Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

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.     

Interleukin-12 induces gene expression in interleukin-2 stimulated human T lymphocytes

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses by inducing the differentiation of Th1 cells. To better clarify the molecular basis of IL-12 action, we compared the gene expression in human T lymphocytes activated by IL-2 and IL-12. mRNAs from T lymphocytes activated by either IL-2 alone or IL-2 plus IL-12 were transcribed into cDNAs. A differential mRNA display was conducted. As a result, differential display of five cDNA fragments was obtained. Sequence analysis suggests that they had high homology with recorded genes as found by a computer search against GenBank. Two full genes of the five fragments were cloned, which activation-induced C-type lectin and glucose transporter-like protein. Interestingly, these proteins were expressed in the T cells stimulated by IL-2 and IL-12, but not in the T cells stimulated by IL-2 alone. These results suggest that C-type lectin and glucose transporter-like protein may play an important role in the T lymphocyte activation induced by IL-12.     

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.     

Immunosuppreession induced by Salmonella infection is correlated with augmentation of interleukin-2 receptor α chain expression in murine splenic lymphocytes

Abstract In a previous study, we observed that the suppression of T-cell proliferation induced by Salmonella cell-free extract was associated with augmentation of IL-2 receptor (IL-2R) α chain expression. In this study, we also observed this kind of augmentation of IL-2Rα in Salmonella -infected mice. Phytohaemagglutinin (PHA)-stimulated proliferation of murine spleen cells was significantly suppressed when the mice were infected with Salmonella typhimurium . However, expression of the α chain but not the β chain of IL-2R in lymphocytes was augmented by the infection. Analysis of the IL-2R-positive cell-populylation showed that the augmentation of IL-2Rα was not specific to certain cell subpopulations. Furthermore, the inhibition of PHA-stimulated murine spleen cell proliferation and the augmentation of IL-2Rα expression induced by the infection in lymphocytes was completely reversed by treatment with anti-interferon-γ monoclonal antibody (anti-IFN-γ Ab). These results suggest that the suppression of T-cell proliferation induced by Salmonella infection was associated with augmentation of IL-2Rα expression in an IFN-γ production-dependent manner in the same way as the suppression of T-cell proliferation induced by Salmonella cell-free extract.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.     

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.     

Induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore: Suppression by inhibitors of fatty acid lipoxygenase

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [³H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5–2 μg/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.     

Regulation of transglutaminase 1 gene expression by 12-O-tetradecanoylphorbol-13-acetate, dexamethasone, and retinoic acid in cultured human keratinocytes.

Transglutaminase 1 (TG1) is an enzyme that is expressed at the late stage of terminal differentiation of keratinocytes and catalyzes the epsilon-(gamma-glutamyl)lysine cross-linking reaction to form a highly insoluble cell envelope. To elucidate the mechanism of TG1 gene expression in keratinocytes, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), dexamethasone, 1,25-dihydroxyvitamin D3, and retinoic acid on the levels of TG1 mRNA in cultured normal human epidermal keratinocytes (NHEK). Treatment of NHEK with TPA, up to 10 nM, markedly increased the levels of TG1 mRNA in a dose-dependent manner. The effect by treatment with 1 nM TPA reached a peak after 16 h of incubation (20-fold above the basal level). In contrast, phorbol had no effect on TG1 gene expression. The induction of TG1 mRNA expression by TPA was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and staurosporine. Dexamethasone at a concentration of 1 microM also increased the TG1 mRNA levels, but the maximum induction was observed (3-fold above the basal level) after 72 h of incubation. The effect of dexamethasone was not suppressed by H-7. Moreover, 1 microM of retinoic acid completely inhibited the induction of TG1 mRNA by both TPA and dexamethasone. 1,25-Dihydroxyvitamin D3 showed no effect on the TG1 mRNA levels. From these results, we suggest that the expression of TG1 gene may be upregulated by protein kinase C and glucocorticoid receptor systems and down-regulated by the retinoic acid receptor system.     

Failure of tumor promoter 12-O-tetradecanoylphorbol-13-acetate to displace estradiol from its specific receptor

A permanent cell line derived from rat endometrium which contains a specific, low capacity, high affinity estrogen binding protein in cytosol and nuclear fractions (estrogen receptor) is available. Extracts of cells from this line did not appreciably bind the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate. The result suggests that the action of the promoter does not involve translocation to the cell nucleus via binding to the specific estrogen receptor.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

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.     

Specific stimulation of histone H2B and H4 phosphorylation in mouse lymphocytes by 12-O-tetradecanoylphorbol 13-acetate

This report demonstrates that the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate rapidly stimulates the phosphorylation of histones H2B and H4 in a cell cycle-independent manner. This effect was observed in primary cultures of BALB/c mouse splenocytes, a population of noncycling, G0 cells which are not stimulated to divide by 12-O-tetradecanoylphorbol 13-acetate treatment alone. The biological nature of this cell system allowed the analysis of histone phosphorylation in the absence of a background of cell cycle-dependent changes and in response to a nonmitogenic agent. The phosphorylation of H2B was determined with high resolution through the use of two-dimensional gel electrophoresis. In contrast to 12-O-tetradecanoylphorbol 13-acetate, the mitogen from pokeweed did not induce stimulation of H2B and H4 phosphorylation, but did, however, elicit increases in the phosphorylation of histones H1, H2A, and H3, in parallel with changes in rate of DNA synthesis.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.     

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.