have found that calphostin-C induces substantial serine and threonine phosphorylation of the epidermal growth factor (EGF) receptor in a light-dependent fashion in the EGF receptor-hyperproducing squamous carcinoma cell line NA. Tryptic phospho-peptide mapping and phospho-amino acid analysis revealed that calphostin-C–-enhanced phosphorylation was on threonine 669, serine 671, serine 1046/1047, and serine 1166. However, caiphostin-C did not inhibit phosphorylation of the 80 K protein, a cytosolic major substrate of PKC (MARCKS). Staurosporine, a potent PKC inhibitor with affinity for the catalytic domain of PKC, inhibited phosphorylation of the 80 K protein and 12-O-tetradecanoyl-13-phorbol acetate induction of EGF receptor phosphorylation but did not inhibit the calphostin-C induction of the EGF receptor phosphorylation. These results suggest that the target of calphostin-C in vivo is different from that of staurosporine and thus calphostin-C in vivo does not inhibit PKC. Furthermore, calphostin-C enhanced the internalization of phosphorylated EGF receptor. Thus, calphostin-C apparently activates a novel signal transduction pathway which involves phosphorylation and internalization of the EGF receptor via light-dependent mechanism. © 1994 Wiley-Liss, Inc.     

Protein kinase C activation in a 3T3 cell variant morphologically unresponsive to tumor-promoting phorbol esters

To investigate the mechanism of the morphological changes induced in cells by tumor-promoting phorbol esters, we isolated a 3T3 cell variant which was morphologically unresponsive to phorbol esters and analyzed the activation of protein kinase C induced by the phorbol esters in it. The variant resembled the parent cells in its activation and appeared to have been altered at some step distal to the early events of protein kinase C activation.     

Phorbol esters and related analogs regulate the subcellular localization of beta 2-chimaerin, a non-protein kinase C phorbol ester receptor

The novel phorbol ester receptor beta2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein kinase C (PKC) isozymes that is involved in phorbol ester and diacylglycerol binding. We have previously shown that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in a phospholipid-dependent manner (Caloca, M. J., Fernandez, M. N., Lewin, N. E., Ching, D., Modali, R., Blumberg, P. M., and Kazanietz, M. G. (1997) J. Biol. Chem. 272, 26488-26496). In this paper we report that like PKC isozymes, beta2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of alpha1 (n)- and beta1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of beta2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, beta2-chimaerin translocates to the Golgi apparatus after phorbol ester treatment, as revealed by co-staining with the Golgi marker BODIPY-TR-ceramide. Structure relationship analysis of translocation using a series of PKC ligands revealed substantial differences between translocation of beta2-chimaerin and PKCalpha. Strikingly, the mezerein analog thymeleatoxin is not able to translocate beta2-chimaerin, although it very efficiently translocates PKCalpha. Phorbol esters also promote the association of beta2-chimaerin with Rac in cells. These data suggest that chimaerins can be positionally regulated by phorbol esters and that each phorbol ester receptor class has distinct pharmacological properties and targeting mechanisms. The identification of selective ligands for each phorbol ester receptor class represents an important step in dissecting their specific cellular functions.     

Tumor-promoting phorbol esters and cell proliferation stimulate secretion of basement membrane (type IV) collagen-degrading metalloproteinase by human fibroblasts

The secretion of a type IV collagen-specific proteinase is stimulated in cultured human skin fibroblasts by the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) and during cell proliferation. Exposure of the cells at the late log phase of growth to 10(-9) to 10(-6) M TPA resulted in the secretion of type IV collagenase activity to the medium, this effect being reversible. Incubation of intact type IV procollagen with TPA-induced fibroblast medium protein produced six peptides, four of which corresponded in size to the fragments produced by a type IV collagen-specific collagenase (Fessler, L., Duncan, K., Fessler, J., Salo, T., and Tryggvason (1984) J. Biol. Chem. 259, 9783-9789). The TPA-induced type IV collagen-degrading enzyme could be activated by trypsin, was inhibited by EDTA, but was not affected by soybean trypsin inhibitor, N-ethylmaleimide, aprotinin, or cysteine. Therefore, in human skin fibroblasts, TPA can induce a type IV collagen-specific, metal-dependent collagenase as was previously described in some invasive tumor cells. Furthermore, another metalloprotease is apparently secreted under the same conditions of TPA exposure. The production of metal-dependent, type IV collagen-degrading activity was also studied at different stages of cellular proliferation. In early log phase, a significant amount of enzyme activity was observed in the control cell medium; this activity disappeared during both late log and stationary growth phases. This activity could be markedly increased by the addition of 10(-8) M TPA to the culture medium. The production of matrix-degrading proteinases is therefore likely to be associated with rapid cell proliferation in both transformed and untransformed cells.