Modulation of human T-cell differentiation markers by 12-O-tetradecanoylphorbol-13-acetate

The analysis of seven differentiation markers following incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined in the human leukemic T-cell line MOLT-3. Significant changes were observed in the activity of the markers terminal deoxynucleotidyl transferase (TdT). spontaneous proliferation and the ability of these cells to bind sheep erythrocytes. Levels of human thymus-leukemia-associated antigen (HThy-L) recently identified as a low molecular weight form of adenosine deaminase (ADA), were reduced by about 50%. No significant changes were observed in ecto-5'-nucleotidase [5'-NT) activities, in the proliferative response to PHA, or in the expression of IA-like antigens. These data and the time kinetics of the changes suggest that following incubation of these T-lymphoblasts with TPA there is a sequential loss of TdT, loss of the capacity for spontaneous proliferation, and the appearance of receptors for sheep erythrocytes. Subsequently there is a decrease in the level of HThy-L/ADA. This sequence appears to follow that proposed for prethymic precursor T-cell differentiation following activation with thymic epithelium.     

Growth of tumor colonies mediated by phorbol ester and 13-cis retinoic acid in mixed cultures

The aim of the study was to investigate the influence of the substances modifying cell phenotype (phorbol ester and 13-cis retinoic acid) on the growth of mouse neuroblastoma N2a cells in the mixed culture system. The results were compared with monoculture system and with tumor multicellular spheroids system where neoplastic cells are cultivated in the close contact from all sides. The suppression of the pleyotypic effect of phorbol ester was observed when N2a cells were cocultured with 3T3 fibroblasts. On the contrary, the close contact of neoplastic cells to each other (spheroids) do not change the stimulatory effect of phorbol ester on tumor cells proliferation. 13-cis retinoic acid suppressed neoplastic cells growth in all cell culture systems we used.     

Ultrastructural studies on cell fusion induced by Epstein-Barr virus or N-butyrate and 12-O-tetradecanoylphorbol-13-acetate

Summary The morphological changes which occur in Raji cells during EBV induced fusion are described. Of particular interest is the formation of local contacts between cells, at these points the plasmalemmae of the two cells become disorganized and cytoplasmic bridges are formed.With 3 Figures     

Pharmacological modification of 12-O-tetradecanoylphorbol-13-acetate induced inflammation and epidermal cell proliferation in mouse skin

Topical application of TPA to mouse skin causes oedema (2–6 h) neutrophil influx (3–24 h) and epidermal cell proliferation (24–48 h). Topical application of a cyclooxygenase inhibitor (indomethacin) dual cyclooxygenase and lipoxygenase inhibitors (phenidone and BW 755C) a selective lipoxygenase inhibitor (AA 861), protein synthesis inhibitors (cycloheximide and actinomycin D) or a glucocorticosteroid (prednisolone) inhibited oedema and neutrophil influx. Systemic administration of an inhibitor of microtubule assembly (colchicine) also prevented neutrophil influx and oedema. These results suggest that the inflammatory response to TPA depends on an interaction between a protein and products of arachidonic acid metabolism to produce a neutrophil dependent oedema. Epidermal cell proliferation was inhibited by topical administration of prednisolone, indomethacin, BW 755C and cycloheximide but not systemically administered methotrexate. This suggests that inhibition of the early inflammatory response to TPA prevents the subsequent epidermal proliferation.     

Cell differentiation and cell cycle effects on human promyelocytic leukemia cells induced by 12-O-tetradecanoylphorbol-13-acetate

As has been reported, doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) as small as 1 to 100 ng/ml induced human promyelocytic leukemia HL-60 cells to differentiate terminally into macrophage-like cells rather than toward cells of the granulocytic series. This differentiation was accompanied by the appearance of monocyte/macrophage markers and by the disappearance of myeloid markers from the view point of enzyme cytochemistry. Contrasted to untreated HL-60 cells, TPA-treated cells increased in cell size and showed increased phagocytotic activities against opsonized sheep blood red cells and activated yeast. Nitroblue tetrazolium-positive cells increased rapidly after TPA exposure. The alterations of the cell cycle traverse of HL-60 cells by TPA were analyzed by [3H]thymidine autoradiography, flow microfluorimetry, and mitotic cell counting. TPA sequentially caused (a) inhibition of cells to move from G1 to S near G1/S boundary in G1; (b) temporary inhibition in G2; (c) growth arrest of most cells in G1 within 2 to 3 days after TPA exposure.     

Activation of T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate is resistant to inhibition by cyclosporin A

Cultured T lymphocytes from pig blood can be activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Activation is additive with that induced by the mitogenic lectin phytohaemagglutinin (PHA). Activation by TPA differs from that induced by PHA or other mitogenic lectins in that it is not inhibited even by high concentrations of the immunosuppressive drug cyclosporin A (CS-A). Neither co-culture of lymphocytes with PHA and TPA nor addition of culture supernatants from TPA-stimulated cultures affected the sensitivity to CS-A of the response to PHA.     

Identification by monoclonal antibodies of T-cell subpopulations activated by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Two major T-cell subpopulations obtained from human peripheral blood were studied their capacity to be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). The subpopulations were identified and separated from each other by their reactivity either to the monoclonal antibody, OKT4 or OKT8, followed by complement-dependent lysis. The fraction enriched in T8+ cells showed approximately a 1.2-2.0-fold greater proliferative response to TPA (20 ng/ml) than did the T4+ cell subset. Optimal activation required the presence of monocytes. Comparison of the mitotic activity showed that the sum of each T-cell subset was less than a mixture of the two. This indicates either a unilateral or cooperative interaction of mitogenesis in the presence of TPA.     

Induction of suppressor macrophages by 12-O-tetradecanoylphorbol-13-acetate in phorbol ester-sensitive (SENCAR) and resistant (B6C3F1) mice

Following topical application of 8 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) twice in one week, the ability of splenic macrophages (M phi s) isolated from phorbol ester-sensitive (SENCAR) and resistant (B6C3F1) mice to suppress the phytohemagglutinin (PHA)-induced lymphocyte blastogenesis and NK activity mediated by spleen cells from naive animals was determined. In B6C3F1 mice, suppression of lectin-induced lymphocyte blastogenesis was mediated by M phi s from TPA-dosed animals. Alternatively, in TPA-dosed SENCAR mice, induction of M phi s suppressive to lectin responses was not apparent. In addition, suppressor M phi s did not mediate the decreased splenic natural killer (NK) activity that is characteristically observed in TPA-dosed SENCAR mice. Therefore, it is proposed that the decreased PHA responsiveness and NK activity observed in vivo in TPA-dosed SENCAR mice may be the result of a decreased proportion of lectin-responding T cells and NK cells in the spleen as a result of proliferation of inflammatory cell precursors.     

Suppression of the in vitro specific antibody response by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the mouse

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has several effects on the immune system. The effect of TPA on the in vitro antibody response to sheep red blood cells (sRBC) was studied. Spleen cells treated in vitro with TPA resulted in a dose-dependent inhibition of the IgM antibody forming cell (AFC) response with an IC50 of 0.74 nM. Nontumor promoting analogs at concentrations up to 1000 nM had no effect on this system. 2-Mercaptoethanol (2ME), which is known to augment macrophage function in this assay, did not reverse the inhibition. Spleen cells were separated into plastic adherent cells (ADC) and nonadherent cells (NAC). When NAC were treated with TPA and cultured with untreated ADC, the inhibition was seen. However, treating the ADC alone did not result in an inhibition. These results show that TPA will suppress the in vitro antibody response in the mouse and that the suppression is due to a selective effect on the plastic nonadherent cell population. The study of these effects of TPA may allow us to gain insight into the mechanism of its tumor promoting activity and to use it as a tool to study the modulation of the immune system.     

Effects of antiinflammatory agents on edema and DNA synthesis induced by 12-O-tetradecanoylphorbol-13-acetate in the guinea pig

Inflammation and hyperplasia are frequently associated in skin diseases. In order to verify this relationship, we studied the antagonistic effect of different classes of antiinflammatory agents on the inflammatory and hyperplasiogenic responses elicited by one topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the ear of the guinea-pig. Edema and DNA synthesis were chosen as relevant parameters. All antiinflammatory agents tested significantly inhibited DNA synthesis induced by TPA. Moreover, all compounds except quinacrine and phenylbutazone also inhibited edema formation. In conclusion, our results demonstrate that while edema and hyperplasia are frequently associated, this is not always the case.Chargé de Recherche INSERM     

The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate,enhances the evoked quanta release of acetylcholine at the frog neuromuscular junction

The possible role of protein kinase C in the regulation of quantal transmitter release was studied at the frog neuromuscular junction by using the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a compound known to mimic the effects of the physiological activator of the enzyme, endogenous diacylglycerol. The main effect of the phorbol ester was to increase the quantal content,m, of the endplate potential. The initial values ofm were adjusted over a wide range by changing the Ca²⁺ concentration of the extracellular medium, and the TPA-induced fractional increase inm was significantly greater at junctions with a lower initial quantal content. On the other hand, the absolute increases inm induced by the phorbol ester were positively correlated with the square root of the initial quantal content. The possible physiological significance of this correlation is discussed in view of the well known relationship between extracellular Ca²⁺ concentration and the quantal content of the end plate potential.     

Influence of insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) on influenza virus multiplication

Insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) interfere with the multiplication of fowl plague virus, an influenza A virus, in primary chick embryo cells. Specifically the production of the viral glycoproteins hemagglutinin and neuraminidase are affected by the drugs. A decrease or omission of glucose from the culture medium enhances this effect, which is in agreement with the idea that these drugs act on virus replication via a shortage of glucose in the host cell. Virus replication in cells of different organs is affected to different extents by insulin and TPA.     

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on axonal elongation and fasciculation

Summary Ultrastructural examination of neurons treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) confirmed our previous finding that TPA promoted neurite differentiation. At the low concentration of 16 nM TPA, the outgrowth of long neurites was correlated with the increased appearance of membrane-filled varicosities and filopodial extensions along the axons. In contrast, treatment with high concentrations of TPA (160 nM) produced dense outgrowths which were shorter in length and organized as thick fascicles. Increased neurite fasciculation appeared to result from the enhanced side-to-side interactions of neighboring neurites by a neural cell adhesion molecule. Axons within these fascicles were retracted and appeared congested with cytoskeletal and membranous components. Treatment with the antibody to the neural cell adhesion molecule defasciculated the thick outgrowths and permitted further axonal elongation.     

Skin carcinomas and micronucleus induction in epidermal keratinocytes following 12-O-tetradecanoylphorbol-13-acetate and mezerein treatment.

Phorbol esters are chemical compounds which have the ability to promote carcinogenesis in the skin of mice. In the HRA/Skh mouse strain, an initiation-promotion assay substantiated evidence that mezerein acts as a complete promoter. Complete promotion with 9.0 nmol mezerein induced a total of 53 papillomas in 28 mice within 40 weeks, while 1.5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA), using the same application regime, resulted in a total of 298 papillomas in 58 mice. However, 7.6% of the papillomas in the mezerein group progressed to carcinomas, compared with 0.7% in the TPA group. Approximately the same molar concentrations of TPA and mezerein induced a dose-dependent and reproducible increase in micronuclei in supra-basal cells derived from the epidermis of treated mice. In contrast, the latter compound was less toxic to these cells and induced higher numbers of micronuclei at the higher concentrations.     

Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C

We have previously demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) activates human T-cell leukemia virus type-I long terminal repeat (LTR) in Jurkat cells by a protein kinase C (PKC)-independent mechanism involving a posttranslational activation of Sp1 binding to an Sp1 site located within the Ets responsive region-1 (ERR-1). By employing the PKC inhibitor, bisindolylmaleimide I and cotransfecting the reporter LTR construct with a vector expressing PKC-alpha, we demonstrated, in the present study, that this effect of TPA was not only independent of, but actually antagonized by, PKC. Electrophoretic mobility shift assays together with antibody-mediated supershift and immuno-coprecipitation analyses, revealed that the posttranslational activation of Sp1 was exerted by inducing the formation of Sp1-p53 heterocomplex capable of binding to the Sp1 site in ERR-1. Furthermore, we demonstrated that Jurkat cells contain both wild-type (w.t.) and mutant forms of p53 and we detected both of them in this complex at variable combinations; some molecules of the complex contained either the w.t. or the mutant p53 separately, whereas others contained the two of them together. Finally, we showed that the Sp1-p53 complexes could bind also to an Sp1 site present in the promoter of another gene such as the cyclin-dependent kinase inhibitor p21(WAF-1), but not to consensus recognition sequences of the w.t. p53. Therefore, we speculate that there might be several other PKC-independent biological effects of TPA which result from interaction of such Sp1-p53 complexes with Sp1 recognition sites residing in the promoters of a wide variety of cellular and viral genes.     

Enhanced expression of human Ia antigens by chronic lymphocytic leukemia cells following treatment with 12-O-tetradecanoylphorbol-13-acetate

Using a panel of monoclonal antibodies which detect framework determinants of human-Ia à and β polypeptides and the DC-specific monoclonal antibody Genox 353, the in vitro effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of Ia antigens by cells from chronic lymphocytic leukemia (CLL) patients have been studied. In all subjects studied a great increase in expression of framework Ia determinants was found following culture of CLL cells for 90 h with TPA at 10^?7 g/ml. TPA treatment of CLL cells also induced increased expression of Genox 353⁺ antigens, in some cases where Genox 353 binding was either negligible or not detectable in cells cultured in the absence of TPA.     

The effect of 12-O-tetradecanoylphorbol-13-acetate on hormone-induced differentiation of rat parotid gland in organ culture

Parotid glands of 5-day-old rats were maintained in DM-153 synthetic, serum-free medium in organ culture for 24 to 48 hr. Differentiation of acinar cells in vitro, as revealed by an increase in amylase activity in the gland and in the culture fluid, as well as by the immunocytochemical demonstration of amylase in the acinar cells, was accelerated by insulin, prednisolone, and l-thyroxine added to the culture medium. The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (10^?7M) inhibited both the unstimulated and hormone-stimulated differentiation of the gland without causing cellular degeneration or necrosis.     

Morphological changes and neural cell adhesion molecule expression in mouse cerebrum primary cultures following long-term exposure to phorbol ester

The effect of phorbol 12-myristate 13-acetate (PMA) on development of brain cells in culture was investigated. Chronic treatment of brain cells from fetal ICR mouse with PMA resulted in a time-dependent loss of specific [3H]phorbol 12,13-dibutyrate binding, generally called 'down-regulation'. In contrast to control culture, neurons cultured in PMA (162 nM)-containing medium exhibited aggregation and neurite fasciculation, and then the neural cell adhesion molecule (N-CAM) was expressed selectively on neurons. Also, the number of astrocytes, which were positively stained with anti-glial fibrillary acidic protein antibody, decreased by treatment with PMA. These results suggest that the normal development of cultured brain cells is interrupted by PMA treatment, which may involve protein kinase C (PKC) in the differentiation of neural cells and cell-cell (neuron-neuron, neuron-astrocyte etc.) interaction as PKC is believed to be a receptor for PMA and is down-regulated by chronic treatment with PMA.     

On the role of hydroxyl radical and the effect of tetrandrine on nuclear factor--kappaB activation by phorbol 12-myristate 13-acetate

Nuclear factor kappaB (NF-kappaB) is considered to be an important target for therapeutic intervention because of its role in the regulation of proinflammatory and profibrotic mediators. The present study examined the role of hydroxyl (*OH) radical and the effect of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Stephania tetrandra, on NF-kappaB activation by a tumor promoter, phorbol 12-myristate 13-acetate (PMA) in human lymphoid T cells (ie, Jurkat cells). Exogenous superoxide dismutase (SOD) enhanced the NF-kappaB activation by PMA, while catalase blocked it. Formate, a scavenger of *OH radical, also was inhibitory, as was deferoxamine, a metal chelator. These data suggest an important role of *OH radical in PMA-induced NF-kappaB activation. Incubation of the cells with tetrandrine prior to the stimulation of the cells was found to inhibit PMA-induced NF-kappaB activation. Tetrandrine activity was so potent that 50 microM of tetrandrine was sufficient to inhibit activation of NF-kappaB completely. Electron spin resonance (ESR) spin trapping was used to investigate the antioxidant action of tetrandrine using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Tetrandrine is an antioxidant for both *OH and superoxide (O2-)radicals. The reaction rate constant of tetrandrine with *OH is 1.4 x 10(10) M(-1)sec(-1), which is comparable with several well established antioxidants, such as ascorbate, glutathione, and cysteine. The Fenton reaction (Fe(II) + H2O2-->Fe(III) + *OH + OH-) and xanthine/xanthine oxidase were used as sources of *OH and O2- radicals. The free radical scavenging activity of tetrandrine is responsible for its inhibition of PMA-induced NF-kappaB activation.