Tumor-promoting Phorbol Ester 12-O-tetradecanoylphorbol-13-acetate Research Articles

Identification and characterization of the nonphosphorylated precursor of pp17, a phosphoprotein associated with phorbol ester induction of growth arrest and monocytic differentiation in HL-60 promyelocytic leukemia cells.

Treatment of HL-60 promyelocytic leukemia cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), causes rapid phosphorylation and dephosphorylation of pp17, a 17-20-kDa, pI 5.5 cytosolic protein, as an early event in a response sequence leading to growth arrest and terminal differentiation into monocytes (Feuerstein, N., and Cooper, H. L., (1984) J. Biol. Chem. 259, 2782-2788). In the present study, we have identified the nonphosphorylated precursor to pp17 by tryptic peptide mapping of single proteins recovered from two-dimensional gels. The pI of the precursor, p17, was 5.9, and the apparent Mr of both p17 and pp17 was 18,400 by SDS-polyacrylamide gel electrophoresis (one dimension). p17 was shown to be a major cytosolic protein, comprising about 0.5% of steady state labeled protein in that fraction. Both p17 and pp17 were found exclusively in the cytosol (detergent-released SOL) and were not detected in membranes, cytoskeleton, or nuclei. In untreated cells, about 90% of the protein was present in the nonphosphorylated form. Upon TPA treatment, pre-existing p17 was rapidly phosphorylated to pp17. After 15 min, the two forms were nearly equal in quantity. This corresponds to phosphorylation, within that period, of about 0.2% of total cytosolic protein, represented by a single species. The maximum level of pp17 was reached within 1 h, with pp17 exceeding p17 by about 25%. Quantitatively, therefore, the phosphorylation of p17 to pp17 is one of the most prominent early biochemical responses to TPA treatment. Available data indicate that p17 predominates in rapidly proliferating cells, while phosphorylation to pp17 occurs where cell growth is modified by TPA or other agents. Thus, the p17/pp17 system is potentially a major mechanism for intracellular propagation of growth regulatory signals. We propose the name, prosolin, for this prominent cytosolic protein.

A novel type of phorbol ester-dependent protein phosphorylation in the particulate fraction of mouse epidermis

In a Triton X100-extract from the particulate fraction of mouse epidermis but also of other murine tissues, the phosphorylation of a protein with the relative molecular mass of 82,000 (p82) is found to be dependent on phosphatidyl serine and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Unlike protein kinase C-catalyzed phosphorylation, p82 phosphorylation is neither observed in the presence of high concentrations of Ca 2+ and phosphatidyl serine alone nor after addition of exogenous protein kinase C. Dioctanoylglycerol and the “incomplete” promoter 12-O-retinoylphorbol-13-acetate are also capable of stimulating p82 phosphorylation, whereas the non-promoting phorbol ester 4-O-methyl-TPA is at least 100-fold less active in this respect.

Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation.

Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction.

Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters.

Rat pheochromocytoma cells (clone PC12) display cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF) and therefore provide a useful model system with which to study the role of these receptors in the regulation of proliferation and differentiation. In this paper PC12 cells are demonstrated to possess two classes of EGF receptors, a high-affinity class with 7,600 sites per cell and an apparent dissociation constant (Kd) of 0.05 nM, and a low-affinity class with 62,000 sites per cell and a Kd of 14.1 nM. These findings are contrary to literature data (Huff et al., 1981; Vale and Shooter, 1983) but can be explained in part by differences in experimental conditions. Binding studies at 37 degrees C compared with room temperature demonstrated similar affinities of both classes, but during prolonged incubation at 37 degrees C, the binding capacities of both classes decreased. Furthermore the high-affinity class was sensitive to lectins, such as concanavalin A (Con A), and to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Both compounds caused a decrease of the affinity of the high-affinity class without affecting the low-affinity class. At high concentrations of Con A or TPA, a decrease of the apparent number of binding sites of the low-affinity class was also observed. The similarities between the characteristics of EGF binding and NGF binding in PC12 cells are striking and will be discussed.

Studies on the mechanism of phorbol ester-induced inhibition of intercellular junctional communication.

Intercellular gap-junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate lyase-deficient human fibroblasts and argininosuccinate synthetase-deficient Chinese Hamster V79 cells. As previously shown, in this system junctional communication is completely inhibited by the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the absence of extracellular calcium, TPA inhibition was less pronounced. However, synergism with calcium ionophore A23187 could not be demonstrated. Chlorpromazine, trifluoperazine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester partially antagonised the effect of TPA. No antagonism was demonstrable between calmidazolium and TPA. Treatment of co-cultures with exogenous phospholipase C or 1-oleoyl-2-acetylglycerol (OAG) resulted in communication inhibition, suggesting that protein kinase C activation is involved in the mechanism of phorbol ester-mediated communication inhibition. However co-cultures which had been made refractory to TPA by prolonged exposure to high concentrations remained sensitive to inhibition by phospholipase C and OAG. These results suggest either that diacylglycerol can produce other effects independent of protein kinase C activation, or that refractoriness to phorbol esters is not simply due to a decrease in the amount of protein kinase C.

Protein kinase C regulation of the receptor-coupled calcium signal in histamine-secreting rat basophilic leukaemia cells

It has been proposed that protein kinase C mediates cellular responses evoked by external stimuli, leading to alterations in internal free calcium concentrations. We have shown previously that histamine-secreting rat basophilic leukaemia cells (RBL-2H3), which degranulate on aggregation of the receptors for immunoglobulin IgE, contain a Ca2+- and phospholipid-dependent protein kinase (kinase C). The partially purified enzyme is activated directly by the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In intact RBL cells, TPA potentiates histamine release induced by the Ca2+-ionophore A23187 (similar to the synergy reported for platelets, neutrophils and rat peritoneal mast cells). Although TPA at concentrations below 15 nM synergizes with the antigen, higher TPA concentrations inhibit secretion. This selective inhibition suggested that kinase C is involved in both the activation and termination of the secretory process. To examine this possibility, we have determined the effect of TPA on changes in free cytosolic Ca2+ concentration during antigen-induced release. We report here that TPA completely blocks the increase in Ca2+ concentration induced by antigen. Our results strongly suggest that protein kinase C is involved in the regulation of receptor-dependent Ca2+ signalling.

Cyclosporin a inhibits biological effects of tumor promoting phorbol esters

The antilymphocytic and antiphlogistic agent cyclosporin A (CsA) inhibits in vivo various effects induced by the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These include the edema of the mouse ear, the alkaline phosphatase (AP) activity and the ornithine decarboxylase (ODC) activity in mouse epidermis as well as the generation of a specific arachidonic acid (AA) metabolite in mouse epidermis. AA metabolism in an epidermal cell-free system of mouse epidermis was not suppressed by CsA. According to thin layer chromatography the TPA-induced and as yet unidentified AA metabolite exhibits a polarity between that of 5-HETE and 12-/15-HETE. Studies with inhibitors indicate it to be a lipoxygenase product.

Synergistic effect of a tumor-promoting phorbol ester and a hypoglycemic sulfonylurea upon insulin release.

The tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the hypoglycemic sulfonylurea gliclazide both stimulate insulin release from rat pancreatic islets incubated at a low concentration of glucose (2.8 mM). The increment in secretion rate evoked by the combination of these two secretagogues was at least 7 times higher than the sum of increments evoked by each agent alone. TPA augmented insulin release evoked by gliclazide more efficiently than that induced by glucose. The enhancing action of TPA could not be attributed solely to changes in the capacity of gliclazide to inhibit 86Rb outflow, to provoke an exchange between influent 40Ca and effluent 45Ca, or to stimulate the net uptake of 45Ca by the islets. Nevertheless, the facilitation by TPA of gliclazide-induced insulin release suggests that a biophysical interference with the handling of cations at the plasma membrane level represents the primary site of action of hypoglycemic sulfonylureas in the pancreatic B cell.

Retinoids and inhibition of ornithine decarboxylase activity.

The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a dose-dependent induction (up to 300-fold) of ornithine decarboxylase activity in the epidermis of intact mice within 5 hours after the application to the skin of from 1 to 10 nmoles of the ester in 0.2 ml of acetone. Enzyme activity regresses with a half-life of 17 minutes, the shortest known for any enzyme. The induction is inhibited in a dose-dependent manner by a single application of from 0.034 to 3.4 nmoles of retinoic acid within an hour before to an hour after phorbol ester treatment. Of over thirty natural and synthetic retinoids that were tested for this property, all-trans-retinoic acid was most effective. The ability of retinoic acid and its congeners to inhibit tumor promotion by the phorbol ester correlated with the effect of dose, structure, and time of treatment on the induction of ornithine decarboxylase. It appears that a permanently increased level of the enzyme and its product, putrescine, may be one of the essential components of the mechanism of tumor promotion.

Effect of phorbol esters on guniea pig skin in vivo.

When topically applied to guniea pig ear skin the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced inflammation and epidermal hyperproliferation which could be inhibited by indomethacin. This inhibition could be reversed both by prostaglandins E and F. Five minutes after TPA treatment an increase in the level of prostaglandin E but not of prostaglandin F was observed in the epidermis. The non-promoting phorbol ester 4-O-methyl-TPA also stimulated epidermal cell proliferation but this stimulation was not inhibited by indomethacin. The above results are in agreement with those already reported in the mouse system with these two compounds. Ornithine decarboxylase (ODC) activity has been evaluated in the epidermis of guniea pig ear after topical application of 20 nmol of TPA. No increase was noted. This is in contrast with the well documented activation of ODC in mouse skin treated with TPA. Since TPA acts as a promoter in the mouse whereas both croton oil and TPA have no promoting action in the guinea pig, the above result supports the view that ODC activationis related to promotion, and provides a possible explanation for the resistance of this animal species to promotion. This resistance is further documented by the fact that no "dark cells" were found in guinea pig ear skin.