UMLS:C0029463 - FACTA Search

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Query: UMLS:C0029463 (osteosarcoma)
16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Formycin 5'-triphosphate (FoTP), a fluorescent analog of ATP, is shown to be a substrate for the membrane-bound adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rat osteosarcoma cells. The formation of the adenylate cyclase reaction product, 3',5'-cyclic formycin monophosphate (cFoMP), was followed by the conventional radioimmunoassay (RIA) procedure used to detect cAMP and by an assay procedure in which the reaction product was separated from the substrate by reverse-phase high-pressure liquid chromatography (HPLC) and the reaction product was detected by fluorometry. Because the HPLC--fluorometric procedure can determine the amount of cFoMP present in the reaction mixture within 6 min, the enzymatic conversion of FoTP to cFoMP can be followed directly during the course of a typical 15-min incubation. The amount of cFoMP detected by this procedure was found to be within 2% of the values obtained by the RIA. The rate of product formation with FoTP was similar to that observed with ATP and the activity of the enzyme was enhanced about 5-fold with guanyl-5'-yl imidodiphosphate when either ATP or FoTP was used as the substrate. Kinetic studies revealed values for the Vmax of 120 pmol/min per mg of protein and apparent Km values of 220 microM with both substrates. In addition to suggesting that the recognition of the substrate by the adenylate cyclase may not require a specific chemical structure of the 5-membered ring of the base or a unique configuration about either the glycosyl or the C(5')-C(4') bond, the results of this study are consistent with the idea that the cytotoxicity observed with the adenosine analog formycin may be the result of its metabolism to cFoMP. Furthermore, these studies indicate that the fluorescent analog FoTP can be used, in combination with HPLC, to provide an alternative, nonradioactive direct method for the assay of adenylate cyclase catalytic activity.
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PMID:Formycin 5'-triphosphate, a fluorescent analog of ATP, as a substrate for adenylate cyclase. 694 Dec 84

Protein phosphorylation was compared in normal human cells and human osteogenic sarcoma cells. The phosphorylation of endogenous cellular protein substrates was measured by two independent methods, incubation of homogenized cells with [gamma-32P]ATP or labeling of intact cells with Na2H32PO4. Phosphorylated proteins were identified by SDS-polyacrylamide gel electrophoresis and autoradiography. The stained protein bands of all four osteosarcoma cell lines were nearly identical to those of the normal cells. However, each of the osteosarcoma cell lines showed autoradiographic evidence of enhanced phosphorylation in many different protein bands which was neither cyclic AMP-dependent nor a function of cellular growth rate or density. When normal and tumor cell homogenates were mixed prior to incubation with [gamma-32P]ATP, the resulting phosphoprotein patterns resembled those obtained with the tumor cells alone. In addition, a surgically derived osteogenic sarcoma was cultured and an established line obtained; another portion of the fresh tumor was immediately homogenized and used in a phosphorylation assay. The same enhanced phosphorylation pattern was obtained with the homogenized fresh tumor as with the cell line established from it. These results suggest that human osteogenic sarcoma cells are able to perform a significantly increased amount of phosphorylation of endogenous cellular protein substrates when compared to normal human cells.
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PMID:Human osteogenic sarcoma cells exhibit enhanced protein phosphorylation. 694 94

Extracellular ATP acting through purinoceptors may be an important factor in the modulation of bone turnover. In this study we cloned and sequenced the P2U purinoceptor from osteoclastoma, confirming the recently published human sequence. Furthermore, by the reverse transcriptase-linked polymerase chain reaction (RT-PCR) and Southern blotting we demonstrated expression of P2U receptor mRNA in bone, primary cultures of human bone-derived cells, and two osteosarcoma cell lines, Saos2 and Te85. P2U receptor transcripts were identified in alkaline phosphatase-positive human bone-derived cells isolated by flow cytometry providing strong evidence for the expression of the P2U purinoceptor in mature osteoblasts. P2U receptor transcripts were also detected in a purified giant cell population isolated from osteoclastoma, indicating that this receptor is also expressed by osteoclasts. These data suggest that purinergic agonists may play a role in the regulation of bone metabolism.
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PMID:Identification and cloning of human P2U purinoceptor present in osteoclastoma, bone, and osteoblasts. 748 91

1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) has been shown to increase cytosolic calcium and inositol triphosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1 alpha,25-(OH)2D3-induced changes in nuclear calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei was assessed. 1 alpha,25(OH)2D3, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1 beta,25-(OH)2D3, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1 alpha,25-(OH)2D3, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium within 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effect of ATP on nuclear calcium was not additive with 1 alpha,25-(OH)2D3, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1 alpha,25-(OH)2D3 and ATP rapidly increase inositol triphosphate levels in nuclei isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid increase nuclear calcium. Thus, the 1 alpha,25-(OH)2D3 and ATP effects on nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblast-like cells.
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PMID:1 alpha,25-Dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclear envelope of osteoblasts. 764 19

A monoclonal antibody (OSW2) was prepared by using human osteosarcoma cells. OSW2 was found to be directed toward the 116 (also called 100)- kD protein that uniquely associates to the vacuolar-type proton pump. The antibody specifically localized acidic membrane compartments that could be visualized with acridine orange in many types of human cells. It also reacted with the surface and was internalized along the endosomal pathway. Monitoring the endosome pH by using FITC-dextran and acridine orange suggested that the antibody interfered with low pH. Cell-free experiments indicated that the ATP-dependent acidification was inhibited in endosomes associated with OSW2. In contrast, the antibody gave little effect on the ATPase activity of the solubilized H+ pump. The internalization of OSW2 reduced infectivity of certain enveloped viruses (influenza, SFV, VSV) by 50 to 80%. Inhibition of viral fusion was directly demonstrated by monitoring the fate of octadecylrhodamine-labeled influenza virus fluorescence. These results indicate that the 116 (100)-kD protein is necessary for the control of pH. The antibody represents a novel probe for understanding the role of the endosomal compartments in cellular physiology.
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PMID:Interference with the endosomal acidification by a monoclonal antibody directed toward the 116 (100)-kD subunit of the vacuolar type proton pump. 792 69

An oncogene product, p53, interacts with a simian virus 40-encoded T-antigen, which is an initiation protein for the viral DNA replication and also works as DNA helicase during elongation. Here we examine the interaction of p53 with cellular DNA helicase. A recombinant human wild type p53 fused with glutathione S-transferase was immobilized on glutathione-agarose as a ligand for affinity column. Hela cell extract was applied to the p53 column and the adsorbed proteins were eluted with buffers containing salt, 50% ethylene glycol, and glutathione. The ethylene glycol fraction contained a number of p53 binding proteins, and this fraction showed a DNA helicase activity measured by the displacement of DNA fragment from partially duplexed M13 DNA. The DNA helicase translocated in a 5'-to-3' direction on the single-stranded DNA using ATP as an energy source. The glutathione fraction that contained the p53 glutathione S-transferase fused protein also showed the same activity. The corresponding fractions from a control column carrying glutathione S-transferase showed only a trace amount of activity of DNA helicase. Therefore, the binding may be specific. Furthermore, an anti-p53 antibody column retained a p53-DNA helicase complex when the crude extracts of human placenta and of osteosarcoma cells were applied. These results indicate that p53 physically interacts with DNA helicase in vitro as well as in vivo.
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PMID:Anti-oncogene product p53 binds DNA helicase. 795 81

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR-106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3-h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter. 839 31

In certain neurons, alternative RNA processing generates calcitonin gene-related peptide (CGRP) from the same gene that encodes the hormone calcitonin. As CGRP-containing nerve fibers are prominent in skeleton, we evaluated the effects of CGRP on osteoblasts. Because the vasodilatory effect of neural CGRP in smooth muscle probably involves inhibition of unstimulated Ca2+ uptake, we examined the acute effects of CGRP on this parameter in rat osteoblastic cells. CGRP inhibits 45Ca2+ uptake in both UMR 106 osteosarcoma and RCOB-3 osteoblastic cells. This inhibition is rapid (0.5 min), occurs with an EC50 of 1 nM, and cannot be demonstrated in the presence of 0.1 mM diltiazem, a blocker of voltage-dependent Ca2+ channels. Depolarization of bone cells with high extracellular potassium (K+) also blocks the effect of CGRP on 45Ca2+ uptake, suggesting a central role for K+ channels in mediating this action. In agreement with this hypothesis, the effect of CGRP is blocked by 1 microM glybenclamide, a specific inhibitor of ATP-sensitive potassium (K(ATP)) channels, or by pretreatment of cells with 1 mM iodoacetic acid to deplete intracellular ATP. Blocking Ca2+-activated potassium channels with 1 mM tetraethylammonium does not prevent CGRP's effect. Pinacidil, a specific activator of K(ATP) channels, mimics CGRP's effect. Both CGRP and pinacidil also produce a small significant stimulation of cellular Ca2+ efflux in UMR 106 cells. These data suggest that inhibition of diltiazem-sensitive Ca2+ channels occurs secondary to the hyperpolarization engendered by CGRP activation of K(ATP) channels in osteoblastic cells, an effect similar to that of CGRP on smooth muscle cells.
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PMID:Calcitonin gene-related peptide rapidly inhibits calcium uptake in osteoblastic cell lines via activation of adenosine triphosphate-sensitive potassium channels. 860 12

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mM Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nM parathyroid hormone (PTH), 5 nM prostaglandin E2 (PGE2) or 0.1 mM dibutyryl cAMP + 1 microM forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mM K) the channel has a conductance of 246 +/- 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (Po) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the Po vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mM ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca2+) by MG-63 cells was stimulated in the presence of PTH and PGE2 an effect inhibited by Nitrendipine (10 microM). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mM Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mM Ca Ringer and 10 microM Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.
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PMID:Activation of maxi-K channels by parathyroid hormone and prostaglandin E2 in human osteoblast bone cells. 866 78

In cell culture, human osteoblasts and the osteosarcoma cell line MG-63 express annexins I, II, IV, V and VI. Small proportions of annexins IV and V are lost from MG-63 cells into the culture medium in a sedimentable form. however, the bulk of these annexins is intracellular. In non-confluent cells 3 days after passaging, annexin IV and annexin V are strongly present throughout the nucleus and are also present in the cytoplasm. On elevation of the intracellular calcium concentration with the lonophore ionomycin, the intranuclear pools of annexin IV in 38 +/- 4% of cells and annexin V in 70 +/- 5% of cells show relocation to the nuclear membrane within 40 s. Extracellular ATP, which causes a transient increase in the cytosolic free calcium concentration by acting at P2-purinoceptors, also causes relocation of the intranuclear pool of annexin IV in 22 +/- 4% of cells and of annexin V in 38 +/- 8% of cells. After stimulation no significant reversal of the relocation is observed. Elevation of intracellular calcium with ionophore and ATP also causes relocation of the cytoplasmic pools of annexins IV and V. The results support a role for annexins at cellular membranes in response to elevation of cytosolic calcium levels.
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PMID:Calcium-induced relocation of annexins IV and V in the human osteosarcoma cell line MG-63. 874 77

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