Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells.     

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.     

The effects of cAMP,Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine responses inHelix neurons

1. Using internal perfusion and concentration-clamp procedures applied to Helix neurons, the effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine Cl-dependent responses were determined. 2. Intracellular cAMP (10(-4) M) depressed those acetylcholine responses which were blocked by ouabain but had no effect on ouabain-insensitive acetylcholine responses. In the presence of elevated intracellular cAMP, ouabain had no further depressant effect on these acetylcholine responses. Both elevated cAMP and ouabain reduced the acetylcholine response without altering the current-voltage curves. 3. An increase in intracellular Ca2+ concentration depressed the amplitude of current induced by application of acetylcholine in neurons with ouabain-sensitive responses and shifted the dose-response relationship to the right. However, elevated Ca2+ did not reduce the maximal response induced by acetylcholine, nor did it prevent the reduction of that response by ouabain. 4. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase C activity, caused depression of both the ouabain-sensitive and the ouabain-insensitive acetylcholine responses. The inhibitory effect of TPA was markedly enhanced after addition of ATP to the intracellular medium and was greatly reduced by cooling to 5 degrees C. The blocking effect of ouabain, however, reexamined in the presence of TPA. 5. These observations are consistent with the hypothesis that the depression of acetylcholine induced Cl--responses in Helix neurons is a result of an increase in intracellular cAMP concentration but is unrelated to activation of protein kinase C or increases in intracellular Ca2+.     

Dissociation of TPA-induced down-regulation of T cell antigens from protein kinase C activation

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) has diverse effects on lymphoid cell function. Two of the early effects were the induction of early activation antigen EA1 and the down-regulation of certain T cell differentiation antigens (CD3, CD4, CD7). The mechanisms of these TPA effects were investigated. It was confirmed that EA1 expression was dependent on protein kinase C (PKC) activation. Synthetic diacylglycerols were capable of inducing EA1 expression. In addition, inhibition of PKC by the kinase inhibitor, H7, led to the inhibition of EA1 expression induced by TPA and synthetic diacylglycerols. In contrast, down-regulation of T cell differentiation antigens by TPA was not dependent on PKC activation. Synthetic diacylglycerols did not induce down-regulation of T cell antigens and H7 had no effect on the down-regulation of T cell antigens induced by TPA. These data would suggest that TPA exerted its effects on T cell function by mechanisms in addition to the activation of PKC alone. One possible mechanism would be the activation of the calmodulin-dependent pathway(s) since its inhibition resulted in the reversal of TPA-induced down-regulation of the T cell differentiation antigens.     

E2F modulates keratinocyte squamous differentiation: implications for E2F inhibition in squamous cell carcinoma

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 (E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation-insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.     

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.     

Topological regulation of cell-membrane phosphoinositidase C.

Although the translocation of protein kinase C and phospholipase A2 are well documented, no information is available about the possible down-modulation of transmembrane phospholipase C. We found that TPA induced a dose-dependent (10-200 nM) and time-dependent (15 min-6 h) down-modulation of transmembrane phosphoinositidase C (PLC-PI) on lymphoid cells (CEM-CM3 and WIL2-NS) and epitheloid carcinoma cells (HeLa S3) but not on human fibroblasts (MRC-5). Cell-surface expression of PLC-PI on intact cells was assayed by flow cytometry using saturating concentrations of polyclonal anti-PLC-PI antibodies and phycoerythrin-conjugate. A control phorbol-ester which does not activate protein kinase C (PKC) had no internalization effect on PLC-PI. PKC inhibitors staurosporine (2.5 nM) and H-7 (10 microM) partially inhibited the TPA effect. Cytochalasin B (40 micrograms/ml) did not modify the TPA-induced PLC-PI down-modulation. The effect of TPA on PLC-PI seems quite specific since no internalization was induced by TPA on transmembrane phosphatidylcholine-preferring PLC expression. These results show that TPA can translocate the membrane-bound PLC-PI, probably by PKC activation.     

Induction of Epstein-Barr virus lytic cycle by tumor-promoting and non-tumor-promoting phorbol esters requires active protein kinase C.

Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.     

The action of the tumour promoter, TPA, on mutagenesis induced by different agents (UV light, chemical and viral mutagens)

The present paper deals with effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the frequency of induced mutations to 6-mercaptopurine (6MP) and ouabain resistance in Chinese hamster and mouse cells. UV light, bovine adenovirus 3(BAV-3) and 5-bromodeoxyuridine (BrdU) were used as mutagens. TPA was shown to raise the frequency of gene mutations induced by UV light and BAV-3 but it did not enhance the mutagenic effect of BrdU. We also examined the ability of BAV-3 and BrdU to induce tumours in mice. BrdU was shown to have no carcinogenic effect. The results suggest that TPA enhances the mutagenic effect only for carcinogenic mutagens.     

12-O-tetradecanoylphorbol-13-acetate induces the enhancer function of human T-cell leukemia virus type I

Phorbol esters were employed in studies on the molecular mechanism of the induction of expression of human T-cell leukemia virus type I (HTLV-I) by a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Experiments using the chloramphenicol acetyltransferase (CAT) system showed that CAT expression directed by the long terminal repeat (LTR) of HTLV-I was induced by TPA, but not by 4 alpha-phorbol-12,13-didecanoate, which is not an activator of protein kinase C, and that like other known enhancers, irrespective of its position and orientation, a 230-bp fragment in the U3 region of the HTLV-I LTR confers susceptibility to induction by TPA.     

Mutagens and the tumor promoter

We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and saccharin on the frequency of induced mutations of resistance to 6-mercaptopurine and ouabain in Chinese hamster and mouse cells. UV-rays, bovine adenovirus-3 (BAV-3) and 5-bromdeoxyuridine (BrdU) were used as mutagens. In the case of BAV-3 and BrdU, we investigated, apart from the mutagenic effect, the tumor-inducing activity of these mutagens in mice, BrdU proved to have no carcinogenic effect. The data about the influence of TPA on the mutagenic effect of the three different mutagens indicate that TPA increases the frequency of the gene mutations induced by UV-rays and BAV-3. The results of the study of BrdU and TPA combined action revealed the fact that TPA does not increase the mutagenic effect of BrdU. We demonstrated that saccharin also possesses the promoter activity; it increases the mutagenic effect of BAV-3. The results described above lead to the assumption that TPA influence on the mutagenic effect only takes place when carcinogenic mutagens are used.     

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.     

Dual activity of pyrrolidine dithiocarbamate on kappa B-dependent gene expression in U937 cells: I. Regulation by the phorbol ester TPA.

Pyrrolidine dithiocarbamate (PDTC) has been widely used as an inhibitor of the nuclear factor-kappa B, (NF-kappa B) signalling pathway. Here, we show that kappa B-dependent reporter gene expression induced by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) is potentiated by PDTC in the human pro-monocytic U937 cell line. The stimulatory effect of PDTC on kappa B-dependent gene expression was shown with a 4 x kappa B chloramphenicol acetyltransferase construct and required an intact kappa B element in the human immunodeficiency virus long terminal repeat (HIV-1 LTR). Unexpectedly, an HIV-1 LTR construct with a mutation of the activator protein 2 (AP-2) binding site located between the two kappa B elements was unresponsive to the stimulatory effect of PDTC with TPA. The stimulation or inhibition of kappa B-dependent gene expression was dependent on PDTC pre-treatment and the concentration of TPA. No stimulatory effect on HIV-1 LTR activity was observed with the metal chelator dipyridyl or the anti-oxidant N-acetyl-L-cysteine. These results are consistent with the hypothesis that PDTC treatment potentiated kappa B-dependent gene expression in a manner dependent on the concentration of TPA.     

Cytokeratins as serum markers in egyptian bladder cancer. A comparison of CYFRA 21-1, TPA and TPS

Cytokeratins (CKs) have been shown to be overexpressed in bladder cancer and to be valuable as tumor markers. The present study was designed to evaluate the single and combined use of three cytokeratin fragments, CYFRA 21-1, TPA, and TPS, in serum of Egyptian bladder cancer patients. The study subjects comprised 40 healthy controls, 30 patients with benign bladder diseases, and 60 patients with histologically confirmed primary bladder cancer. The cutoff was set at 95% specificity versus benign bladder diseases, resulting in cutoff values of 2.93 ng/mL for CYFRA 21-1, 158 U/L for TPA and 143.7 ng/mL for TPS. With 41% true positive results CYFRA 21-1 had a higher sensitivity than TPA (32%) and TPS (27%). Evaluation by histological findings revealed a highest sensitivity of CYFRA 21-1 (46%) in transitional cell carcinoma (TCC) followed by TPA (27%) and TPS (21%). Also in adenocarcinoma CYFRA 21-1 showed the highest sensitivity (38%) followed by TPA (32%) and TPS (28%). A high percentage (41.6%) of Egyptian bladder cancers is represented by squamous cell carcinoma (SCC). In this population TPS showed the highest sensitivity (69%), followed by CYFRA 21-1 (54%) and TPA (41 %). The sensitivity of each of the three markers increased with advancing tumor stage and increasing tumor grade. Combined use of two of the three markers did not raise the sensitivities obtained by single determination of CYFRA 21-1. The present study suggests that serum CYFRA 21-1 could be a marker of choice in bladder cancer.     

Differentiation of HL-60 cells by phorbol ester is correlated with up-regulation of protein kinase C-alpha.

To clarify the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of HL-60 cells, we investigated the correlation between the effects of protein kinase C (PKC) inhibitors on the induction of markers of TPA-induced differentiation and those on suggested critical steps of the differentiation. H-7, sphingosine, and trifluoroperazine significantly suppressed TPA-induced cell adhesion but their effects on the induction of acid phosphatase and nonspecific esterase differed among the inhibitors. The three inhibitors failed to affect on TPA-induced annexin I expression. In contrast, staurosporine markedly suppressed the induction of all these markers. The effects of the inhibitors on some suggested critical steps of the differentiation, a rapid phosphorylation of specific proteins, a rapid membrane association of PKC, and down-regulation of PKC at 18 h after addition of TPA, were not correlated with those on the differentiation marker induction. Only the effect of the inhibitors on up-regulation of PKC-alpha was closely correlated with TPA-induced annexin I expression; staurosporine inhibited up-regulation of PKC-alpha but other inhibitors did not similarly affect the induction of annexin I expression. These results suggest that PKC-alpha is intimately related to macrophage-like differentiation of HL-60 cells by TPA.