Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.     

Luteinizing hormone stimulates the production of prostacyclin by isolated ovarian cell

Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF₁α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF₁α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE₂ were devoid of effect. The time-dependent in vitro production of 6-keto-PGF₁α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.     

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation

GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.     

Histamine stimulates calcium-mediated protein phosphorylation in a colonic epithelial cell line

Protein phosphorylation responses in intact enterocytes were examined by stimulating 32Pi-labeled T84 cell monolayers with histamine and resolving proteins by two-dimensional gel electrophoresis. Histamine increases 32P-incorporation into two acidic proteins of Mr 83,000 and of Mr 29,000, designated p83 and p29. Labeling of p83 and p29 is also increased in cells exposed to ionomycin, but not in cells exposed to vasoactive intestinal peptide under conditions resulting in cAMP-mediated secretion and cAMP-stimulated protein phosphorylation. When T84 cell fractions are incubated with [gamma-32P]ATP, labeling of p83 is stimulated by Ca++, but not by cAMP. Thus, histamine stimulates Ca++-mediated protein phosphorylation during the regulation of Cl- secretion.     

Triiodothyronine stimulates cystatin C production in bone cells

Thyroid hormones increase cystatin C levels in vivo. To study whether 3,3',5-triiodo-l-thyronine (T(3)) stimulates the production of cystatin C in vitro, we used a T(3)-responsive osteoblastic cell line (PyMS) which can be kept in serum-free culture. We compared the effects of T(3) on cystatin C mRNA expression (by Northern) and on protein release (by Western and ELISA) with those of dexamethasone (dex). Triiodothyronine increased cystatin C mRNA expression and cystatin C accumulation in culture media in a dose- and time-dependent manner, 1.5-fold at 1 nmol/l after 4d; dex (100 nmol/l) was more potent and increased cystatin C accumulation 3-fold after 4d. Triiodothyronine but not dex stimulated glucose uptake. Our in vitro findings explain in vivo observations. Triiodothyronine-induced increase in the production of cystatin C may be related to an increased cell metabolism and proteolysis control demand.     

Interleukin 3 stimulates phosphatidylcholine turnover in a mast/megakaryocyte cell line

The hemopoietic growth factor, interleukin 3, has been shown to activate protein kinase C without causing hydrolysis of inositol phospholipids. The potential involvement of phosphatidylcholine hydrolysis as an alternative source of diacylglycerol was investigated in an interleukin 3-dependent murine mast/megakaryocyte cell line, R6-XE.4. Treatment of these cells with interleukin 3 rapidly stimulated both the release of water-soluble choline metabolites and the resynthesis of phosphatidylcholine. Therefore, a phosphatidylcholine cycle may operate as part of the signal transduction pathway in cells responding to interleukin 3.     

Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

Phorbol myristic acetate stimulates LAF production by the macrophage cell line,P388D

Phorbol myristic acetate markedly stimulated LAF production by the murine macrophage cell line, P388D₁. This effect was both time and concentration dependent. Other analogs, such as phorbol didecanoate and to a lesser extent, phorbol dibenzoate also enhanced LAF production, but the parent compound, phorbol, was inactive. The PMA induced supernatant LAF had a molecular weight of 16,000 daltons and exhibited charge heterogenity on DEAE cellulose. Since PMA is a small compound of defined structure which induces the production of large amounts of LAF it should be a useful tool with which to probe the biology and biochemistry of LAF.     

GRF (somatocrinin) stimulates release of neurotensin, calcitonin and cAMP by a rat C cell line

We reported previously that GRF stimulates release of neurotensin (NT) by a clonal line of rat C cells (44-2 C). We report here that GRF stimulates calcitonin (CT) and cAMP release in these cells. For release experiments, replicate cultures are incubated for 5-180 min in serum-free defined medium. CT, NT and cAMP are measured by RIA. At a maximally effective concentration of GRF (.1-1.0 nM), there is a 2-3 fold stimulation of CT release at 60-90 min with peak release at 180 min. In contrast, GRF causes a rapid 4-6 fold increase of NT release within 5-15 min. In 44-2 C cells there is a 4-40 fold stimulation of cAMP release by GRF. We conclude that in 44-2 C cells GRF stimulates release of NT and cAMP and show for the first time the effect of this peptide on release of CT.     

Dexamethasone enhances trichosanthin-induced apoptosis in the HepG2 hepatoma cell line

AimsTrichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with antitumor activities for various cancers. In this paper, we aimed to investigate whether dexamethasone, an important synthetic member of the glucocorticoid steroids, in combination with TCS can be a potential therapy in treating hepatoma.Main methodsCell viability was investigated using MTT assay, and apoptosis was evaluated with Hoechst 33258 staining. Western blot analysis was used to examine the changes in the expression levels of IκB-α, NF-κB p65 subunit and Cox-2. Additionally, we took advantage of dominant-negative IκB (IκB-DM) over-expression and chemical inhibitor PDTC to inhibit NF-κB activation.Key findingsOur results demonstrated that dexamethasone could enhance TCS-induced apoptosis in the hepatoma cell line HepG2, decreasing IC50 values from in excess of 200 μg/ml to 50 µg/ml. In addition, our results demonstrated that TCS could induce rapid degradation of IκB-α, nuclear translocation of NF-κB and decrease of COX-2 expression in HepG2 cells. Inhibition of NF-κB by biological (IκB-DM) or chemical inhibitor (PDTC) increased HepG2 cells' sensitivity to TCS, resulting in cell viability rate decreasing and apoptotic rate increasing. Simultaneously, dexamethasone increased the level of IκB-α protein and effectively inhibited TCS-induced degradation of IκB-α.SignificanceThese results suggest that dexamethasone could enhance trichosanthin-induced apoptosis in the HepG2, at least in part, by inhibiting the NF-κB signaling pathway and thus strengthening the antitumor effects of TCS, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma.     

Bombesin stimulates the in vitro growth of a human gastric cancer cell line

Bombesin (BBS) and its mammalian equivalent, gastrin-releasing peptide (GRP) exhibit diverse biological functions, including that of a neurotransmatter, a regulator of gastrointestinal hormone release, and a trophic factor for various normal and neoplastic tissues. Bombesin stimulates the growth of normal cells of the stomach, pancreas, and bronchial epithelium as well as cells in breast cancer, gastrinoma, and small cell lung cancer. The purpose of this study was to determine whether BBS regulates the growth of a human gastric cancer cell line (SIIA) in vitro, and if so, to examine the mechanisms of signal-transduction that are involved. We found that BBS stimulated the growth of SIIA cells in vitro. The GRP receptor antagonists, BIM 26189 and BIM 26226, had no effect on growth of SIIA cells. Although these antagonists blocked the BBS-induced increase of [Ca²⁺]_i, they failed to block the growth-stimulatory effect of BBS. BBS stimulated intracellular tyrosine phosphorylation of multiple proteins, with a predominant protein of apparent molecular weight of 125 kDa. Inhibition of intracellular tyrosine kinases by tyrphostin blocked the growth-stimulatory effect of BBS on SIIA cells. These results indicate that BBS exerts its trophic effect on SIIA cells through a receptor(s) linked to tyrosine kinase pathway, but not to the phospholipase C (PLC) pathway. © 1994 Wiley-Liss, Inc.     

Recombinant gamma-interferon stimulates iodide uptake and cyclic AMP production by the FTRL5 thyroid cell line

The effect of recombinant rat gamma-interferon (gamma-IFN) on iodide uptake and cAMP production by rat thyroid cells in vitro was studied using the continuously growing, functional FRTL5 cell line. Both functions were stimulated by gamma-IFN at concentrations of 1-10 U/ml. Iodide uptake was dependent on protein synthesis, since it was blocked by cycloheximide treatment, but was not dependent on growth factors in calf serum routinely used for FRTL5 cell culture. These results show that gamma-IFN can stimulate thyroid cell function as well as aberrant Ia expression in vitro.     

Ecdysteroid production by a continuous insect cell line

The spent medium from ten established cell lines was extracted and tested for ecdysteroids by radioimmunoassay. Of the seven lepidopteran lines tested, only IAL-TNDI and MRRL-CH showed evidence of ecdysteroid production. However, the results were erratic and difficult to evaluate and these lines were dropped from further consideration. However, of the three cockroach cell lines tested, one, UMBGE 4, produces ecdysteroid and consistently releases virtually all of it into the medium. The main ecdysteroid was identified as ecdysone and the increase was logarithmic during the first 11 days of the subculture, with a decrease from day 11 to day 14. UMBGE 4 is a vesicle cell line which also tested positive for chitin synthesis. When the pH of the medium was lowered from pH 7.4 to pH 6.3, both the chitin synthesis and the ecdysone synthesis dropped by roughly 50%.     

Dietary thyrotropin-releasing hormone stimulates growth rate and increases the insulin: glucagon molar ratio of broiler chickens

The effects of thyroid manipulation on growth, feed efficiency, and plasma hormone levels were determined in rapidly growing chickens. Beginning at 3 weeks of age, eight broiler cockerels were provided with control feed (CF) or feed containing either 1 ppm of triiodothyronine (T3), 1 ppm of thyroxine (T4), 0.3% propylthiouracil (PTU), or 5 ppm of thyrotropin-releasing hormone (TRH) for 3 weeks. Blood samples were taken at 4, 5, and 6 weeks for determination of plasma levels of growth hormone, insulin-like growth factor, T3, T4, insulin, glucagon, glucose, and nonesterified fatty acids. Dietary TRH increased (P less than 0.05) the growth rate of chickens by 14% when compared with the CF group. Plasma growth hormone levels were reduced (P less than 0.05) 65% by dietary T3 and 33% by treatment with either T4 or TRH when compared with the CF group. Plasma insulin-like growth factor levels were 16% lower (P less than 0.05) in PTU-fed birds than the other treatment groups. Plasma T3 levels were elevated (P less than 0.05) 3-fold by dietary T3 and 38% by TRH whereas plasma T3 in the PTU group was 38% below the average of CF birds. Plasma T4 levels were increased (P less than 0.05) by 12-fold in T4-fed birds, decreased 48% in TRH-fed birds, and nondetectable in birds treated with either T3 or PTU. Compared with the other treatments, dietary PTU increased (P less than 0.01) plasma insulin levels 4.3-fold whereas TRH provided a 2.7-fold increase in plasma insulin. Plasma glucagon levels were 26% higher (P less than 0.05) in T3-fed birds than those fed either T4 or PTU. These observations indicate that thyroid activity plays an important role in regulating secretion of GH and the pancreatic hormones. Furthermore, our study demonstrates the potential use of TRH as an orally active growth promoter for poultry.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.