To The Phorbol Ester 12-O-tetradecanoylphorbol-13-acetate Research Articles

Effect of novel modulators of protein kinase C activity upon chemotherapy-induced differentiation and apoptosis in myeloid leukemic cells.

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol. U937 cells displayed a slight reduction in the number of cells in G(2)/M cell cycle phase after exposure to FTT, SC-10, R59022 and R59949, respectively. In contrast, HL-60 cells demonstrated a largely unaltered cell cycle distribution. Whereas TPA treatment resulted in a strong induction of p21(WAF/CIP1), c-Fos and c-Jun levels, neither one of the novel PKC activators altered expression of these proteins. Consequently, we tested the ability of the activators to cause membrane translocation of PKC. While TPA treatment resulted in translocation of the PKC isoforms alpha, delta and epsilon, SC-10 and FTT failed to induce alterations in the PKC content of the membrane and cytosolic fractions, respectively. Expression of the beta(2)-integrin CD11c that is induced during TPA-mediated differentiation remained unaltered after exposure to SC-10 and was partly reduced after treatment with FTT. To further investigate the effect of these activators upon apoptosis in leukemic cells, HL-60 and U937 cells were treated with 1-beta-D-arabinofuranosylcytosine (Ara-C) or etoposide (VP-16). Whereas TPA strongly reduced apoptosis in Ara-C- or VP-16-treated U937 cells, little if any reduction was observed after pretreatment with either FTT, SC-10, R59022 or R59949, respectively, in these cells. In contrast, TPA enhanced apoptosis in Ara-C- or VP-16-treated HL-60 cells. Interestingly, FTT and SC-10 demonstrated a protective effect in Ara-C-treated HL-60 cells. Taken together, these data suggest that the novel PKC activators FTT, SC-10, R59022 and R59949 exhibit modest biological effects upon leukemic blast cells, and are not capable of enhancing the apoptotic response of these cells to cytotoxic drugs.

Mitochondria-targeting drugs arsenic trioxide and lonidamine bypass the resistance of TPA-differentiated leukemic cells to apoptosis

Exposure of U937 human leukemic cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces their differentiation into monocyte/macrophage-like cells. This terminal differentiation is associated with a resistant phenotype to apoptosis induced by the topoisomerase II inhibitor etoposide. The inhibition occurs upstream of the mitochondrial release of cytochrome c and the activation of procaspase-2, -3, -6, -7, -8, and -9. By using cell-free systems, it was demonstrated that the mitochondrial pathway to cell death that involves mitochondrial membrane depolarization, cytochrome c release and cytosolic activation of procaspases by cytochrome c/dATP remains functional in TPA-differentiated U937 cells. Accordingly, 2 drugs recently shown to target the mitochondria, namely lonidamine and arsenic trioxide, bypass the resistance of TPA-differentiated U937 cells to classical anticancer drugs. Cell death induced by the 2 compounds is associated with mitochondrial membrane depolarization, release of cytochrome c and Smac/Diablo from the mitochondria, activation of caspases, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. Moreover, the decreased glutathione content associated with the differentiation process amplifies the ability of arsenic trioxide to activate the mitochondrial pathway to cell death. Similar results were obtained by comparing undifferentiated and TPA-differentiated human HL60 leukemic cells. These data demonstrate that mitochondria-targeting agents bypass the resistance to classical anticancer drugs induced by TPA-mediated leukemic cell differentiation. (Blood. 2001;97:3931-3940)

Differential response of mature TrkA/p75(NTR) expressing human and pig oligodendrocytes: aging, does it matter?

A differential morphological response of mature oligodendrocytes (OL) isolated from human and pig brains to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and to the nerve growth factor (NGF) was observed. In both cases, OL regenerate their processes; however, the rate and the extension of the process formation of human OL were behind that of pig OL. Presumably, the advanced age of the human tissue in these experiments might have contributed to this decrease in process formation, an effect that was already observed for rat OL [Yong et al. (1991) J Neurosci Res 29:87-99]. The less effectivity of NGF via TrkA, which was immunocytochemically shown in human OL, and of TPA via the protein kinase C (PKC) pathway, may have its common focus on the mitogen-activated protein kinase (MAPK) cascade. In this context, it was noted that only a few studies on aging of mature OL are available. It is conceivable that age-related changes in the properties of OL could be an important factor for their cellular responsiveness during longer lasting demyelinating diseases such as multiple sclerosis. Hence, this review would like to provide a basis for future investigations on the aging of mature OL. The data presently available suggest a preliminary classification of mature OL into three categories.

Expression of PGF(2alpha) receptor mRNA in normal, hyperplastic and neoplastic skin.

Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis was used to determine the level of expression of prostaglandin F(2alpha) (FP) receptor mRNA in various mouse tissues, including normal, hyperplastic and neoplastic mouse epidermis. Steady-state concentrations of FP receptor mRNA were low in normal and hyperplastic epidermis. The response of the epidermis to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was biphasic in that FP receptor mRNA was increased immediately after treatment, followed by a long-lasting down-regulation at later time points. FP receptor mRNA was down-regulated in the majority of papillomas obtained by the mouse skin carcinogenesis initiation-promotion protocol. In carcinomas, FP receptor mRNA expression was similar to that in normal epidermis. The steady-state concentration of FP mRNA was inversely correlated with PGF(2alpha) levels in normal and hyperplastic epidermis and in papillomas, indicating that FP mRNA expression is regulated by this eicosanoid.

Chronic Exposure of LLC-PK1Epithelia to the Phorbol Ester TPA Produces Polyp-like Foci with Leaky Tight Junctions and Altered Protein Kinase C-α Expression and Localization

Acute exposure (up to 4 h) of LLC-PK1epithelial cell sheets to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid decrease of transepithelial electrical resistance (TER) to less than 15% of initial value. As the TPA exposure period is continued by chronically passaging cells in the presence of TPA, TER begins to recover. By 6 weeks of exposure, TER recovers to almost 50% of its initial value, suggesting that tight junctions (TJs) are recovering barrier function even in the continued presence of TPA. Between 6 and 8 weeks, TER then decreases a second time to approximately 20 to 40% of initial values, and TER values remain at this level for at least 18 weeks of exposure. Transepithelial (paracellular) fluxes ofd-mannitol inversely correspond with TER changes. Across chronically treated cell sheets, rates are higher than those across control cell sheets, but lower than those across acutely treated cell sheets. The decrease of TER at 6–8 weeks coincides with the appearance of multilayered, polyp-like foci (PLFs) on the otherwise one cell layer thick epithelium. Electron microscopy shows that the electron-dense dye ruthenium red cannot penetrate across TJs of control cells but passes across all of the TJs of a cell sheet treated acutely with TPA. In chronically treated cultures, ruthenium red penetrates TJs between most cells of PLFs, but not TJs of adjacent morphologically normal epithelium. A clonal subline derived from cells of a PLF (clone PLF-A) is multilayered almost throughout and exhibits ruthenium red penetration across nearly all of its tight junctions, monolayer or multilayer. Acute exposure of control cell sheets to TPA induces activation, translocation, and down-regulation of protein kinase C-α (PKC-α). In chronically TPA-treated and clone PLF-A cells, total PKC-α levels are reduced even further and almost all remaining PKC-α is found in the membrane-associated and Triton-insoluble fractions. Immunofluorescence shows that PKC-α expression is restricted to the PLFs in chronically TPA-treated cells and is more homogeneously distributed in clone PLF-A cultures. In summary, the data show that chronic treatment of epithelial cells with a tumor promoter induces the formation of abnormal cell architecture (PLFs) associated with increased leakiness of TJs and membrane translocation of PKC-α. Recovery of barrier function in portions of chronically TPA-treated cultures does not correlate with up-regulation of PKC-α nor translocation back to the cytosolic compartment.

Effects of 12-O-tetradecanoylphorbol-13-acetate on human papillomavirus type 16-positive keratinocytes at different stages of transformation.

Normal human keratinocytes grown under serum-free conditions can be triggered to differentiate by exposure to serum or to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that TPA treatment of human papillomavirus (HPV) type 16-immortalized cells in culture induced formation of cornified envelopes indicative of squamous differentiation. Concurrent with differentiation, TPA inhibited the expression of HPV 16 E6 and E7 mRNA transcripts. Adaptation of the immortalized cells to growth in serum-containing medium led to the selection of a subpopulation of HPV-transformed cells that was resistant to TPA-induced differentiation. In this cell line, a transient suppression of HPV transcripts was observed at 5 h, whereas in differentiation-resistant, carcinoma-derived lines, TPA had little effect on HPV oncogene expression. c-myc transcripts were suppressed for the duration of exposure to TPA in only the differentiation-competent cells; c-fos and c-jun were transiently induced in all cell lines. Transforming growth factor-alpha mRNAs were also increased approximately eightfold as HPV 16-immortalized cells were induced to differentiate. These results demonstrate that, in HPV 16-immortalized keratinocytes, acquisition of resistance to inducers of squamous differentiation is accompanied by altered regulation of cell growth and gene expression.

Modulation of regulatory and catalytic subunit levels of cAMP-dependent protein kinase A in anterior pituitary cells in response to direct activation of protein kinases A and C or after GnRH stimulation.

We have previously shown that direct activation of protein kinase A (PKA) and protein kinase C (PKC) induced changes in the expression of genes coding for PKA RII beta and C alpha subunit isoforms in cultured anterior pituitary cells, suggesting the possibility of interconnected regulation at this point. To evaluate whether the cell content of PKA protein subunits could be similarly altered, the catalytic (C) and regulatory type I (RI) and type II (RII) subunits were identified by Western blot analysis using specific immunoaffinity-purified antibodies. Activation of PKA by the permeant cyclic adenosine monophosphate (cAMP) analogue 8-Br-cAMP induced a dramatic time- and concentration-dependent decline of C subunit to a residual level that may represent 10-15% of that in untreated cells. The most profound decrease occurred during the first 5 h following treatment with 0.5-2 mM analogue (by 65 +/- 14 and 79 +/- 5%, respectively). Under identical conditions, RII was increased by about 40% at the higher concentrations, while RI increased slightly, but only at low concentrations (below 1 mM 8-Br-cAMP), and then gradually decreased. Exposure of cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) also resulted in decreased levels of the PKA C subunit, however, with a different concentration-dependent profile. In particular, a decline in PKA C was most pronounced (60%) at a low concentration of TPA (10 nM) as compared with the concentrations equal to or above 20 nM (40% decrease). TPA at 10 nM also depressed notably (by 25%) the level of RII subunit, but higher concentrations were essentially ineffective, although a slight and statistically not significant elevation of the cell subunit content was observed as for RI. Simultaneous activation of both PKA and PKC pathways resulted in further depletion of PKA C and an important loss (50%) of RII, a subunit which was enhanced by the activation of either system alone. Finally, gonadotropin-releasing hormone, a neuropeptide that has the potentiality to activate both PKA and PKC signaling in gonadotropes, was able to alter PKA subunit cell content: PKA C was significantly reduced at either a subliminal (0.1 nM) or maximal (10 nM) concentration, whereas RII increased at the low concentration and decreased at the high concentration. In conclusion, these data demonstrate that the pituitary cell contents of RI, RII, and C subunits of PKA are regulated under the activation of PKA itself as well as PKC in a manner that can exhibit further alteration when both systems come simultaneously into play. Changes in the PKA subunit levels in certain cases may correlate with a variation of the mRNAs suggesting multiple control mechanisms, including an alteration of gene expression and changes in subunit degradation, synthesis, and/or turnover. These data, together with those obtained in the presence of gonadotropin-releasing hormone, provide further support for a hormonally induced interplay between PKA and PKC signaling pathways at the crucial level of PKA in the pituitary gland including gonadotropes.

Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.

LONG‐WAVE ULTRAVIOLET RADIATION INDUCES PROTEIN KINASE C IN NORMAL HUMAN KERATINOCYTES

Skin tumor promotion by phorbol ester is believed to be mediated by the phospholipid-dependent ser/thr kinase, protein kinase C (PKC). Long-wave ultraviolet radiation (320-400 nm, UVA), which has also been shown to promote skin tumors, induces elevated levels of PKC in murine fibroblasts, suggesting that UVA may promote the development of basal and squamous cell skin cancers by a mechanism involving PKC. To examine UVA effects on PKC in a model relevant to skin, we maintained normal human epidermal keratinocytes (NHEK) in serum-free medium and exposed the cultured cells to various doses of UVA or to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Fifty minutes after exposure to UVA (5-20 J/cm2), PKC activity was elevated up to three-fold in NHEK cytosolic fractions, and membrane-associated PKC activity was elevated up to two-fold by UVA. The TPA treatment induced a 10-fold increase in membrane-associate PKC activity only. Immunoblot analysis suggested that a UVA-induced increase in PKC protein occurred. Both UVA and TPA reduced the cell number by 50-75% in the first 24-48 h; however, irradiated cultures began to recover at 72 h post-UVA due to an increased proliferative rate beginning after 48 h. Treatment with TPA induced a high level of differentiation as measured by cornified envelope formation. Ultraviolet A irradiation exposure was not followed by increased differentiation. These findings suggest that acute UVA exposure elevates PKC activity in human keratinocytes and may act through PKC to promote actinic skin cancer. The molecular mechanism is like to differ from that of the phorbol esters, however.

Liver tumor promotion: Effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor-?1 expression

Phenobarbital (PB) added to the medium of cultured rat hepatocytes alters epidermal growth factor (EGF) dependent mitogenesis in a biphasic manner; PB concentrations less than 1.5 mM are growth stimulatory but higher concentrations significantly inhibit normal hepatocyte proliferation. In contrast, the growth of putative preneoplastic cells is inhibited less by high concentrations of PB. Mechanistic studies designed to test the ability of PB to alter the early events of EGF signal transduction demonstrate that PB neither competes with EGF for binding to the EGF receptor nor alters EGF-induced receptor down-regulation. However, pretreatment with PB (greater than 1 mM) results in a transient inhibition of EGF binding to hepatocytes. The kinetics of this effect are similar to those obtained when hepatocytes are exposed to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a skin tumor promoter and activator of Ca2+/phospholipid-dependent protein kinase C. However, several observations suggest that distinct mechanisms mediate the responses to these two tumor promoters. First, the inhibitory effects of PB and TPA on EGF binding are additive. Also down-regulation of EGF receptors in response to TPA occurs with hepatocytes, A431 epidermal carcinoma cells, HepG2 hepatoma cells, and rat liver epithelial cells, but only hepatocytes are sensitive to PB. Furthermore, translocation of protein kinase C to the membrane occurs in hepatocytes treated with TPA but not in those treated with PB. The chronic treatment of rats with PB further sensitizes hepatocytes to EGF receptor down-regulation by in vitro PB while desensitizing them to EGF receptor down-regulation by TPA. This latter effect is correlated with a decreased ability of TPA to induce translocation of protein kinase C to the membrane. PB significantly increases the intracellular concentration of TGF-beta 1 in periportal hepatocytes but not in putative preneoplastic cells. TGF-beta 1 may therefore have an important function in regulating early stages of cell cycle progression in proliferating hepatocytes.

Failure of F-Met-Leu-Phe to Induce Chemotaxis in Differentiated Promyelocytic (HL-60) Leukemia Cells

Treatment of HL-60 promyelocytic leukemia cells with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) induced these cells to differentiate toward mature neutrophilic granulocytes (PMN). DbcAMP was found to produce a rapid time- and dose-dependent inhibition of HL-60 cell proliferation, reaching a maximum after 48 hr treatment of the cells with 250-500 microM. This was associated with morphologic alterations consistent with maturing PMN. Using immunofluorescence and flow cytometry, we found that dbcAMP-treated HL-60 cells expressed some, but not all, surface markers characteristic of mature phagocytes. Thus receptors for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP); the monocyte/granulocyte markers My-8, Mo-1, and Leu-15; as well as the monocyte markers Mo-2 and My-4 were identified on the cells. In contrast, dbcAMP-treated cells did not express the PMN-specific antigen DL1.2. We also found that dbcAMP-treated HL-60 cells produced hydrogen peroxide in response to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, the magnitude of this response was significantly less than that observed in mature PMN. In contrast, fMLP-stimulated levels of hydrogen peroxide production were comparable in both cell types. In addition, despite the fact that dbcAMP-treated cells expressed fMLP receptors, they did not exhibit directed migration toward this chemotactic peptide. Taken together, these data suggest that dbcAMP-induced differentiation of HL-60 cells is incomplete.

Abnormal Protein Kinase C Down Regulation and Reduced Substrate Levels in Non-Phorbol Ester-Responsive 3T3-TNR9 Cells

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.

Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells.

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.

Autoregulation of Epstein-Barr virus putative lytic switch gene BZLF1

Expression of the Epstein-Barr virus (EBV) BZLF1 gene in latently infected lymphocytes is sufficient to trigger the viral lytic cycle. As shown in the accompanying report (E. Flemington and S.H. Speck, J. Virol. 64:1217-1226, 1990), the promoter for the BZLF1 gene (Zp) contains two distinct types of elements (ZI and ZII [an AP-1-like domain]) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. Although Zp can be activated with TPA in an EBV-negative Burkitt's lymphoma cell line (Ramos), its activity is considerably lower than in EBV-positive cell lines which can be induced with TPA. Here we show that the protein product of the BZLF1 gene (ZEBRA) can transactivate its own promoter by a mechanism which involves direct binding to a region distinct from the ZI and ZII element. Moreover, we show that this region is composed of two distinct ZEBRA-binding-transactivation domains. Interestingly, these two domains are not homologous, and while one domain (ZIIIA) is similar to previously described ZEBRA-binding domains, the second (ZIIIB) is a higher-affinity site which bears no detectable homology to the consensus ZEBRA recognition sequence. We also show that transactivation is independent of the otherwise essential ZII domain, suggesting that ZEBRA binding may functionally replace or supercede the need for a functional ZII domain. This observation supports a model for activation of the lytic cycle whereby synthesis of a critical level of ZEBRA signals commitment to BZLF1 transcription and initiation of the lytic cascade.

Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.

Vascular responsiveness to phorbol esters in coarctation-hypertensive rats

Recent observations suggest that a phospholipid-sensitive, calcium-dependent protein kinase affects the contractile responses of vascular smooth muscle. Protein kinase C activators such as the tumor-promoting phorbol esters have been used as tools to study protein kinase C function in various intact cells. The present study characterizes vascular reactivity to protein kinase C activation in rats made hypertensive by coarctation of the abdominal aorta. Thoracic aortic strips from hypertensive rats developed greater force than arteries from normotensive rats in response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thoracic aortae from hypertensive rats were more responsive (lower threshold dose) to the phorbol ester than those from normotensive rats. Additionally, arteries from hypertensive rats were more responsive to the contractile effects of mezerein, a non-phorbol ester activator of protein kinase C. Removal of the endothelium did not eliminate the difference in responsiveness to TPA in thoracic aortae from normotensive and hypertensive rats. The threshold dose of TPA in abdominal aortae from hypertensive rats was not different from that in normotensive rats. However, the maximal response to 10(-6) mol/l TPA after 60 min in abdominal aortae from hypertensive rats was significantly less than that in aortae from normotensive rats. Thus, contractile responses to TPA appear to be influenced by arterial pressure per se. The inhibitory effects of the calcium antagonist, verapamil, in thoracic aortae from hypertensive rats were greater than in those from normotensive rats. Verapamil inhibited TPA-induced contractions in abdominal aortae from hypertensive rats to the same extent as in those from normotensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A phorbol ester tolerant (PET) variant of HL-60 promyelocytes.

The promyelocytic leukaemia cell line HL-60 differentiates to a macrophage-like cell when exposed to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and other agents which activate protein kinase C. To investigate this phenomenon we developed an HL-60 variant which does not differentiate when exposed to TPA. HL-60 cells were exposed to the mutagen ethyl methanesulphonate and were cloned in soft agar in the presence of a normally lethal concentration of TPA. One colony of cells that proliferated in TPA was obtained. The cells of this phorbol ester tolerant (PET) line have retained their resistance to TPA for several years without selective pressure. They are somewhat larger than their phorbol ester sensitive (S) parent, but they are otherwise morphologically similar. When PET-cells are exposed to TPA their growth is arrested for approximately 48 h. Thereafter, they resume their original rate of replication at all concentrations of TPA tested. S-cells undergo changes typical of HL-60 when exposed to TPA; they aggregate, stop growing, adhere to the flask and die. The PET-cells appeared to be as sensitive as S-cells to other agents which differentiate HL-60 such as retinoic acid, dimethysulphoxide, and 1,25-dihydroxyvitamin D3, as determined by rate of proliferation in culture, Wright's stain, nitroblue tetrazolium reduction, and induction of the ectoenzyme NAD-glycohydrolase. TPA-induced protein phosphorylation was studied using one- and two-dimensional polyacrylamide gel electrophoresis. Several proteins increased their incorporation of 32P when S- and PET-cells were exposed to TPA, the most prominent of which were the two previously described nuclear matrix proteins of 80 kd and 33 kd. There was no difference in the protein phosphorylation pattern in S- and PET-cells, nor in how this pattern changed on TPA exposure. Fluorescent activated cell sorting and karyotypic analysis revealed PET-cells to be a hypotetraploid variant of S-cells, with approximately 80 chromosomes, including a marker chromosome iso(1p) not found in the S-cells. Identification of the biochemical lesion responsible for this TPA resistance in PET cells will provide clues concerning the mechanism of this important pathway for the induction of cell differentiation.

Concanavalin A prevents phorbol-mediated redistribution of protein kinase C and β-adrenergic receptors in rat glioma C6 cells

Exposure of rat glioma C6 cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an activation of protein kinase C where-in the enzyme rapidly became membrane-bound ( T 1 2 of 15 min). This translocation of protein kinase C from cytosol to membrane was followed by a sequestration of cell surface β-adrenergic receptors and a loss of isoproterenol-stimulated adenylate cyclase activity. We had reported previously that prior exposure of rat glioma cells to concanavalin A prevents the TPA-mediated sequestration of receptors and desensitization of adenylate cyclase (Kassis et al., 1985). We now show that the concanavalin A treatment also prevents the translocation and activation of protein kinase C. These results are further evidence that in the TPA-treated cells, sequestration of β-adrenergic receptors is mediated by membrane-bound protein kinase C.

Voltage-activated Na+ currents and their suppression by phorbol ester in clonal insulin-producing RINm5F cells

The whole-cell configuration of the patch-clamp technique was applied on the clonal insulin-producing cell line RINm5). Thus attempts were made to characterize voltage-activated inward and outward membrane currents and to examine to what extent these were affected by both long-term and acute exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Current responses to voltage-clamp steps up to -40 mV were small. A pulse to -28 mV evoked an inward current, and slowly activating outward currents developed at potentials above -20 mV. The inward current had a V-shaped current-voltage relationship, reaching a peak between -10 and 0 mV, whereas the outward current increased linearly at potentials beyond -20 mV. It was demonstrated that the inward currents are carried primarily by Ca2+ and Na+ and the outward current by K+. After long-term exposure to TPA, there was a suppression of Na+ currents in one-third of the cells, whereas the Ca2+ and K+ currents were unaffected. Acute exposure to the phorbol ester increased the Ca2+ currents with little effect on the Na+ currents. The extent to which the differences in effects on membrane currents initiated by respective acute and long-term exposure to TPA may reflect two separate mechanisms of protein kinase C activation, the latter related to regulation of differentiation of the RINm5F cells, merits further investigation.