Phorbol esters, but not the hormonal form of vitamin D, induce changes in protein kinase C during differentiation of human histiocytic lymphoma cell line (U-937)

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1,25-dihydroxycholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possible through a different mechanism of that of phorbol ester.     

An inhibitory effect of tumour promoters on human epithelial cell growth can be dissociated from an effect on junctional communication

Studies with rodent cells have indicated that the abilities of various tumour promoters to inhibit metabolic cooperation correlate with their potencies as mitogens. Here we have examined the effects of the most potent phorbol ester tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), on metabolic cooperation and growth of human epidermal cells transformed by SV40 (SVK14 cells). In this system, TPA inhibits Junctional communication and at the same concentration also inhibits growth in a reversible fashion. These effects appear to be mediated by binding of phorbol ester to a single class of high affinity binding site with a Kd similar to that reported for rodent cells (K_d = 20.9 nM at 4 °C). Further studies on the effects of phorbol esters on other human epithelial cell lines reveal that the inhibitory effects of TPA on growth and metabolic cooperation may be completely dissociated. Alternative mechanisms by which TPA may exert its growth-inhibitory effects are discussed.     

Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm,Manduca sexta: The cytosolic enzyme

A calcium and diacylglycerol-activated and phospholipid-dependent protein kinase (protein kinase C; PKC) in the cytosol of the pupal brain of the tobacco hornworm Manduca sexta has been characterized. Two peaks of PKC activity were separated by DEAE-cellulose chromatography, both of which were dependent upon Ca²⁺, phosphatidylserine and 1,2 diolein. Maximal enzyme activity was obtained in the presence of 0.7 mM Ca²⁺ and 200 μg/ml of phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) enhanced PKC activity and calcium sensitivity markedly in the presence of phospholipids. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate substituted for diacylglycerol in the activation of PKC. By utilizing the differential inhibition of PKC and cyclic AMP-dependent protein kinase by trifluoperazine and protein kinase inhibitor, both enzymes were measured accurately in dilute, crude cytosol preparations using the common substrate histone H1.     

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.     

Schistosoma mansoni: evidence for protein kinase-C-like modulation of muscle activity

The phorbol esters, phorbol-12,13-dibutyrate, phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate, as well as mezerin at concentrations as low as 10 nM produce a spastic paralysis of the schistosome musculature. The action of these protein kinase-C activators is dependent on the sites of esterification and is stereo-specific since phorbol-13,20-diacetate, phorbol-12,13,20-triacetate, 20-oxo, 20-deoxy-beta-phorbol-12,13-dibutyrate, alpha-phorbol-12,13-didecanoate, and alpha-phorbol are inactive. A phospholipid and phorbol ester-dependent protein kinase is identified. This kinase is stimulated by all of the phorbol esters that increase muscle tone but is not stimulated by phorbol esters that do not affect muscle tone. A high affinity, stereo-specific phorbol ester receptor is identified. Dose-response curves of phorbol-12,13-dibutyrate-induced muscle tension and -stimulated kinase activity and receptor binding indicate that these responses are mediated by the same system. These results indicate that protein kinase-C-like enzyme may play an important role in modulating activity of the schistosome musculature.     

Differential effects of norepinephrine and phorbol ester on alpha-1 adrenergic receptor number and surface-accessibility in DDT1 MF-2 cells

The short (5-60 min) and long (24 hrs) term effects of norepinephrine (10 uM) and the phorbol ester, 12-0-tetradecanoyl phorbol-13-acetate (10 nM), on total cellular and surface-accessible alpha-1 adrenergic receptor number were determined in DDT1 MF-2 smooth muscle cells. The density of alpha-1 adrenergic receptors was determined with [3H]-prazosin in a crude cellular homogenate (total cellular receptors) and in intact cells at 4 degrees C (surface-accessible receptors). Under basal conditions, all receptors were accessible to the cell surface at 4 degrees C. Short term norepinephrine exposure caused an approximately 40% decrease in surface-accessible binding without a change in total receptor number. Long term norepinephrine exposure caused a further decrease in surface-accessible binding, and an approximately 30% decrease in total receptor number. In contrast, phorbol ester had no effect on surface-accessible or total receptor number with either short or long term exposure. These data suggest that sequestration of cell surface alpha-1 adrenergic receptors is an early step in the process of agonist-mediated down-regulation. In DDT1 MF-2 cells, phorbol ester, alone, does not mimmick the effect of agonist on receptor sequestration or number.     

Polyamines and terminal deoxynucleotidyl transferase expression In KM 3 pre-B cell line during phorbol ester induced differentiation.

The aliphatic polyamines, putrescine, spermine and spermidine belong to a category of molecules implicated in DNA replication. Their synthesis is strongly activated during the G₁ period and they have been implicated in the regulation of cell proliferation and differentiation. Terminal transferase is a DNA polymerase present in pre-T and pre-B cells and its expression can be modulated by phorbol ester treatment. In this study we have monitored the relationship of intracellular polyamine levels with terminal deoxynucleotidyl transferase down-regulation induced by 12-0-tetradecanoyl phorbol myristate 13-acetate treatment in the human pre-B KM-3 cell line. Phorbol myristate acetate can cause an increase, at 4 and 8 hours of differentiation, of intracellular levels of putrescine as well as a decrease in terminal deoxynucleotidyl transferase synthesis showing the probable involvement that polyamines have in the differentiation process.     

Tumour promoter-mediated reversible inhibition of cell-cell communication (electrical coupling) : Relationship with phorbol ester binding and de novo macromolecule synthesis

A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [³H]phorbol-12,13-dibutyrate ([³H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 × 10⁵ PDBu molecules were bound to each cell. The binding of [³H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4α-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [³H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary.     

Decreased incidence of gap junctions between Chinese hamster V-79 cells upon exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate

Previous studies have shown that metabolic cooperation between Chinese hamster V-79 cells is inhibited by the tumor-promoting phorbol esters, but not by those phorbol esters inactive in tumor promotion. Metabolic cooperation is believed to be mediated by gap junctions. Therefore, in the present study we have used freeze-fracture and quantitative morphological techniques to examine the effect of the most potent tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of the inactive analog, 4α-phorbol-12,13-didecanoate (4α-PDD) on the frequency of gap-junctional contacts between V-79 cells. In both control and in 4α-PDD treated cultures the junctions were frequent, though rather small in size. In contrast, in the TPA-treated cultures, gap junctions were few, and the area of membrane occupied by gap junctions was reduced more than 20-fold from that found for controls. These results suggest that the TPA-induced inhibition of metabolic cooperation between V-79 cells is a consequence of a decrease in the number of gap junctions.     

The kinetics of fibronectin synthesis and release in normal and tumor promoter-treated human lung fibroblasts

Summary The present study is a detailed kinetic analysis of the synthesis, release and multimerization of fibronectin (FN) in normal and tumor promoter-treated human lung fibroblasts. Pulse/chase and surface labeling experiments were performed to follow the fate of both newly synthesized and pre-existing cell-surface FN over time. The majority of FN (80%) left the intracellular compartment within one hour of synthesis. However, the rate of direct secretion was very low and after one hour, 70% of newly synthesized FN was still at the cell surface. This material was primarily dimeric. Dimeric and multimeric (very high molecular weight) FN was detectable at the cell surface and in the medium 4 hours after synthesis. Pulse-labeled FN multimer levels peaked at 12 hours and declined thereafter. After 24 hours, 85% of pulse-labeled FN had been shed into the medium and the labeled FN remaining at the cell surface was primarily multimeric. Surface labeling experiments confirmed that the majority of FN resides at the cell surface prior to release into the medium.One hour treatment with the phorbol ester tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), stimulated a nine-fold increase in release of preexisting, dimeric cell-surface FN (¹²⁵I-labeled). The major effect of longer term TPA treatment up to nine hours was continued depletion of dimeric cell-surface FN. Increased release of cell-surface multimeric FN was also stimulated by TPA, but to a much lesser extent. Release of newly synthesized (pulse-labeled) dimeric FN was also stimulated by TPA though much less than pre-existing FN, and TPA treatment produced a small decrease in the steady-state level of multimeric FN. Thus, preexisting cell-surface IN and newly synthesized FN differ dramatically in their susceptibility to TPA treatment.Abbreviations FN fibronectin - HLF human lung fibroblasts - PMSF phenylmethylsulfonylfluoride - TPA 12-O-tetradecanoyl phorbol-13-acetate - DMSO dimethylsulfoxide - MEM minimal essential medium - ELISA enzyme linked immunosorbent assay - PBS phosphate-buffered saline - BSA bovine serum albumin - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DOC deoxycholate - DTT dithiotreitol     

A protein kinase C-like activity in Escherichia coli

The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80-85,000 in E. coli and the alpha- and gamma-isozymes, but probably not the beta-isozyme, of eukaryotic PKC.     

Effects of phorbol-12,13-diesters on prostaglandin production and phospholipase activity in canine kidney (MDCK) cells

Two tumor promoting phorbol-12,13-diesters, 12-0-tetradecanoyl-phorbol-13-acetate and phorbol-12,13-didecanoate, at concentrations of 10^?9 to 10^?10 M, stimulated prostaglandin production by dog kidney (MDCK) cells cultured in serum-supplemented medium. The non-tumor producing phorbol diester, 4α-phorbol-12,13-didecanoate, at a concentration of 10^?7 M, had no effect. The two biologically active phorbol diesters, but not the non-tumor promoting analog, stimulated deacylation of the cellular phospholipids of MDCK cells radioactively labelled with [³H]arachidonic acid. Most of the arachidonic acid was converted into prostaglandins.     

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 Ca2+ and phosphatidyl serine alone nor after addition of exogenous protein kinase C. Dioctanoylglycerol and the "incomplete" promoter 12-0-retinoylphorbol-13-acetate are also capable of stimulating p82 phosphorylation, whereas the non-promoting phorbol ester 4-0-methyl-TPA is at least 100-fold less active in this respect.     

Differential effects of the tumor promoter phorbol-12-myristate-13-acetate on the morphological and biochemical differentiation of N-18 mouse neuroblastoma cells

The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) was found to have differential inhibitory effects on the expression of morphological and biochemical differentiation of N-18 mouse neuroblastoma cells. PMA completely inhibited neurite extension and associated growth characteristics and partially inhibited the increased expression of R1 cAMP-binding protein; PMA had no effect on the induction of acetylcholinesterase activity in cells prompted to differentiate either by treatment with 1 mM dibutyryl cAMP or by serum deprivation. 4-alpha-Phorbol-12, 13-didecanoate, an inactive analogue of phorbol ester tumor promoter, was without effect. The implications of these findings concerning the mechanism of action of phorbol ester tumor promoters in the control of cell differentiation are discussed.     

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.     

Calcitonin-induced increase in phosphate accumulation in LLC-PK1 cells probably through protein kinase C activation

To assess the role of protein kinase C and cAMP on the calcitonin-induced alteration of phosphate accumulation by renal tubular cells, the effects of phorbol esters, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and DBcAMP on the phosphate accumulation in LLC-PK1 cells were investigated. Calcitonin stimulated phosphate accumulation with a concomitant increase in cAMP production. Phorbol esters and 1-oleoyl-2-acetyl-glycerol, activators of protein kinase C, also stimulated the phosphate accumulation. Furthermore, H-7, an inhibitor of protein kinase C, inhibited a calcitonin-induced increase in phosphate accumulation significantly. Although DBcAMP by itself did not increase the phosphate accumulation, it enhanced the stimulatory effect of 12-0-tetradecanoyl phorbol-13-acetate on the phosphate accumulation. Accordingly, protein kinase C as well as cAMP might be involved in the calcitonin-induced increase in phosphate accumulation in LLC-PK1 cells.     

Ca2+-Atpase inhibitor,cyclopiazonic acid,releases Ca2+ from intracellular stores in rbl-2h3 mast cells and activates a Ca2+ influx pathway that is permeable to sodium and manganese

Cyclopiazonic acid has been reported to inhibit the Ca²⁺-ATPase of intracellular calcium stores in some nonexcitable cell types, such as myeloid cells and lymphocytes. The present study examines the effects of cyclopizonic acid on rat basophilic leukemia (RBL) cells, a mucosal mast cell line. Addition of cyclopiazonic acid to fura-2-loaded RBL cells evoked a biphasic increase in free ionized intracellular calcium. Release of stored calcium accounted for the first phase of this response. The second phase was determined to be calcium entering through an influx pathway activated by cyclopiazonic acid. The influx pathway was selective for calcium, But was somewhat permeable to manganese. However, in a Ca²⁺-free solution containing EGTA, sodium ions permeated freely. This influx pathway appears to be identical to that which is activated by antigen, the physiological stimulus to the cells. Cyclopiazonic acid also induced secretion when combined with the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate, which activates protein kinae C. © 1995 Wiley-Liss, Inc.     

Phorbol ester-induced regulation of transferrin receptors in human leukemia K562 cells

The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13-acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20% of that in untreated cells. Pulse-chase experiments showed that turnover of the receptors in TPA-treated cells was accelerated over that in untreated cells. These results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors.     

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells

Phospholipase A₂ activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A₂ activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-³H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7–14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E₁ did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A₂ activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A₂, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A₂ activity was also related to previously found differences in the ability of the clones to develop desensitization to β-adrenergic hormones or prostaglandin E₁.