Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0029463 (osteosarcoma)
 16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues. To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88). The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa. The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells. The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34). In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34). No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells. The biologic activity of recombinant chicken PTH-(1-88) purified from E. coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells. Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells. The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.
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PMID:Full-length chicken parathyroid hormone. Biosynthesis in Escherichia coli and analysis of biologic activity. 184 86

Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.
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PMID:Expression of adenylate cyclase-coupled osseous parathyroid hormone and parathyroid hormone-like peptide receptors in Xenopus oocytes. 184 24

Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with the protein kinase C(PK-C) activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) results in a time dependent (1-12h) desensitization of PTH-stimulated cAMP production. Compared to controls, PMA-treated cells showed 50% decrease of PTH-stimulated cAMP production. PK-C inhibitor, H-7 significantly blocked this PMA-induced desensitization. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was decreased by about 20% in PMA-treated cells. H-7 treatment completely restored receptor binding in PMA-treated cells. These data suggest that PK-C might act directly on PTH receptor which is coupling to adenylate cyclase, and induce desensitization.
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PMID:Phorbol ester induces desensitization of PTH-stimulated cyclic AMP production by decreasing the PTH receptor binding in UMR-106 cells. 185 Oct 4

Micromolar concentrations of aluminum sulfate consistently stimulated [3H]thymidine incorporation into DNA and increased cellular alkaline phosphatase activity (an osteoblastic differentiation marker) in osteoblast-line cells of chicken and human. The stimulations were highly reproducible, and were biphasic and dose-dependent with the maximal stimulatory dose varied from experiment to experiment. The mitogenic doses of aluminum ion also stimulated collagen synthesis in cultured human osteosarcoma TE-85 cells, suggesting that aluminum ion might stimulate bone formation in vitro. The effects of mitogenic doses of aluminum ion on basal osteocalcin secretion by normal human osteoblasts could not be determined since there was little, if any, basal secretion of osteocalcin by these cells. 1,25 Dihydroxyvitamin D3 significantly stimulated the secretion of osteocalcin and the specific activity of cellular alkaline phosphatase in the human osteoblasts. Although mitogenic concentrations of aluminum ion potentiated the 1,25 dihydroxyvitamin D3-dependent stimulation of osteocalcin secretion, they significantly inhibited the hormone-mediated activation of cellular alkaline phosphatase activity. Mitogenic concentrations of aluminum ion did not stimulate cAMP production in human osteosarcoma TE 85 cells, indicating that the mechanism of aluminum ion does not involve cAMP. The mitogenic activity of aluminum ion is different from that of fluoride because (a) unlike fluoride, its mitogenic activity was unaffected by culture medium changes; (b) unlike fluoride, its mitogenic activity was nonspecific for bone cells; and (c) aluminum ion interacted with fluoride on the stimulation of the proliferation of osteoblastic-line cells, and did not share the same rate-limiting step(s) as that of fluoride. PTH interacted with and potentiated the bone cell mitogenic activity of aluminum ion, and thereby is consistent with the possibility that the in vivo osteogenic actions of aluminum ion might depend on PTH. In summary, low concentrations of aluminum ion could act directly on osteoblasts to stimulate their proliferation and differentiation by a mechanism that is different from fluoride.
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PMID:Aluminum stimulates the proliferation and differentiation of osteoblasts in vitro by a mechanism that is different from fluoride. 192 12

We showed recently that the initial peak cytosolic ionized calcium ([Ca2+]i) response to PTH (2-min exposure) is preserved relative to the cAMP response in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) desensitized by 72-h exposure to PTH. We attempted in the present studies to determine the mechanisms for preservation of the [Ca2+]i response and to explore the effects of longer PTH rechallenges. The [Ca2+]i response to a 20-min perifusion with rat PTH [rPTH-(1-34)] was monitored by aequorin luminescence in both naive and PTH-desensitized ROS 17/2.8 cells. The responses of both naive and desensitized cells consisted of two phases: an initial peak, followed by an intermediate plateau that was sustained in the presence of PTH. We observed in the naive cell populations synchronous oscillations in [Ca2+]i concentration during this second phase (amplitude, 10-60 nM; frequency, 1-3/100 sec). These oscillations were maintained through extracellular calcium (EC Ca2+) entry; the initial peak was the result of Ca2+ release from intracellular stores. In desensitized cells, these two phases could not be clearly separated with respect to Ca2+ source, but, as we showed before, exhibited an enhanced dependence on EC Ca2+ entry for the response to PTH. Nevertheless, in the desensitized cells, the sustained [Ca2+]i response was diminished in magnitude and showed little oscillatory behavior. Brief exposure to neomycin sulfate, an inhibitor of phosphoinositide turnover, attenuated the PTH-induced [Ca2+]i rise in both naive and desensitized cells. Protein kinase-C activity did not appear to be required for either phase of the PTH-induced [Ca2+]i response. Exposure to cholera toxin attenuated the [Ca2+]i response to hormone in both naive and desensitized cells, more markedly in the latter. Cholera toxin treatment dramatically increased basal cAMP levels in both cell preparations; PTH-stimulated cAMP production was unchanged in naive cells, but increased nearly 4-fold in desensitized cells. We propose that the preserved PTH-induced peak [Ca2+]i rise in desensitized cells results primarily from the diminished regulation of EC Ca2+ entry by the cAMP response limb. The attenuated sustained oscillatory behavior observed in desensitized cells upon rechallenge with hormone may be the result of reduced phosphoinositide turnover and reduced Ca2+-stimulated Ca2+ release. Thus, the [Ca2+]i response to PTH in osteoblast-like cells is complex and modulable and seems to provide a number of ways to regulate intracellular metabolism under various conditions. We speculate that this plasticity of the [Ca2+]i response to PTH is related to the pleiotropic actions of the hormone on cells of the osteoblast lineage.
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PMID:Parathyroid hormone (PTH)-induced intracellular Ca2+ signalling in naive and PTH-desensitized osteoblast-like cells (ROS 17/2.8): pharmacological characterization and evidence for synchronous oscillation of intracellular Ca2+. 195 83

[Nle8,18,Tyr34]bPTH-(1-34)amide (NlePTH) was biotinylated using sulfosuccinimidyl 6-(biotinamido)hexanoate, in dimethyl sulfoxide, and the multiple resulting peptides peaks were separated by reverse-phase high performance liquid chromatography. Their biological activities were compared with those of NlePTH, the parent compound, in radioreceptor and cAMP accumulation bioassays using rat osteosarcoma 17/2.8 cells; the earliest two eluting products, bioPTH 1 and 2, were equipotent, a third, bioPTH 3, was only 10% as potent, and the remaining, later eluting derivatives all were less than 0.1% as active. Competitive avidin binding assays using [3H]biotin suggested that bioPTH 1 and 2 had a single biotin congener per molecule, while bioPTH 3 contained two biotin residues. Upon Edman degradation, bioPTH 1 contained biotin on the lysine at position 13 of NlePTH; bioPTH 2's biotin was on the lysine at position 26 (or 27) and bioPTH 3 had biotins on lysines at both positions 13 and 26 (or 27). Avidin tagged with 125I, peroxidase, or fluorescein isothiocyanate was detected on bone-derived cells which had been incubated initially with bioPTH 2 (1, 10, and 100 nM) for 4 h, but not when NlePTH (1 microM) was added with bioPTH 2. A fluorescence-activated cell sorter detected a symmetrical shift in fluorescence of bone-derived cells incubated with 10 nM of bioPTH 2 and 10 micrograms/ml fluorescein isothiocyanate-avidin. Addition of a 30-fold molar excess of NlePTH, or omission of bioPTH 2, completely reversed this fluorescence shift, and no shift in fluorescence was seen with cells lacking PTH receptors. This fully active, high affinity biotinylated PTH-derivative should prove useful in the study of PTH receptor-bearing cells.
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PMID:Characterization of fully active biotinylated parathyroid hormone analogs. Application to fluorescence-activated cell sorting of parathyroid hormone receptor bearing cells. 215 27

Past work described the partial purification and characterization of a novel serine protein kinase activity designated protein kinase N (PKN) that is activated by nerve growth factor (NGF) in cultured PC12 cells [Rowland et al. (1987) J. Biol. Chem. 262; 7504-7513]. We have now devised a rapid, sensitive technique for partially purifying and assaying PKN activity in cell extracts. This methodology was applied to the IARC-EW-1 osteosarcoma and several additional non-neuronal cell lines that possess NGF receptors but that lack both morphological and a variety of additional biochemical responses to NGF. In each case, NGF significantly elevated PKN activity. The assay also revealed activation of PKN activity in IARC-EW-1 cells by additional agents, including epidermal growth factor, fibroblast growth factor, phorbol ester, and a cAMP analog. Also tested were an NGF-receptor-deficient PC12 cell variant and sublines thereof into which human NGF receptors had been introduced [Hempstead et al. (1989) Science 243; 373-375]. Acquisition of the NGF receptors resulted in NGF-activatable PKN activity. These findings indicate that detection of PKN activity may serve as a sensitive means to test NGF responsiveness in cells lacking macroscopic responses to the factor and that non-neuronal cells may be useful for studying primary signaling events in the NGF mechanism of action.
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PMID:Nerve growth factor (NGF) responses by non-neuronal cells: detection by assay of a novel NGF-activated protein kinase. 215 63

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.
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PMID:Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists. 216 26

We investigated the differentiation-inducing effects of dibutyryl cyclic AMP (dBc AMP) on several cultured osteosarcoma cell lines (DUNN, MOLONEY, OST, FBJ). 1. Cell growth rates of all the osteosarcoma cell lines were reduced by 3mM dBc AMP. 2. Both alkaline phosphatase activity and 45Ca2(+)-uptake were promoted by 3mM dBc AMP. 3. There was, in each cell line except for the OST cells, a marked enlargement of the cell processes under the light microscopic observation. At the electron microscopic level, there were also many findings indicating an increase in cell functions. These results suggest that the differentiation may be induced by cAMP in osteosarcoma cells, and that the differentiation therapy by cAMP-related drugs is promising for a clinical application.
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PMID:[Experimental study on maturational therapy of osteosarcoma cell]. 216 18

A human osteosarcoma cell line, HuO9, was established from a tumor that was heterotransplanted into athymic nude mice. Antiserum against nude mouse spleen cells was added to the early passage cultures to eliminate the host fibroblastic cells. The cell line retained a high activity of liver/bone/kidney-type alkaline phosphatase (ALP) and secreted osteocalcin, i.e., bone gamma-carboxyglutamic acid-containing protein (BGP), into the medium. The addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) increased the ALP activity as well as the level of BGP secreted into the medium. The ALP of 1,25(OH)2D3-treated cells has the same inhibition characteristics to heat and amino acids as that of untreated cells. Synthetic human parathyroid hormone stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) approximately 100-fold within five minutes. However, the stimulation was not observed with a synthetic human thyrocalcitonin. When HuO9 cells were transplanted into the back of a nude mouse, a tumor with an abundant osteoid formation and mineralization was produced. The results indicate that the HuO9 cell line expresses well-differentiated osteoblastic phenotypes. HuO9 is the first established human cell line to produce BGP, and it provides a useful model for the studies of osteoblasts and the regulatory mechanisms of BGP production.
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PMID:A newly established human osteosarcoma cell line with osteoblastic properties. 217 70


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