Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells.

Western blot analysis indicated that WB cells contained multiple proteins of molecular mass 42-47 K which reacted with anti-connexin 43 antibody. Incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5 min caused the inhibition of dye transfer and the appearance of reactive material of 50 K which was abolished by acid phosphatase. Longer term incubation with TPA caused loss of connexin 43 protein.     

Phorbol ester tumor promoters specifically stimulate choline phospholipid metabolism in human leukemic cells

Human hairy cell leukemia (HCL) cells in culture showed a marked increase in both [1-14C]acetate and [14C]choline incorporation into phosphatidylcholine (PC) when treated with a 10 nM concentration of 12-O-tetradecanoylphorbol 13-acetate (TPA) for 3 h. Dramatic morphological changes occurred and synthesis of most phospholipids was stimulated. However, the most dramatic increase was seen in the [14C]acetate labeling of both long- and short-chain fatty acid-containing sphingomyelins (from 200-425% of control levels), sphingomyelin being especially enriched in HCL cells. Negligible incorporation of [14C]choline into sphingomyelin was observed and phospholipase inhibitor (U10029A) studies indicated that PC was the major source of sphingomyelin choline. These changes were most clearly seen by autoradiography of two-dimensional thin-layer chromatography plates. Chronic myelogenous leukemia (CML) blasts, which did not respond morphologically to TPA, showed no increased phospholipid synthesis under the same conditions and increases in sphingomyelin synthesis were modest. Other non-TPA-responding leukemic cells were similarly refractive. However, one out of four acute monomyelocytic leukemic (AMMoL) cells studied responded morphologically in a manner identical to HCL cells and exhibited the same dramatic increase in sphingomyelin synthesis. Data are presented which suggest that TPA may also stimulate PC phospholipase C activity in addition to activating the calcium-dependent protein kinase by mimicking diacylglycerol.     

Phorbol ester tumor promoters induce epidermal transglutaminase activity

Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.     

Phorbol ester induces diaphragmed fenestrae in large vessel endothelium in vitro

The endothelium of visceral capillaries is fenestrated, whereas that of large vessels is not. We show here that the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate (PMA), triggers formation of diaphragmed fenestrae in endothelial cells cultured from the calf pulmonary artery and the human umbilical vein. This demonstrates that large vessel endothelial cells have the potential to express, in response to exogenous signals, a structural specialization uniquely associated with the endothelium of visceral capillaries in vivo.     

Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA.

We report the unexpected result that Escherichia coli isolates containing a multicopy plasmid (pDT1.5) carrying the manganese-superoxide dismutase gene sodA were more sensitive than the wild type to paraquat-mediated growth inhibition. The pDT1.5 locus responsible for the paraquat-sensitive phenotype was delimited to a 0.6-kilobase segment by transposon Tn5 mutagenesis. Moreover, superoxide dismutase activity was the same as in the wild type in strains carrying pDT1.5::Tn5 insertions mapping to the 0.6-kilobase locus. These data identify the 0.6-kilobase segment as the locus of sodA and establish an association between growth inhibition by paraquat and the function of the plasmid-borne sodA gene.     

Insulin antiapoptotic signaling involves insulin activation of the nuclear factor kappaB-dependent survival genes encoding tumor necrosis factor receptor-associated factor 2 and manganese-superoxide dismutase.

We recently showed that the antiapoptotic function of insulin requires nuclear factor kappaB (NF-kappaB) activation (Bertrand, F., Atfi, A., Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., and Cherqui, G. (1998) J. Biol. Chem. 273, 2931-2938). Here we sought to identify the NF-kappaB-dependent survival genes that are activated by insulin to mediate this function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-kappaB activation since it was abolished by overexpression of dominant-negative IkappaB-alpha(A32/36) and mimicked by overexpression of the NF-kappaB c-Rel subunit. TRAF2 contributed to insulin protection against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression of dominant-negative TRAF2-(87-501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased basal and insulin-stimulated NF-kappaB activities. All effects were inhibited by IkappaB-alpha(A32/36), suggesting that an amplification loop involving TRAF2 activation of NF-kappaB is implicated in insulin antiapoptotic signaling. We also show that insulin increased manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-kappaB activation and that Mn-SOD contributed to insulin antiapoptotic signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin activates the NF-kappaB-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-kappaB in the antiapoptotic function of insulin.     

Phorbol ester induces tyrosine phosphorylation in normal and abnormal human B lymphocytes

Tumor-promoting phorbol esters have been found to bind and activate phospholipid/Ca2+-dependent or C-kinase, and several of their effects, including proliferative responses in lymphocytes, have been assumed to be related to activity of this enzyme. However, phorbol esters have also recently been found to stimulate tyrosine phosphorylation in certain other cell types, and we therefore studied tyrosine kinase activity in normal and chronic lymphocytic leukemia (CLL) peripheral blood B lymphocytes stimulated with phorbol ester. High levels of tyrosine labeling were observed in unstimulated cells with major endogenous substrates of 75K, 66K, 43K, and 28K in Triton-soluble material, and of 56K to 61K in Triton-insoluble material; this profile was essentially similar in normal and CLL B cells. Treatment with phorbol ester for time periods varying from 20 min to 48 hr led to qualitative increases in tyrosine labeling of these phosphoproteins, as measured both in vitro and in intact cells "in vivo." Although the relative abundance of tyrosine phosphorylation as a percentage of total labeling was variable due to concomitant enhancement of serine and threonine phosphorylation, exogenous peptide substrate assays confirmed the increased tyrosine kinase activity quantitatively. Enhanced tyrosine phosphorylation was succeeded or accompanied in both normal and abnormal B cells by cellular activation, as judged by increased [3H]thymidine uptake, and terminal differentiation of CLL cells. These findings provide further evidence implicating tyrosine kinases in B lymphocyte activation.     

Phorbol ester induces loss of VIP stimulation of adenylate cyclase and VIP-binding sites in HT29 cells

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected by PMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.     

Induction of manganese-superoxide dismutase by membrane-binding drugs in Escherichia coli.

Treatment of exponentially growing cells of Escherichia coli with membrane-binding drugs such as chlorpromazine (CPZ) and procaine resulted in an induction of manganese-superoxide dismutase (Mn-SOD). A slight decrease was observed in the amount of Fe-SOD. The induction of Mn-SOD required de novo synthesis of this enzyme, since it was suppressed by rifampin. The treatment did not cause the induction of Mn-SOD when performed under anaerobic conditions. In E. coli cells with a sodA-lacZ operon fusion, CPZ and procaine induced beta-galactosidase in the presence of oxygen, whereas it was not expressed and was not induced by CPZ and procaine under anaerobic conditions. Although CPZ reduced the ability of cell suspensions to take up oxygen, it increased the cyanide-resistant fraction of the total respiration. Therefore, it appeared likely that the induction of the sodA gene was a response to an increase in superoxide radical production mediated by these membrane-binding drugs in E. coli cells, possibly by disruption of the electron transport systems in the cell membranes.     

Phorbol ester induces inhibition of arachidonate incorporation into phospholipids in human neutrophils

PMA is known to enhance calcium ionophore A-23187 induced arachidonate release in human neutrophils. Mechanism of enhancement by PMA is not clear. We have found that neutrophils pretreated with PMA showed significant reduction in labeled arachidonate uptake. Decrease in arachidonate uptake following PMA treatment was attributed, at least in part, to inactivation of arachidonoyl CoA synthase and arachidonoyl CoA lysophosphatide acyltransferase, two key enzymes involved in arachidonate incorporation into phospholipids. These results suggest that PMA may induce protein kinase C activation which in turn may cause inactivation of the two enzymes involved in incorporation of arachidonate resulting in greater availability of arachidonate which is liberated by A-23187 for oxygenation and release into extracellular space. Abbreviations: PMA, 4 beta-phorbol 12-myristate 13-acetate; PDD, 4 alpha-phorbol 12,13-didecanoate; TXB2, thromboxane B2; LTB4, leukotriene B4; PC, phosphatidylcholine; LPC, lysophosphatidylcholine; DMSO, dimethylsulfoxide.     

Induction of manganese-superoxide dismutase in MRC-5 cells persistently infected with an alphavirus, sindbis.

Sindbis virus (SV), a single-stranded positive-sense RNA virus, multiplies in a variety of cells and causes various outcomes of infection. As we described acute infection of SV induces stress response of small heat shock protein HSP27 and activation of mitogen-activated protein kinase (MAP) signaling pathway (Nakatsue, T., et al., Biochem. Biophys. Res. Commun. 253, 59-64, 1998). In contrast to lytic infection in Vero cells, MRC-5 cells, a human fetus lung cell line, resulted in persistent infection by SV. Here we investigated a cellular factor involved in persistent infection of MRC-5 cells infected with SV. Partial sequence analysis of a 25 kilodalton (kDa) protein, accumulated in large amounts in the cells, showed that manganese-superoxide dismutase (Mn-SOD) was induced during the infections. When Mn-SOD was overexpressed in Vero cells, 20% of the cells survived more than one month, in contrast with the death of 99% of the vehicle-transfected Vero cells at 48 h after infection with SV. These data strongly suggest that a cellular factor which regulates the oxidative pathway modulates the outcome of SV infection.     

Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors

We have previously shown that the tumor promoter 4 beta-phorbol 12-myristate 13-acetate (PMA) induces capillary endothelial cells grown to confluency on the surface of three-dimensional collagen gels to invade the underlying matrix and to form capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell, 42:469-477, 1985). Since angiogenesis frequently occurs within a fibrin-rich extracellular matrix, we have examined the ability of PMA-treated endothelial cells to invade fibrin gels. Control endothelial cells grown on fibrin gels formed a confluent monolayer on the gel surface and did not invade the underlying matrix. Treatment of the cultures with PMA resulted in a progressive lysis of the substrate without invasion of the fibrin matrix. However, if the cells were treated with PMA either in the presence of fibrinolytic inhibitors (Trasylol, epsilon-aminocaproic acid) or in the absence of detectable plasminogen, dissolution of the substrate was prevented, and the endothelial cells invaded the fibrin gel, forming vessel-like tubular structures similar to those previously observed with collagen gels. These results demonstrate that the invasive and morphogenetic events induced by PMA do not necessarily require an interaction between endothelial cells and collagen fibrils but can also occur with other biologically relevant substrata. They also suggest (1) that invasion may occur via a plasmin-independent mechanism and (2) that in vivo, neutralization of excess proteolytic activity may play an important permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.     

Localization of urokinase-type plasminogen activator receptor on U937 cells: Phorbol ester PMA induces heterogeneity

The binding of human urokinase-type plasminogen activator (u-PA) to the surface of the human monocytic cell line U937 was studied by immunological detection of bound u-PA or binding of biotinylated diisopropyl fluorophosphate-inactivated human u-PA visualized by light or electron microscopy. Untreated U937 cells showed a characteristic binding pattern, with the majority of the u-PA bound to the microvillar-containing protruding pole of the cells. After treatment with the phorbol ester PMA, the resulting adherent cell population was very heterogeneous with respect to both cellular morphology and u-PA binding. The bound u-PA was distributed on both the dorsal and the substrate side of the cells, and the patches of bound u-PA could not be correlated to any typical membrane conformations or cell-cell or cell-substratum contacts. When a monoclonal antibody directed against the amino-terminal fragment (ATF) of u-PA was used, the results were identical regardless of whether intact u-PA or ATF was used for binding to the cells. In contrast, when a monoclonal antibody recognizing the non-receptor-binding protease domain of u-PA was used, bound ATF showed no staining, while bound intact u-PA was stained as efficiently as above. The alteration of u-PA receptor distribution following treatment with PMA could be related to the changes in glycosylation and ligand affinity of the purified u-PA receptor previously described following PMA treatment of U937 cells.     

Reduced expression of manganese superoxide dismutase in cells resistant to cytolysis by tumor necrosis factor.

Tumor necrosis factor (TNF) induces synthesis of manganese superoxide dismutase (MnSOD). It was previously shown that overexpression of MnSOD protected some mammalian cells from TNF cytotoxicity. The purpose of this study was to establish whether MnSOD was increased in cells selected for resistance to cytolysis by TNF in combination with cycloheximide. Melanoma SK-MEL-109 and HeLa cell-resistant variants were selected by repeated treatments with TNF and cycloheximide. The SK-MEL-109 variants had relatively low levels of MnSOD that were inducible by TNF. Surprisingly, the HeLa variants had very low levels of MnSOD that were poorly inducible by either TNF or interleukin-1 alpha. Therefore, an elevated level of MnSOD was not required to protect these cells from TNF-mediated cytolysis. The HeLa variants were more sensitive than parental cells to superoxide radical (O2-) generating compounds, such as paraquat or xanthine/xanthine oxidase. Pretreatment of these variants with TNF did not provide protection against damage by superoxide radicals.     

Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.     

Phorbol ester tumor promoter modulation of alloantigen-specific T lymphocyte responses

The phorbol diester tumor promoter PMA was studied for its effects on T lymphocyte activation in allogeneic human mixed lymphocyte cultures. Lymphocyte proliferation was significantly enhanced in cultures with PMA at concentrations greater than or equal to 12.5 ng/ml, and interferon-gamma production was synergistically increased in cultures with PMA at concentrations greater than or equal to 5 ng/ml. In contrast, the generation of HLA-specific cytotoxic T lymphocyte activity was completely inhibited in cultures with PMA at concentrations greater than or equal to 5 ng/ml. Fluorescence-activated cell sorter analysis with OKT mouse monoclonal antibodies indicated these PMA effects on lymphocyte activation are associated with an increase in the number of activated OKT4-positive T lymphocytes.     

Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells.

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.     

Phorbol ester induces differential membrane-association of protein kinase C subspecies in human platelets

Human platelets contained proteins which cross-reacted with antisera specific for brain protein kinase C-alpha and -beta. When platelets were incubated with 12-O-tetradecanoylphorbol-13-acetate there was a rapid accumulation of protein kinase C-alpha in the particulate fraction associated with a loss of this subspecies from the soluble fraction. No particulate accumulation or soluble loss of protein kinase C-beta could be detected when platelets were incubated with the phorbol ester.