Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.     

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.     

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: an in situ hybridization study.

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.     

Proliferative responses of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast Pathogenesis

We have explored the relationship of changes in proliferative responses of human mammary epithelial cells to a phorbol ester (TPA) and to 8-Br-cAMP, which modulate the activities of protein kinases A and C (PKA and PKC), with breast tumour progression. Treatment with TPA had no effect on nontumorigenic cell lines established from human fibrocystic biopsies and apparently normal tissue around a tumour. In contrast, TPA strongly inhibited the proliferation of numerous human tumorigenic breast cell lines. Treatment with 8-Br-cAMP decreased the proliferation of all studied nontumorigenic and tumorigenic cell lines. We have also studied the effect of TPA and 8-Br-cAMP on growth of epithelial cells in short-term culture obtained from surgical human mammary biopsies with different states of breast disease. Both drugs enhanced growth of normal breast cells but had no significant effects on cells from biopsies with benign breast disease. In contrast, all examined cuitures from breast cancer biopsies were strongly inhhited by 8-Br-cAMP. Otherwise, TPA had an inhibitory effect only in the case of invasive ductal carcinoma of grade III. Malignant Ha-ras-transformation of nontumorigenic TPA-insensitive breast HBL-100 cells induced an inhibitory effect of TPA. In addition, a TPA-insensitive MCF7 clone was much less tumorigenic in athymic mice than the parental strain shown to be inhibited by TPA. These data suggest that the two intracellular transduction pathways change at different stages of breast pathogenesis. Alterations in the PKA pathway are early events and are probably important to cell immortalization but do not necessarily lead to malignant development. In contrast, changes in PKC pathway are rather later events associated with advanced malignant transformation. © 1994 Wiley-Liss, Inc.     

Phorbol esters and mitogenesis: comparison of the proliferative response of parental and Na+K+Cl- -cotransport-defective BALB/c 3T3 cells to 12-0-tetradecanoylphorbol-13-acetate

The ability of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl- cotransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 microM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30-35% increase in the saturation density of mutant cells compared to the 100-120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H-thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016-1.6 microM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl- cotransport activity decreases the response of these cells to the mitogenic action of TPA.     

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.     

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: An in situ hybridization study

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.     

Protein kinase C and membrane transport: divergent responses of Na+/K+/Cl- cotransport and sugar transport to exogenous diacylglycerol

Even though the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is known to bind to and activate protein kinase C (PKC), it is still not certain that all cellular responses to phorbol esters are necessarily mediated by PKC. In BALB/c 3T3 preadipose cells, TPA has previously been shown to rapidly inhibit Na+K+Cl- -cotransport activity, stimulate 2-deoxyglucose uptake and induce ornithine decarboxylase activity. The cell-permeable diacylglycerol sn-1,2-dioctanoylglycerol (DiC8) was used in order to distinguish between PKC-dependent and -independent responses of BALB/c 3T3 cells. DiC8 modulated 86Rb+ fluxes in BALB/c 3T3 cells in the same manner as TPA: furosemide-sensitive 86Rb+ influx and efflux was inhibited, while in cotransport-defective cells no effect was observed. In contrast, DiC8 did not stimulate 2-deoxyglucose uptake in either parental or cotransport-defective cell lines, even though TPA is a very effective inducer of this transport system in both cell types. Pretreatment of cells with DiC8 did not substantially alter the subsequent induction of 2-deoxyglucose uptake by TPA, although a slight but reproducible reduction in the magnitude of the response was observed in DiC8-pretreated cells. The PKC-dependent phosphorylation of an acidic 80-kDa protein was stimulated by both TPA and DiC8 in parental and cotransport-defective cell lines, suggesting that a gross defect in the primary effector system used by both TPA and diacylglycerols cannot explain any of our results. Ornithine decarboxylase was induced by DiC8 and the K1/2 was approximately the same as that for inhibition of Na+/K+/Cl- cotransport in these cells. Thus, our results suggest that PKC is clearly essential for some phorbol ester membrane transport responses (such as inhibition of Na+/K+/Cl- cotransport), but our results do not allow us to conclude that other responses (such as stimulation of 2-deoxyglucose uptake) necessarily require PKC activation.     

Phorbol esters induce both intracellular translocation and down-regulation of protein kinase C in MCF-7 cells

Exposure of MCF-7 human breast cancer cells to phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) results in a complete inhibition of cell proliferation. We investigated the effects of TPA on protein kinase C activity when cells were exposed to phorbol ester for various lengths of time. TPA induces within 5 min a drastic dose-dependent decrease of the cytosolic protein kinase C activity. The enzyme apparently lost at the cytosolic level was only partially recovered in the particulate fraction. The apparent down-regulation of the translocated enzyme which was only 34% after 1 min reached 72% and 84% after respectively 10 min and 15 min. Moreover, when cells are treated with TPA for longer periods of time, the particulate protein kinase C activity continues to decrease, dropping below control after 1 hour. This progressive decline leads to an almost complete disappearance of protein kinase C activity in MCF-7 cells after 45 hours of TPA treatment. The apparent loss of protein kinase C activity upon short- as well as long-exposure of cells to TPA was not accompanied by a concomitant increase of Ca, PL-independent protein kinase activity. We discuss the implication of these biochemical events in the inhibition of cell proliferation with regard to the respective short- and long-term effects of TPA on protein kinase C activity.     

Phorbol ester induces manganese-superoxide dismutase in tumor necrosis factor-resistant cells.

The effects of phorbol ester (TPA) and other known stimulators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide on induction of mRNA for manganese-superoxide dismutase (Mn-SOD) were investigated in various cell lines. TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines including HeLa cells, in which the other reagents also induced expression of the gene, but did not affect TNF-sensitive cells, in which the other stimulators did not alter expression of the gene. HeLa cells which had been desensitized to TPA by pretreatment with TPA for 24 h expressed Mn-SOD mRNA at a slightly higher level than the cells without TPA treatment. TPA-pretreated cells stimulated with TNF, however, expressed Mn-SOD mRNA at about twice the level of TNF-stimulated, TPA-untreated cells. When protein synthesis was inhibited by cycloheximide during TPA pretreatment, TNF no more enhanced the Mn-SOD mRNA accumulation. These data suggest that at least two separate signal-transducing pathways are involved in expression of this gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulation with TNF, interleukin-1, or lipopolysaccharide and in which a protein factor that can be induced by TPA treatment is involved.     

Phenotypic modulation of chronic lymphocytic leukemia cells by phorbol ester: induction of IgM secretion and changes in the expression of B cell-associated surface antigens

Freshly explanted neoplastic populations from 22 cases of phenotypically well-characterized chronic type B lymphocytic leukemia were studied for their capacity to respond to the phorbol ester TPA in vitro. In all but four cases the secretion of IgM was either induced or increased, often to a high level. In contrast, the export of free immunoglobulin (Ig) light chains, an almost consistent feature of the B lymphocytic leukemias, remained relatively constant after TPA treatment. Parallel changes in leukemic cell surface phenotype were probed with both "conventional" and monoclonal antibodies, revealing some modulation of markers in every case investigated. A diminution in the level of surface Ig (preferentially IgD) and the accumulation of cytoplasmic Ig observed after phorbol ester treatment were accompanied by a corresponding reduction or loss of the B1 antigen and usually of B2 when present. The most consistent change induced by TPA was the appearance of BB-1, a marker of activated B lymphocytes, which was rarely expressed on fresh leukemic cells. Another marker of activated lymphocytes, LB-1, was also often induced or increased in its expression after exposure of the cells to TPA. The magnitude of the TPA response appeared to relate to the stage of maturation arrest of the individual leukemic clones rather than to any clinical parameter explored. The significance of the findings to normal B cell differentiation and their potential clinical utility are discussed.     

Phorbol ester induction of plasmacytoid and hairy cell leukemia features in B-type lymphocytic leukemias: the relation to B-cell differentiation and maturation

Mononuclear cells concentrated from 11 patients with chronic lymphocytic leukemia (CLL), 7 with non-Hodgkin's lymphoma in leukemic phase (NHL), 5 with hairy cell leukemia (HCL), 1 with prolymphocytic leukemia (PLL), and 1 with plasma cell leukemia (PCL) were induced to differentiate with various doses of TPA. The degree of induction was followed for up to 6 days by measuring the expression of surface membrane markers (SmIg and GP-70) and Ig secretion, the induction of tartrate-resistant acid phosphatase (TRAP) and by recording ultrastructural changes as seen by electronmicroscopy. The results show a dose and time dependency of the TPA effect and a great heterogeneity in the cellular response, particularly in cells obtained from B-CLL patients. TPA induced two main features, namely the development of "plasmacytoid" or "hairy cell" leukemia features that clearly depended on the dose and duration of treatment with the phorbol ester. The plasmacytoid features were more frequently encountered with lower doses (1 ng/ml) of TPA and were more evident after shorter exposures to TPA (1-2 days). Nevertheless, the hairy cell features were more striking after incubation with higher concentrations of TPA (10-100 ng/ml) after longer periods of incubation (up to 6 days) with lower doses of TPA. The various features of differentiation measured including cell morphology, surface membrane markers, Ig secretion, and TRAP staining, were frequently independent of each other, suggesting an autonomous pathway of differentiation for some of these features. Furthermore, in most of the cases, hairy cell leukemia features were obtained more frequently following TPA exposure than plasmacytic changes.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.     

Effect of the phorbol ester TPA on PTH secretion. Evidence for a role for protein kinase C in the control of PTH release

Parathyroid hormone (PTH) secretion is stimulated by low extracellular calcium (Ca2+) in association with a reduction in cyosolic Ca2+, indicating that this cell type does not conform to classical models of stimulus-secretion coupling. We used the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate), which directly activates protein kinase C, to investigate the possible role of this enzyme in the unusual secretory properties of the parathyroid cell. TPA causes a dose-dependent stimulation of PTH release inhibited by high extracellular Ca2+ (EC50 = 10 nM) but has relatively little effect on secretion stimulated by low Ca2+. This effect was mimicked by the beta 4-isomer of phorbol 12,13-didecanoate which also activates kinase C, but not by the alpha 4-isomer, which has no effect on this enzyme. TPA does not modify cellular cAMP or cytosolic Ca2+ in the parathyroid cell indicating that its effects on PTH secretion are not mediated indirectly via changes in these second messengers. These results suggest that inhibition of PTH release at high Ca2+ might be related to a reduction in protein kinase C activity which can be overcome when the enzyme is directly activated by TPA.     

The stimulation of phosphorylation of intracellular proteins in GH3 rat pituitary tumour cells by phorbol esters of distinct biological activity

Using a pituitary tumour cell line (GH3), we have studied the phosphorylation of intracellular proteins induced by phorbol esters of diverse biological activity. All the active phorbol esters, including the weakly tumour-promoting but non-platelet aggregatory compound DOPPA, stimulated the phosphorylation of a cytosolic 80 kDa protein. A protein of this molecular mass has been suggested to be a marker of PKC activity. In contrast, only TPA and the non-tumour promoting but highly active phorbol ester SAP A stimulated the phosphorylation of a 130 kDa membrane protein. The results suggest that these phorbol esters activate PKC, but induce the differential phosphorylation of a variety of intracellular proteins.     

Modulation of adenylate cyclase of human platelets by phorbol ester. Impairment of the hormone-sensitive inhibitory pathway

The influence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone-sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory NS protein nor on stimulatory hormone receptors (prostaglandin E1) and the mutual interactions of these components of the stimulatory hormone-sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP-dependent, hormone-sensitive inhibitory pathway to the adenylate cyclase, involving the inhibitory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni-mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni-protein-mediated signal transduction to the adenylate cyclase.