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N C Partridge

Publications and source records attributed to N C Partridge.

At least 55 records · Page 3Linked to original sources
PubMed1989

Parathyroid hormone inhibits collagen synthesis at both ribonucleic acid and protein levels in rat osteogenic sarcoma cells.

Cells of the clonal rat osteogenic sarcoma cell line, UMR 106-01, were used to investigate the regulation of collagen synthesis by PTH in osteoblastic cells. Monolayer cultures of cells were labeled with [3H] proline in order to determine both collagen type and rates of production. Analysis of labeled extracellular polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that UMR 106-01 cells synthesized predominantly type I collagen, accounting for 45.48 +/- 2.09% of the radioactivity incorporated into total protein. After 24-h treatment with bovine PTH (1-34, 10(-8) M), collagen synthesis (i.e. collagenase-digestible protein) was decreased to 29.45 +/- 1.39% of total protein production. This decrease was first observed 12 h after addition of hormone and greatest inhibition was achieved at 24 h. The effect of PTH was dose dependent, with half-maximal inhibition of collagen synthesis occurring at 5 x 10(-10) M after 24-h treatment. In contrast, when steady state levels of mRNA for type I collagen chains were examined by Northern blot analysis, the concentration of PTH that reduced collagen synthesis by 35-45% (10(-8) M), caused a net decrease of approximately 80-96% in the number of procollagen transcripts; a small reduction in beta-actin mRNA levels was also observed. The effect of the hormone on procollagen message level was dose dependent, with significant inhibition observed at 10(-10) M PTH and, as with collagen synthesis, maximal after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Hormonal influences on bone cells.

The methods for establishing osteoblast-rich rat calvarial cell cultures have been described, together with methods for the use of clonal osteogenic sarcoma cells of osteoblast phenotype. The latter clonal lines are useful for several purposes, but all the precautions and quality control measures necessary in the study of clonal lines must be observed. Some of the techniques for studying biochemical responses to hormones in these cells have also been detailed, but clearly others are applicable, including studies of the synthesis of matrix constituents. Osteoclast-like cells have not been considered in this chapter, because osteoclast culture methods have not yet been developed to the degree of purity and reproducibility necessary for this type of biochemical approach.

Adenylyl Cyclases↗
PubMed1987

Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells.

Collagenases that specifically cleave native collagen at neutral pH have been implicated in the maintenance and turnover of connective tissue. In bone, the origin of neutral collagenase has remained equivocal, although recent studies have indicated that it is synthesized by the osteoblast. In the present work, regulation of secretion of neutral collagenase and a collagenase inhibitory activity was investigated using the osteoblastic tumor cell line UMR 106-01 and a variety of bone-resorbing agents. Under basal conditions, UMR 106-01 cells produced very low levels of collagenase but substantial amounts of the inhibitory activity. Exposure to PTH and, to a lesser extent, 1,25-dihydroxyvitamin D3, prostaglandin E2, retinoic acid, and epidermal growth factor stimulated the release of collagenase, an effect not seen with interleukin-1 or heparin. The stimulation of collagenase by PTH was dose dependent, with a half-maximal response occurring at 10(-8) M. Inclusion of isobutylmethylxanthine decreased the concentration of PTH required to produce half-maximal stimulation to 2 X 10(-10) M, indicating action via cAMP. With respect to the inhibitory activity, PTH and epidermal growth factor were the only agents, among those tested, able to enhance its production. Both hormones caused a 50-100% increase over control levels 72 h after hormone administration. There were notable differences in the time courses of production of collagenase and the inhibitor. After treatment with PTH, the enzyme reached maximal concentrations between 12-48 h, but declined to undetectable levels by 96 h. In contrast, the inhibitory activity was secreted in a linear fashion, with the highest concentrations achieved around 72-96 h. These results suggest a complex pattern of regulation of collagenase and inhibitor secretion by the osteoblastic cell, with the steady accumulation of inhibitor perhaps being responsible for the ultimate curtailment of enzyme activity.

Animals↗
PubMed1985

Characterization of an osteoblast-like clonal cell line which responds to both parathyroid hormone and calcitonin.

The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.

Acid Phosphatase↗
PubMed1985

Inhibitory effects of parathyroid hormone on growth of osteogenic sarcoma cells.

The effects of the bone resorbing hormone, parathyroid hormone (PTH), on the growth of malignant osteoblastic cells have been examined. The malignant osteoblastic cells were a clonal line (UMR 106) derived from a transplantable rat osteogenic sarcoma. The predominant effect of PTH at doses above 10(-10) M was an inhibition of replication and DNA synthesis. Replication was decreased by PTH in both the presence or absence of serum and at various cell seeding densities. Both bovine PTH (1-84) and the synthetic hormone, human PTH (1-34), inhibited replication, but with bovine hormone being an order of magnitude more potent. The effects could be observed in as short a time as 6 hours with DNA synthesis and 24 hours with replication.

Animals↗
PubMed1985

Regulation of prostaglandin production by osteoblast-rich calvarial cells.

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were all shown to stimulate PGE2 and 6-keto-PGF1 alpha (the hydration product of PGI2) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE2 and PGI2 (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occurring at approximately 10(-7) M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments in vivo which have indicated that the compounds stimulated PGI2 production. Finally, since the osteoblast is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early times EGF stimulated PGE2 release, however, the predominant effect of the growth factor was an inhibition of both PGE2 and PGI2 production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the osteoblast and that any increase in PG production by these cells may be in response to vascular agents.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Regulation of plasminogen activator production by bone-resorbing hormones in normal and malignant osteoblasts.

The plasminogen activator (PA) activity of clonal rat osteogenic sarcoma cell (phenotypically osteoblast) and of osteoblast-rich rat calvarial cells is shown to be increased by treatment with the bone-resorbing hormones, PTH, 1,25-dihydroxyvitamin D3, prostaglandin E2, and epidermal growth factor. Dose-dependent increases were observed, after a lag period of 4 to 8 h. Stimulated and control PA activities were inhibited by actinomycin D and cycloheximide but not by cytosine arabinoside. Glucocorticoid hormones prevented the hormone stimulation, but other steroids did not. Calcitonin had no effect either on basal or on hormone-treated PA activity. Isobutyl-methylxanthine alone increased PA activity and enhanced responsiveness to PTH and to prostaglandin E2. These data point to a common pathway in the actions upon osteoblasts of several hormones with diverse initial cellular actions and raise the possibility that the PA/plasmin system may contribute to cellular mechanisms of bone turnover.

Animals↗
PubMed1984

Stimulation of plasminogen activator in osteoblast-like cells by bone-resorbing hormones.

Hormonal control of plasminogen activator (PA) was studied in clonal rat osteogenic sarcoma cells which are phenotypically osteoblast, and in osteoblast-rich rat bone cell cultures. The bone-resorbing hormones (parathyroid hormone, prostaglandin E2, epidermal growth factor and 1,25-dihydroxyvitamin D3) stimulated PA activity in both cell types. The relative efficacies of vitamin D metabolites and of prostanoids reflect their relative potencies as stimulators of bone resorption.

Animals↗
PubMed1983

Cyclo-oxygenase products of arachidonic acid metabolism in rat osteoblasts in culture.

The metabolism of arachidonic acid to its cyclo-oxygenase products was studied in monolayer cultures of osteoblast-rich rat calvarial cells and of clonal cell lines from a rat osteogenic sarcoma, enriched in the osteoblast phenotype. Prostanoids were measured by radioimmunoassay after extraction of media and fractionation by high pressure liquid chromatography. In both normal and malignant osteoblasts the major cyclooxygenase product was 6-oxo-prostaglandin F1 alpha, the hydration product of prostacyclin, with lesser amounts of prostaglandin E2 and prostaglandin F2 alpha. No significant thromboxane B2 was detected. Prostaglandins are thought to have a local role in the regulation of bone resorption. These results point to the possible importance of prostacyclin either in bone resorption or in some other local function, e.g., regulation of bone blood flow.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

Epidermal growth factor receptors in clonal lines of a rat osteogenic sarcoma and in osteoblast-rich rat bone cells.

Studies were carried out to identify and characterize the receptors for epidermal growth factor (EGF) in osteoblast-rich newborn rat calvarial cells and in 4 clonal lines derived from a transplantable rat osteogenic sarcoma with a well-characterized osteoblast-like phenotype. The cells were grown in monolayer culture in replicate wells; 40,000-50,000 cpm 125I-labeled mouse EGF with a specific activity of 100-120 microCi/micrograms was added to each well. Binding studies were carried out at 37 degrees C. Binding of 125I-labeled EGF was specific, saturable, reversible, and pH dependent. Maximum binding occurred 2 h after addition of the tracer. Thereafter, cell-bound radioactivity decreased to reach a plateau of 15-20% of maximum binding at 24 h. This observation is consistent with internalization and processing of the receptor-hormone complex as has been shown with other EGF target cells. Scatchard analyses revealed a single class of high-affinity binding sites in the normal and malignant osteoblast-like cells. Dissociation constants (KD) in the clonal lines ranged from 2.3 X 10(-10)M to 4.7 X 10(-10)M with receptor number per cell ranging from 25,000 to 33,000. The calvarial cells had a KD of 2.0 X 10(-10)M with 14,000 receptors per cell. In both the normal and malignant cell strains, EGF was found to increase incorporation of 3H-labeled thymidine into acid-precipitable macromolecules. EGF has been shown to stimulate bone resorption; however, studies in organ cultures have not identified the target cell for EGF. The present results point to an interaction of EGF with osteoblasts.

Animals↗
PubMed1983

Stimulation of DNA synthesis by epidermal growth factor in osteoblast-like cells.

Normal and malignant osteoblast-like cells in culture have been shown to possess specific, high affinity receptors for epidermal growth factor (EGF). In this study, the mitogenic response to EGF was examined in a clonal line of a rat osteogenic sarcoma (UMR 106) and in osteoblast-rich newborn rat calvarial cells. Twenty-four hour treatment of UMR 106 cells with EGF in doses ranging from 10(-12) M to 2 X 10(-8) M stimulated the incorporation of [3H]thymidine and DNA synthesis in a dose-dependent manner. This short-term stimulatory effect was sustained in long-term culture with a dose-dependent increase in cell proliferation by calvarial cells. A lag period of 8 h occurred before significant stimulation of [3H]thymidine incorporation was observed. Commitment to increased incorporation of [3H]thymidine required a minimum of 6 h continuous incubation with EGF. These results establish the osteoblast as a target cell for EGF action on bone.

Animals↗
PubMed1983

Morphological and biochemical characterization of four clonal osteogenic sarcoma cell lines of rat origin.

The ultrastructural and biochemical properties of four clonal osteogenic sarcoma lines, UMR 104, 105, 106, and 108, have been compared with uncloned osteogenic sarcoma cells and normal osteoblast-rich cells derived from newborn rat calvaria. High alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone were used as biochemical markers of osteoblastic cells. Cloning enriched both of these parameters above those of the parent tumor and far higher than that seen in normal cells, suggesting enrichment of the osteoblast phenotype. Both of these properties have been retained through many passages in culture. Morphologically, the clonal lines have also retained the "blast"-like appearance of the uncloned osteogenic sarcoma cells and consist mainly of flat, relatively featureless cells. Many cells with mitotic figures were observed, indicating continuous cell division taking place in the malignant cells. Each clonal line gave rise to characteristic tumors when reinjected into rats. It is concluded that the clonal osteogenic sarcoma lines are highly differentiated tumor lines which have conserved the differentiated properties of the mature osteoblast, making them a suitable model for the study of the effects of hormones on the growth of a differentiated tumor, as well as for the study of hormonal regulation of the osteoblast.

Acid Phosphatase↗
PubMed1982

Selective hormonal activation of cyclic AMP-dependent protein kinase isoenzymes in normal and malignant osteoblasts.

The pattern of cyclic AMP-dependent protein kinase isoenzyme response to acute hormonal activation has been studied in cultured cells derived from rat osteogenic sarcoma and osteoblast-rich cells grown from newborn rat calvaria. Using multiple small anion exchange columns and a batch elution technique, a rapid method of separating the isoenzymes of cyclic AMP-dependent protein kinase was developed and the acute activation by parathyroid hormone and prostaglandin E2 of each isoenzyme was studied. Activation was rapid, being detectable at 5 s, maximal at 15-30 s, and persisting for up to 6 h. Both hormones showed a dose-dependent activation of each isoenzyme in both cell types, but the patterns of response differed. Parathyroid hormone predominantly stimulated isoenzyme I in the clonal osteogenic sarcoma cells but showed equivalent activation of each isoenzyme in calvarial cells. Prostaglandin E2 also predominantly stimulated isoenzyme I in the malignant cells, whereas in the calvarial strain there was a major effect on isoenzyme II with almost no stimulation of isoenzyme I. Half-maximal stimulation of cyclic AMP-dependent protein kinase in the malignant cell strain was achieved for both hormones at concentrations an order of magnitude lower than those in the normal strain. These studies demonstrate selective activation of cyclic AMP-dependent protein kinase isoenzymes by hormones. Furthermore, the nature of the response differs between the normal and the corresponding neoplastic cell types for the same hormone stimulus.

Animals↗
PubMed1982

Activity ratio measurements reflect intracellular activation of adenosine 3',5'-monophosphate-dependent protein kinase in osteoblasts.

Parathyroid hormone, prostaglandin E2, and prostacyclin activate cAMP-dependent protein kinase in osteoblast-rich normal rat calvarial cells and in clonal rat osteogenic sarcoma cells of osteoblastic phenotype. The present study was undertaken to determine the activation of the enzyme in relation to cellular cAMP concentrations at increasing doses of the three hormones and also to test that the activity ratio measurement of the enzyme (ratio of the activity in the absence of cAMP to the activity in the presence of excess cAMP) was a true reflection of intracellular activation of the enzyme. With each hormone, using either normal or malignant osteoblasts, activation of the enzyme took place at hormone concentrations lower than those required to produce detectable changes in cAMP concentrations in the incubations. Stimulation of activity was abolished by addition of the heat-stable inhibitor of cAMP-dependent protein kinase, indicating that activation was of cAMP-dependent protein kinase alone. To demonstrate that protein kinase activation occurred intracellularly and not during sample preparation, charcoal was added at the time of cell disruption to absorb free cAMP. Under these conditions, no change was observed in the concentration of bovine parathyroid hormone required to cause activation of cAMP-dependent protein kinase. Finally, addition of purified cAMP-dependent protein kinase type I or type II to treated cells at the time of lysis did not result in significant activation of added isoenzyme, except at hormone concentrations sufficient to increase the total cAMP concentration of incubations. It is concluded that activity ratio measurement reflects the intracellular state of activation of cAMP-dependent protein kinase in the osteoblast-like cells treated by hormones and, furthermore, that only a fraction of the maximally generated cAMP is necessary for full enzyme activation.

Animals↗
PubMed1981

Adenylate cyclase responsiveness of human insulinomas.

Adenylate cyclase activity was assayed in a crude particulate fraction of one benign and one malignant human insulinoma. Adenylate cyclase of both tumours responded to 5'-guanylyl-imidodiphosphate, sodium fluoride, glucagon and prostaglandin E2, and in addition the adenylate cyclase of the benign tumour responded to isoprenaline. Glucose and prostaglandin I2 (prostacyclin) did not stimulate the adenylate cyclase in either tumour, although prostaglandin I2 stimulated insulin secretion in cultures of the benign tumour. The in vitro responsiveness of the adenylate cyclase to glucagon did not correlate closely with the effect of glucagon on insulin secretion in vivo.

Adenoma, Islet Cell↗
PubMed1981

Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells.

Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.

Acid Phosphatase↗
PubMed1981

Activation of adenosine 3',5'-monophosphate-dependent protein kinase in normal and malignant bone cells by parathyroid hormone, prostaglandin E2, and prostacyclin.

Hormonal activation of cAMP-dependent protein kinase has been studied in cultured cells derived from a rat osteogenic sarcoma and in osteoblast-rich cells grown from newborn rat calvaria. Both cell strains contain adenylate cyclase activities which respond to parathyroid hormone (PTH) and a variety of prostanoids. PTH, prostaglandin E2 (PGE2), and prostacyclin (PGI2) were all capable of activating cAMP-dependent protein kinase(s) in suspensions of the two cell types. Activation was very rapid in all cases, being detectable at 10 sec and maximal between 30-60 sec. Using saturating concentrations of hormones, the protein kinase activity ratio remained elevated (between 0.6-0.9) for up to 35 min after the start of PGE2 stimulation, but declined toward basal activity ratio 5-10 min after stimulation with PTH or PGI2. Each of the hormones caused a dose-dependent increase in activation of cAMP-dependent protein kinase in both cell types. Half-maximal activation of the enzyme occurred at 2 X 10(-9) M bovine PTH for calvarial cells, at 10(-8) M bPTH for osteogenic sarcoma cells, and at 2-4 X 10(-8) M PGE2 and 1-3 X 10(-7) M PGI2 for both cell types. Maximal activation of protein kinase occurred before maximal cAMP accumulated, implying that only a fraction of cAMP is biologically significant. These two cell strains provide a useful means of analyzing postreceptor events in the hormonal regulation of bone cells.

Animals↗