Newly synthesized glycoconjugates from two cell lines derived from rat osteogenic sarcoma: effects of Matrigenin activity from bone

1991 ◽  
Vol 69 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Ravi K. Chopra ◽  
Tassos Anastassiades ◽  
Christian Stephens ◽  
David Lohnes ◽  
Glenville Jones
Keyword(s):  
Cell Lines ◽  
Osteogenic Sarcoma ◽  
Distinct Species ◽  
Enzymatic Digestion ◽  
Proteoglycan Synthesis ◽  
Tissue Cell ◽  
Osteoblastic Cell ◽  
Bovine Bone ◽  
The Media ◽  
Umr 106

An activity isolated from bovine bone was previously shown to stimulate proteoglycan synthesis by several connective tissue cell lines from normal tissues (Matrigenin activity). The effect of this activity on glycoconjugate synthesis by two osteoblastic cell lines, ROS 17/2 and UMR-106, derived from rat osteogenic sarcoma, was examined after labelling of the cells with [3H]glucosamine and [35S]sulfate. The glycoconjugates from the cell layers and the media were separated by DEAE-Sephacel chromatography and the anionic glycoconjugates of the media were further analyzed by chromatography on Sepharose CL-2B and enzymatic digestion of the papain-released glycosaminoglycans. The ROS 17/2 cells secreted at least two distinct species of proteoglycan (one heparan sulfate rich and the other chondroitin sulfate rich), whereas the UMR-106 secreted primarily an anionic glycoprotein. The addition of Matrigenin activity to the ROS 17/2 cells resulted in stimulation of incorporation of radioactivity into the proteoglycan and hyaluronic acid, but in UMR-106 cultures it resulted in decreased incorporation into the anionic glycoprotein. The decrease in incorporation into the anionic glycoprotein from the medium was shown, by alkaline β-elimination, to have occurred mainly in the oligosaccharide fraction, relative to control cultures.Key words: osteogenic sarcoma cells, Matrigenin activity, glycoconjugate synthesis.

Modulation of glucose transport by parathyroid hormone and insulin in UMR 106–01, a clonal rat osteogenic sarcoma cell line

1995 ◽  
Vol 14 (2) ◽  
pp. 263-275 ◽  
Author(s):  
D M Thomas ◽  
S D Rogers ◽  
M W Sleeman ◽  
G M Pasquini ◽  
F R Bringhurst ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Cell Line ◽  
Glucose Transport ◽  
Transport System ◽  
Osteogenic Sarcoma ◽  
Regulatory Subunit ◽  
Sarcoma Cell ◽  
Sarcoma Cell Line ◽  
Glucose Transport System ◽  
Umr 106

ABSTRACT This study characterizes the actions of insulin and parathyroid hormone (PTH) on the glucose transport system in the rat osteogenic sarcoma cell line UMR 106–01, which expresses a number of features of the osteoblast phenotype. Using [1,2-3H]2-deoxyglucose (2-DOG) as a label, UMR 106–01 cells were shown to possess a glucose transport system which was enhanced by insulin. In contrast, PTH influenced glucose transport in a biphasic manner with a stimulatory effect at 1 h and a more potent inhibitory effect at 16 h on basal and insulin-stimulated 2-DOG transport. To explore the mechanism of PTH action, a direct agonist of cAMP-dependent protein kinase (PKA) was tested. 8-Bromo-cAMP had no acute stimulatory effect but inhibited basal and insulin-stimulated 2-DOG transport at 16 h. This result suggested that the prolonged, but not the acute, effect of PTH was mediated by the generation of cAMP. Further studies with the cell line UMR 4–7, a UMR 106–01 clone stably transfected with an inducible mutant inactive regulatory subunit of PKA, confirmed that the inhibitory but not the stimulatory effect of PTH was mediated by the PKA pathway. Northern blot data indicated that the prolonged inhibitory effects of PTH and 8-bromo-cAMP on glucose transport were likely to be mediated in part by reduction in the levels of GLUT1 (HepG2/brain glucose transporter) mRNA.

scholarly journals Proteoglycan synthesis in two murine bone marrow stromal cell lines

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1777-1783 ◽  
Author(s):  
SL Kirby ◽  
SA Bentley
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Dermatan Sulfate ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Proteoglycan Synthesis ◽  
Murine Bone Marrow

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3–2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3–2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3–2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3–2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

Effects of Parathyroid Hormone and Insulin on Proliferation in Osteogenic Sarcoma UMR-106-01 Cells

1998 ◽  
Vol 33 (2) ◽  
pp. 466
Author(s):  
Kyung Moon ◽  
Choon Sung Lee ◽  
Jae Suk Chang ◽  
Key Yong Kim ◽  
Seong Who Kim ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Osteogenic Sarcoma ◽  
Umr 106

scholarly journals Expression of p27Kip1 in Osteoblast-Like Cells during Differentiation with Parathyroid Hormone*

Endocrinology ◽  
1997 ◽  
Vol 138 (5) ◽  
pp. 1995-2004 ◽  
Author(s):  
Takehisa Onishi ◽  
Keith Hruska
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Osteogenic Sarcoma ◽  
S Phase ◽  
G1 Phase ◽  
Osteoblastic Cell ◽  
Cyclin Dependent Kinases ◽  
Kinase A

Abstract PTH is a major systemic regulator of bone metabolism and plays an important role in both bone formation and resorption. PTH either inhibits or stimulates osteoblastic cell proliferation depending on the model that is studied. We analyzed the cell cycle of the UMR-106 cell line, a relatively differentiated osteoblastic osteogenic sarcoma line in which PTH is known to inhibit proliferation but the mechanism of action is unknown. PTH decreased the proportion of cells in S phase and increased the number of G1 phase cells. We examined the effect of PTH on the regulators of the G1 phase cyclin-dependent kinases and found that PTH increased p27Kip1, but not p21Cip1, levels. This effect was mimicked by 8-bromo-cAMP, but not by phorbol 12-myristate 13-acetate. The protein kinase A inhibitor KT5720 abolished the effect of PTH on the increase in p27Kip1 expression. PTH increased CDK2-associated p27Kip1 without affecting the levels of CDK2. CDK2 activity was down-regulated by both PTH and 8-bromo-cAMP treatment. These data suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1, which is mediated through the protein kinase A pathway. The inhibition of G1 cyclin-dependent kinases by p27Kip1 could cause a reduction of phosphorylation of key substrates and inactivation of transcription factors essential for entry into S phase. The inhibition of cell cycle progression through PKA-mediated p27Kip1 induction might play an important role in PTH-induced differentiation of osteoblasts.

scholarly journals Various factors affect lipopolysaccharide sensitization in cell cultures

BioTechniques ◽  
2020 ◽  
Vol 69 (2) ◽  
pp. 126-132
Author(s):  
Nan Yang ◽  
Don D Sin ◽  
Delbert R Dorscheid
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Signal Loss ◽  
The Media ◽  
Fetal Bovine

Commercially available lipopolysaccharide (LPS) is commonly used in research. Although protocols for its use are well established, we experienced a loss of LPS responsiveness in our cell cultures despite no obvious experimental changes. Our cell lines were stimulated with LPS and the media quantified for LPS responsiveness via an IL-8 ELISA. We discovered that the major cause of signal loss was differences in fetal bovine serum (FBS) formulation and concentration. One FBS formulation was notably better at eliciting an IL-8 signal than the second FBS, and 10% FBS in media was better at inducing LPS responsiveness than lower concentrations. We urge researchers to be aware of inherent variations in seemingly commonplace reagents as they may be unexpected sources of inconsistencies.

scholarly journals Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies

2016 ◽  
Vol 35 (2) ◽  
pp. 150-160 ◽  
Author(s):  
Flor M. Pérez-Campo ◽  
Tobias May ◽  
Jeannette Zauers ◽  
Carolina Sañudo ◽  
Jesús Delgado-Calle ◽  
...  
Keyword(s):  
Cell Lines ◽  
Osteoblastic Cell ◽  
Human Osteoblastic Cell
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