Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.     

Activation of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase during the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

We previously reported that the synthesis of NeuAc(alpha 2-3)Gal(beta 1-4)GlcCer (GM3) ganglioside was preferentially enhanced during the differentiation of HL-60 cells into a monocyte/macrophage lineage induced by 12-O-tetradecanoylphorbol-13-O-acetate (TPA). Since exogenously added GM3 ganglioside was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage in a synthetic medium, the functional role of the GM3 ganglioside increase during the differentiation of HL-60 cells has become the subject of much interest. In the present study, we investigated the activity of CMP-NeuAc:lactosylceramide sialyltransferase, which catalyzes the synthesis of GM3 ganglioside from lactosylceramide, in cells undergoing differentiation induced by two different reagents, TPA and 1 alpha,25-dihydroxy-vitamin D3, which induce the differentiation of HL-60 cells into the monocyte/macrophage lineage through different modes of action. We showed that the activation of CMP-NeuAc:lactosylceramide sialyltransferase and the increase in GM3 ganglioside were not related to the differentiated lineage but to the specific action of TPA, i.e. activation of protein kinase C.     

Inhibition of adipose conversion of BALB/c 3T3 cells by interferon and 12-O-tetradecanoylphorbol-13-acetate

The adipose conversion of BALB/c 3T3 preadipose cells is inhibited by interferon; this inhibition is directly correlation with the interferon concentration. In cultures treated with low doses of interferon and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, another inhibitor of adipose conversion (Diamond et al., 1977), the two compounds act synergistically to block differentiation. Several lines of evidence suggest that the compounds differ in the mechanism by which they inhibit adipose conversion.     

Induction of interleukin-2 receptor α-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes

Abstract. The high affinity form of interleukin-2 receptor (IL-2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin-2 (IL-2) is controlled by the expression of the IL-2R α-chain. In this study, we examined the effect of cytochalasin B (CB) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of IL-2 and IL-2R. Northern blot and flow cytometric analysis showed that the IL-2R α-chain was expressed both at mRNA and protein levels. However, IL-2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL-2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([³H]dT) in the presence of IL-2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL-2R α-chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL-2R expression in only a subset of T cells.     

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a protein in human lymphocytes binding satellite DNA

Interactions between a satellite DNA fragment (SH3, 1.8 kb) cloned in pBR325 plasmid and DEAE-protein fractions from human lymphocytes treated with phorbol-12-myristate-13-acetate (TPA) have been studied by filtration technique through nitrocellulose filters. The 0.3 M fraction was found to have a protein which binds SH3 DNA specifically. Cytogenetic studies have shown an increased frequency of chromosome endoreduplications which may be due to the binding of TPA-induced protein to centromeres.     

Differential role of PKC-induced c-Jun in HTLV-1 LTR activation by 12-O-tetradecanoylphorbol-13-acetate in different human T-cell lines

We have previously shown that TPA activates HTLV-1 LTR in Jurkat T-cells by inducing the binding of Sp1-p53 complex to the Sp1 site residing within the Ets responsive region 1 (ERR-1) of the LTR and that this activation is inhibited by PKCalpha and PKCepsilon. However, in H9 T-cells TPA has been noted to activate the LTR in two consecutive stages. The first stage is activation is mediated by PKCetta and requires the three 21 bp TRE repeats. The second activation mode resembles that of Jurkat cells, except that it is inhibited by PKCdelta. The present study revealed that the first LTR activation in H9 cells resulted from PKCetta-induced elevation of non-phosphorylated c-Jun which bound to the AP-1 site residing within each TRE. In contrast, this TRE-dependent activation did not occur in Jurkat cells, since there was no elevation of non-phosphorylated c-Jun in these cells. However, we found that PKCalpha and PKCepsilon, in Jurkat cells, and PKCetta and PKCdelta, in H9 cells, increased the level of phosphorylated c-Jun that interacted with the Sp1-p53 complex. This interaction prevented the Sp1-p53 binding to ERR-1 and blocked, thereby, the ERR-1-mediated LTR activation. Therefore, this PKC-inhibited LTR activation started in both cell types after depletion of the relevant PKCs by their downregulation. In view of these variable activating mechanisms we assume that there might be additional undiscovered yet modes of HTLV-1 LTR activation which vary in different cell types. Moreover, in line with this presumption we speculate that in HTLV-1 carriers the LTR of the latent provirus may also be reactivated by different mechanisms that vary between its different host T-lymphocyte subclones. Since this reactivation may initiate the ATL process, understanding of these mechanisms is essential for establishing strategies to block the possibility of reactivating the latent virus as preventive means for ATL development in carriers.     

Activation of protein kinase C by ganglioside GM3 in the presence of calcium and 12-O-tetradecanoylphorbol-13-acetate

Ganglioside GM3 and 12-O-tetradecanoylphorbol-13-acetate activated protein kinase C as substitutes for phosphatidylserine and diacylglycerol. Hydrophobic gangliosides such as GM4 and GM3 were rather more potent activators of protein kinase C than hydrophilic ones such as GD1a and GT1b. Active tumor promoters such as teleocidin, mezerein, phorbol 12,13-acetate and phorbol 12,13-dibenzoate also activated protein kinase C, but not inactive tumor promoters such as phorbol and 4-alpha-phorbol 12,13-didecanoate.     

Regulation of myeloperoxidase gene expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in human leukemia HL-60 cells

Myeloperoxidase synthesis during induction of differentiation of human promyelocytic leukemia HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. Differentiation was characterized by morphological changes, arrest of cell proliferation, development of cell adherence, and increased secretion of lysozyme. The cellular myeloperoxidase activity decreased early during induction of differentiation by TPA. Pulse-labeling experiments indicated that the rate of myeloperoxidase synthesis decreased to an undetectable level in cells exposed to TPA for 22 h. The relative amounts of myeloperoxidase mRNA in TPA-treated and untreated cells were determined by measuring translatable mRNA activity in a reticulocyte lysate system. Reduction in the myeloperoxidase mRNA level was observed as early as after 3 h treatment with TPA, and no myeloperoxidase mRNA was detected after 24 h. Time course experiments indicated that the time required for 50% reduction of myeloperoxidase mRNA in TPA-treated cells was approximately 5 h. These results suggest that TPA induces decrease of myeloperoxidase activity in HL-60 cells at a pretranslational level.     

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.     

Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL)

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.     

Enhancement of aggregation of Chinese hamster V79 cells in rotation culture by 12-O-tetradecanoylphorbol-13-acetate

A potent mouse skin tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), enhanced the increase in the size of aggregates of Chinese hamster V79 C-2 cells cultured in rotation flasks for 24 h. The effective concentrations of TPA were 1-100 ng/ml. Phorbol used as the negative control did not enhance aggregate formation of V79 C-2 cells. When aggregates that had formed in culture with TPA for 24 h were transferred to normal medium and cultured for another 24 h in rotation culture, aggregate size was not markedly enhanced as compared with that in the control culture. These results suggest that some changes produced in the cell surface by TPA remain irreversible on further culture in normal medium. No such difference in aggregate-forming activity was found in aggregates formed with phorbol. Dimethyl sulfoxide (DMSO) as the solvent had no effect at the concentrations used in these experiments.     

Stimulation of virus production and induction of self-syncytium formation in human T-cell leukemia virus type I- and type II-infected T cells by 12-O-tetradecanoylphorbol-13-acetate.

Treatment of human T-cell leukemia virus type I (HTLV-I)- and HTLV-II-infected T-cell lines with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated virus release. However, this stimulation was mainly detected at 42 to 48 h of treatment, whereas later virus release declined rapidly. During the first 48 h, TPA had no effect on cell growth, but later, the number of viable cells was profoundly lower in the TPA-treated than in the untreated cultures. This shift in virus release and cell number resulted from self-fusion of a large proportion of the virus-producing cells, which seemed to consequently enter into a dying process. This fusion, which resulted in syncytium formation, was strongly inhibited by anti-HTLV-I env monoclonal antibodies. Furthermore, no self-fusion was detected in three different uninfected T-cell lines similarly treated with TPA. On the other hand, stimulation of virus production by 3-methylcholanthrene (3-MC) treatment failed to induce self-fusion in the infected cells. Moreover, no syncytium was detected when these 3-MC-treated infected cells were cocultured with any of the TPA-treated uninfected cells. The effects of TPA on virus production and syncytium formation were both abolished by three different protein kinase C inhibitors. Taken together, these data suggest that the self-fusion observed in these experiments required both enhanced virus production and protein kinase C-phosphorylated viral or/and virally induced cellular component(s).     

Modulatory action of 12-O-tetradecanoylphorbol-13-acetate on bud production inHydra

Summary A low concentration of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.0 ng/ml) induced a transient inhibition of bud production in hydra which were fed daily. However, when hydra were starved following TPA-treatment, they produced further buds. Phorbol (1.0 ng/ml) and dimethyl sulfoxide (0.001%) did not influence bud production under either feeding or starvation conditions. These results indicate that TPA modulates asexual reproduction in hydra.     

Lymphocyte activation by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA).

TPA, a highly active tumor-promoting agent, is an effective mitogen for primate peripheral blood lymphocytes. Optimal stimulation of human lymphocytes was obtained 4 days after the addition of TPA at a concentration of 7.5 ng/ml. Lymphocyte fractionation experiments demonstrated that both T and B cells incorporated 3H-thymidine significantly in response to TPA. Lymphocyte blastogenesis was not due to the reactivation of latent herpesviruses by the tumor promoter, since similar responses to TPA were obtained with virus-genome positive or negative cells. Increased levels of DNA synthesis were observed when TPA was added to marmoset, baboon, rhesus monkey, or chimpanzee peripheral blood lymphocytes. Canine peripheral blood lymphocytes and spleen cells from guinea pigs, rats, and mice were not stimulated by TPA. These observations suggest that TPA-induced lymphocyte blastogenesis may be useful for studies of lymphocyte activation and of the molecular mechanisms of action of tumor-promoting phorbol esters.     

Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: Inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Mouse epidermal cells can be subcultured at 31°C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.