Regulation of activin beta A mRNA level by cAMP.

We demonstrated the presence of five species of the activin beta A mRNA in human placenta and one major RNA associated with two minor RNAs of the activin in the fetal membrane. We investigated the effect of 8-bromo-cAMP (8-Br-cAMP) on accumulation of activin beta A subunit mRNA in human fibrosarcoma HT1080 cells. Although low levels of the activin mRNA were detectable in the untreated cells, the one main RNA species was predominantly accumulated by 8-Br-cAMP. We propose that generation of multiple activin mRNAs in the fetal membrane and cAMP-treated HT1080 cells is presumably due to a cell-specific alternative polyadenylation.     

Inducible gene expression of activin A/erythroid differentiation factor in HL-60 cells.

Treatment of HL-60 cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA) for 48 h induced expression of mRNA of beta A chain of activin A/erythroid differentiation factor. Under the same condition, interferon-gamma caused a slight increase in beta A chain mRNA, whereas 1 alpha, 25-dihydroxyvitamin D3, dimethylsulfoxide and all-trans-retinoic acid failed to induce this mRNA in HL-60 cells. Furthermore, 4 h-treatment with TPA or lipopolysaccharide (LPS) induced a marked increase in beta A chain mRNA levels in interferon-gamma-pretreated HL-60 cells. In the cells pretreated with 1 alpha, 25-dihydroxyvitamin D3, TPA and LPS induced as little increase in beta A chain mRNA as in the control cells. Neither alpha nor beta B chain mRNA was detected in any sample. These results indicate that interferon-gamma has a priming effect on the activation of activin A/erythroid differentiation factor gene by TPA or LPS in HL-60 cells.     

Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos

Mesoderm development in Xenopus laevis depends on inductive cell interactions mediated by diffusible molecules. The mesoderm inducer activin is capable of redirecting the development of animal explants both morphologically and biochemically. We have studied the induction of four regulatory genes, Mix. 1, goosecoid (gsc), Xlim-1 and Xbra in such explants by activin, and the influence of other factors on this induction. Activin induction of gsc is strongly enhanced by dorsalization of the embryo by LiCl, while expression of the other genes is only slightly enhanced. The protein synthesis inhibitor cycloheximide (CHX) inhibits the activin-dependent induction of Xbra partially, while induction of Mix. 1 and Xlim- 1 is essentially unaffected. In contrast, gsc shows strong superinduction in the presence of activin and CHX, and can be induced in animal explants by CHX alone. Induction and superinduction by CHX have previously been observed for immediate early genes in a variety of systems, notably for the activation of c-fos expression by serum stimulation, but have not been reported in early amphibian embryos. © 1993Wiley-Liss, Inc.     

Biphasic effects of dibutyryl cyclic adenosine 3',5'-monophosphate on synergistic stimulation of DNA synthesis by diacylglycerol, and the ionophore A23187 in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetylglycerol (OAG) and the ionophore A23187 for 8 h, [3H]-thymidine incorporation into the acid-insoluble fraction of the cells was stimulated synergistically. Further addition of dibutyryl cAMP caused a biphasic effect on the synergistic stimulation. Dibutyryl cAMP augmented the synergistic stimulation when A23187 was at the concentration of 0.075 micrograms/ml, but inhibited it when the ionophore was at 0.25 micrograms/ml. At the higher concentration of A23187, dibutyryl cAMP stimulated the [3H]thymidine incorporation when culture was for 4 h, but inhibited it when culture was for 8 h. The results were the same when 12-0-tetradecanoylphorbol-13-acetate (TPA) was used instead of OAG. Butyrate could replace dibutyryl cAMP for stimulation of [3H]thymidine incorporation in combination with TPA and A23187, but not with OAG and A23187 at the lower ionophore concentration. Dibutyryl cAMP but not butyrate stimulated ornithine decarboxylase induction caused by TPA and A23187. These results suggest that the effect of dibutyryl cAMP on DNA synthesis induced by OAG and A23187 was biphasic and depended on the concentration of A23187 and on the time of culture, and that the stimulation mechanism of butyrate is different from that of dibutyryl cAMP.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Phorbol ester induces manganese-superoxide dismutase in tumor necrosis factor-resistant cells.

The effects of phorbol ester (TPA) and other known stimulators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide on induction of mRNA for manganese-superoxide dismutase (Mn-SOD) were investigated in various cell lines. TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines including HeLa cells, in which the other reagents also induced expression of the gene, but did not affect TNF-sensitive cells, in which the other stimulators did not alter expression of the gene. HeLa cells which had been desensitized to TPA by pretreatment with TPA for 24 h expressed Mn-SOD mRNA at a slightly higher level than the cells without TPA treatment. TPA-pretreated cells stimulated with TNF, however, expressed Mn-SOD mRNA at about twice the level of TNF-stimulated, TPA-untreated cells. When protein synthesis was inhibited by cycloheximide during TPA pretreatment, TNF no more enhanced the Mn-SOD mRNA accumulation. These data suggest that at least two separate signal-transducing pathways are involved in expression of this gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulation with TNF, interleukin-1, or lipopolysaccharide and in which a protein factor that can be induced by TPA treatment is involved.     

A fish antimicrobial peptide, tilapia hepcidin TH2-3, shows potent antitumor activity against human fibrosarcoma cells

As part of a continuing search for potential anticancer drug candidates from antimicrobial peptides of marine organisms, tilapia (Oreochromis mossambicus) hepcidin TH2-3 was evaluated in several tumor cell lines. The results indicated that TH2-3, a synthetic 20-mer antimicrobial peptide, specifically inhibited human fibrosarcoma cell (HT1080 cell line) proliferation and migration. The way in which TH2-3 inhibited HT1080 cell growth was then studied. TH2-3 inhibited HT1080 cell growth in a concentration-dependent manner according to an MTT analysis, which was confirmed by a soft-agar assay and AO/EtBr staining. Scanning electron microscopy revealed that TH2-3 caused lethal membrane disruption in HT1080 cancer cells, and a wound healing assay supported that TH2-3 decreased the migration of HT1080 cells. In addition, c-Jun mRNA expression was downregulated after treatment with TH2-3 for 48–96 h compared to the untreated group. These findings suggest a mechanism of cytotoxic action of TH2-3 and indicate that TH2-3 may be a promising chemotherapeutic agent against human fibrosarcoma cells.     

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: An in situ hybridization study

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.     

Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)