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Biomedical subjects

G A Rodan

Publications and source records attributed to G A Rodan.

At least 163 records · Page 9Linked to original sources
PubMed1987

Growth stimulation of rat calvaria osteoblastic cells by acidic fibroblast growth factor.

Purified acidic fibroblast growth factor (aFGF) from bovine brain stimulates the proliferation of calvaria-derived osteoblastic cells. Maximum stimulation, relative to corresponding controls, was seen at 0.2% serum (2- to 3-fold), and no stimulation was seen in the absence of serum or under serum replete conditions. The effect was dose-dependent with an ED50 of around 750 pg/ml (47 pM). aFGF (5 ng/ml) sustained the growth of calvaria cells in culture during multiple passages (72 days) at 0.2% serum. In DNA synthesis assays aFGF produced 2- to 4-fold stimulation; insulin-like growth factor I had a slight effect on DNA synthesis on its own, but enhanced the effect of aFGF 2-fold. In cells fully stimulated by epidermal growth factor (5-fold), aFGF had no further effect. Stimulation of DNA synthesis peaked at 5 ng/ml, while higher concentrations were inhibitory. Recombinant aFGF (bovine sequence) also stimulated cell proliferation (1.5-fold), and its potency was augmented by heparin (50 micrograms/ml), about 2-fold. Using simultaneous histochemical staining for alkaline phosphatase activity and [3H]thymidine nuclear uptake we found that aFGF stimulates DNA synthesis to the same extent in alkaline phosphatase-rich (osteoblastic) and alkaline phosphatase-poor (nonosteoblastic) cells. However, after cell division there is a significant decrease in PTH-responsive adenylate cyclase (2- to 3-fold) and in alkaline phosphatase levels (4- to 8-fold). These findings indicate that aFGF is mitogenic to rat calvaria osteoblastic cells, its action requires additional factors, and its growth stimulation is associated with a reduction in phenotypic expression.

Adenylyl Cyclases↗
PubMed1986

Type-beta transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells.

TGF-beta modulates growth and differentiation in many cell types. MC3T3E1 is a clonal non-transformed murine bone cell line which differentiates in culture. We tested the effect of porcine TGF-beta on the proliferation and differentiation of MC3T3E1 cells in monolayer cultures by following cell number, and alkaline phosphatase activity. TGF-beta treatment (2 ng/ml) altered the shape of MC3T3E1 cells from cuboidal to elongated/spindle-shape. TGF-beta inhibited the growth of MC3T3E1 by up to 40% (P less than 0.02) in a dose-dependent manner with half maximal inhibition at 1 ng/ml. Growth inhibition depended on serum concentration, maximal inhibition occurring at 2% serum. Expression of alkaline phosphatase, which peaks in vitro when the cells reach confluence, was strongly inhibited by TGF-beta, in a dose-dependent manner with half maximal inhibition at around 0.05 ng/ml and complete inhibition at 2 ng/ml. Alkaline phosphatase inhibition was irreversible after 24 hours exposure to TGF-beta.

Alkaline Phosphatase↗
PubMed1986

Clonal differences in prostaglandin synthesis among osteosarcoma cell lines.

This study compares the metabolism of [14C]-arachidonic acid between PGE2 synthesizing (ROS 17/2.8) and nonsynthesizing (ROS 25/1) osteosarcoma cell lines. In both cell lines: (a) 90% of [14C]-arachidonic acid was taken up at 24 h. (b) More than 90% of the label was associated with phospholipids. (c) [14C]-arachidonic acid was rapidly taken up by phosphatidylcholine which reached the highest specific activity around 5 h while the labeling of other phospholipids was still increasing at 24 h. (d) Twenty-four hours after addition of [14C]-arachidonic acid only 4% of the label was associated with triacylglycerols in ROS 25/1 and 0.3% in ROS 17/2.8 cells. The calcium ionophore A23187 enhanced the release of [14C]-arachidonic acid from phospholipids in the PGE synthesizing osteoblastic cells (ROS 17/2.8 and 2/3) but had no effect in nonosteoblastic cells (ROS 24/1 and 25/1). ROS 17/2.8 and 2/3 cells converted the released arachidonic acid as well as exogeneously added arachidonic acid into PGE2. PGE2 synthesis depended on arachidonic acid concentration. Among bone resorbing agents, parathyroid hormone and 1,25(OH)2D3 had no effect on PGE synthesis, whereas thrombin and rabbit serum stimulated PGE2 production. The effect of rabbit serum was abolished by heat inactivation. The findings of this study indicate that the difference in PGE production between the osteoblastic and nonosteoblastic osteosarcoma cells are due mainly to differences in arachidonic acid conversion to PGE2.

1-Methyl-3-isobutylxanthine↗
PubMed1986

The effect of 1,25-dihydroxyvitamin D3 on the cytoskeleton of rat calvaria and rat osteosarcoma (ROS 17/2.8) osteoblastic cells.

1,25-dihydroxyvitamin D3 produces pronounced shape changes in fetal rat calvaria and osteosarcoma-derived (ROS 17/2.8) osteoblastic cells, characterized by retracting processes and cell rounding followed by aggregation of cells. The 1,25(OH)2D3 effect on ROS 17/2.8 morphology was determined morphometrically on scanning electron micrographs. The hormone effect was found to be dose dependent between 10(-12) and 10(-9) M. The shape changes appeared 12 h after hormone (10(-10) M) addition and were present in 80% of the ROS 17/2.8 cells and in 50% of the calvaria cells at 72 h. Cycloheximide at 1 microM, inhibited the hormone-dependent change in morphology. The 1,25(OH)2D3 effects were partially mimicked by 10(-8) M 25(OH)D3 but not by 10(-10) M 25(OH)D3 or 10(-11)-10(-8) M 24,25(OH)2D3. 1,25-dihydroxyvitamin D3 also increased cell proliferation twofold at 14 days in serum-free medium. 1,25(OH)2D3 treatment produced changes in microfilament organization, visualized with rhodamine-conjugated phalloidin. Microfilaments were localized at the terminal attachment points and in the perinuclear region, and few if any, were seen in the retracting processes themselves. Estimation of cytoskeletal actin and myosin by gel electrophoresis of Triton X-100 nonextractable proteins showed a 30% reduction in these proteins in the hormone-treated cells. Microtubules visualized by indirect immunofluorescence showed no major changes in organization. Both colchicine and cytochalasin D altered the hormone-induced shape change, suggesting that both microfilaments and microtubules were required for this process. Thus, 1,25(OH)2D3 had pronounced effects on cell shape in osteoblastic cells, probably via de novo protein synthesis. These changes lead to rearrangement of the cytoskeleton, primarily the microfilaments.

Actins↗
PubMed1986

Glucocorticoid treatment facilitates cyclic adenosine 3',5'-monophosphate-dependent protein kinase response in parathyroid hormone-responsive osteogenic sarcoma cells.

Late passage cultures of a clonal osteogenic sarcoma line (ROS 17/2.8) failed to respond to PTH with activation of cAMP-dependent protein kinase isoenzymes despite showing a sensitive and dose-dependent increase in cAMP after treatment with the hormone. When cells were treated with hydrocortisone or dexamethasone, protein kinase responsiveness to PTH was readily demonstrated; such treatment also resulted in enhanced cAMP production. Forskolin preincubation resulted in a cAMP response to PTH of similar magnitude to that seen with hydrocortisone but no activation of cAMP-dependent protein kinase occurred. Thus, the effect of glucocorticoid cannot be explained merely by the increased amplitude and sensitivity of the cAMP response which developed with glucocorticoid treatment in these cells. The data indicate that cellular activation of cAMP-dependent protein kinase does not automatically follow cAMP generation and that information transfer can be restored by pharmacological means.

Adenylyl Cyclases↗
PubMed1986

Dexamethasone effects on beta-adrenergic receptors and adenylate cyclase regulatory proteins Gs and Gi in ROS 17/2.8 cells.

Treatment of ROS 17/2.8 cells with dexamethasone (dex) increases (-)isoproterenol (ISO)-, PTH-, cholera toxin-, guanine nucleotide-, NaF-, and forskolin-stimulated adenylate cyclase activity. Enhanced hormone stimulation was first apparent 12 h after dex addition. (-)-[3H]Dihydroalprenolol binding, displaceable by ISO, increased up to 2-fold in dex-treated cells. This effect depended on protein synthesis and closely paralleled the extent and time course of the increase in adenylate cyclase stimulation. In dex-treated cells there was also an increase in the maximum velocity of guanyl-5'-yl imidodiphosphate-stimulated adenylate cyclase, a decrease in the lag time for guanyl-5'-yl imidodiphosphate enzyme activation in the presence of ISO from 3 to 1 min, increased stimulation of adenylate cyclase by cholera toxin, and increased labeling of 47,000 and 42,000 mol wt proteins by [32P]NAD in the presence of cholera toxin. [32P]NAD ribosylation in the presence of pertussis toxin resulted in the labeling of 40,000 mol wt protein, which was also increased by 20-50% in dex-treated cells. However, pertussis toxin treatment did not augment or reduce the effect on hormone stimulation, although it increased the cAMP response to PTH and (-)ISO. These findings suggest that dex increases (-)ISO stimulation of adenylate cyclase in ROS 17/2.8 cells by jointly increasing the number of hormone receptors and the abundance of Gs, the guanine nucleotide binding regulatory protein.

Adenosine Diphosphate Ribose↗
PubMed1985

Regulation of creatine kinase activity in rat osteogenic sarcoma cell clones by parathyroid hormone, prostaglandin E2, and vitamin D metabolites.

We have previously shown that both parathyroid hormone (PTH) and prostaglandin E2 (PGE2) stimulate the activity of creatine kinase BB (CKBB) in rat bone cells in culture. Therefore, morphologically distinct rat osteogenic sarcoma cells in culture were tested for stimulation of CKBB activity by hormones that regulate skeletal tissues. PTH stimulated CKBB in the osteoblast-like clone ROS 17/2; 1 alpha,25(OH)2D3 inhibited this activity while PGE2, CT and 24R,25(OH)2D3 had no significant effect. PGE2 stimulated CKBB activity in the fibroblast-like clone ROS 24/1, which was unresponsive to PTH, CT and Vitamin D metabolites. 24R,25(OH)2D3 as well as PGE2 (but not PTH, CT or 1 alpha 25(OH)2D3) stimulated CKBB in clone ROS 25/1, suggesting that this fibroblast-like clone has some chondroblast-like character. Both PTH and PGE2 stimulated the brain type isoenzyme of CK (CKBB), although the osteogenic sarcoma cell clones contain a significant proportion of the muscle type of CK (CKMM). Thus, increased CKBB activity can serve as an additional characteristic marker for the action of steroid and polypeptide hormones and for prostaglandins.

24,25-Dihydroxyvitamin D 3↗
PubMed1985

Effects of thionaphthene 2-carboxylic acid and related compounds on bone resorption in organ culture.

We have compared the effects of thiophene 2-carboxylic acid (TCA) and a number of sulfur- and nitrogen-containing analogs for their ability to inhibit bone resorption in organ cultures of fetal rat long bones. Four compounds,--thionaphthene-2-carboxylic acid (TNCA), dibenzo-thiophene-4-carboxylic acid, indole-2-carboxylic acid and carbazole-1-carboxylic acid--caused a dose-related inhibition of PTH-stimulated bone resorption, although TCA was ineffective in this system. TNCA at 3 X 10(-4) M or 10(-4) M was the most potent inhibitor of PTH-stimulated bone resorption and was selected for further study. TNCA also inhibited stimulation of resorption by prostaglandin E2 and 1,25-dihydroxyvitamin D. Unlike calcitonin, the effect of TNCA was persistent and did not show escape. Moreover, TNCA could inhibit resorption in bones that had previously escaped from calcitonin. TNCA did not appear to be a nonspecific toxin, since it did not decrease incorporation of [3H]thymidine or [3H]proline into fetal rat long bones. The fact that resorption in unstimulated cultures was only decreased when the control rates were high also argues against nonspecific toxicity. Moreover, this suggests that TNCA will be most effective under conditions of accelerated bone resorption when an inhibiting effect is most desirable.

Animals↗
PubMed1985

Glucocorticoid regulation of alkaline phosphatase in the osteoblastic osteosarcoma cell line ROS 17/2.8.

Dexamethasone increased alkaline phosphatase levels up to 7-fold in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. This effect was associated with reduced cell growth and took place over several days in culture. The increase in enzyme activity was dose dependent, (half-maximum near 1 nM, with a hormone specificity suggesting glucocorticoid receptor mediation). Dexamethasone also increased enzyme activity in ROS 2/3 cells, but not in two nonosteoblastic osteosarcoma cell lines, indicating that among these cell lines, the effect is specific for osteoblast-like cells. Moreover, enzyme activity in both control and dexamethasone-treated cells correlated directly with levels of radioimmunoassayable bone-type isoenzyme. Increases in alkaline phosphatase activity in response to dexamethasone were detectable after about 5 h and were inhibited by both actinomycin D and cycloheximide. Thus glucocorticoids appear to increase de novo enzyme synthesis in ROS 17/2.8 cells. Finally, the cAMP-elevating agents PTH, isoproterenol, and 8-bromo-cAMP, which were previously shown to reduce alkaline phosphatase activity in osteoblast-like cells, antagonized the effects of dexamethasone. Moreover, in the presence of dexamethasone, lower concentrations of these agents were required for inhibitory effects on alkaline phosphatase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Thionapthene-2-carboxylic acid: a new antihypercalcemic agent.

Thiophene-2-carboxylic acid (TCA) was previously shown to be hypocalcemic in the rat. We have compared TCA with thionapthene-2-carboxylic acid (TNCA), an analog which is a more potent inhibitor of bone resorption in vitro, for the ability to decrease serum calcium concentration in vivo. In normocalcemic rats on a low calcium diet, TNCA (2 mmol/kg) produced a larger and more prolonged decrease in calcium concentration than TCA. In rats bearing the Walker 256 carcinosarcoma, which became hypercalcemic, TNCA produced a dose-related decrease in serum calcium concentration at 0.3-1.2 mmol/kg. TNCA reduced serum calcium concentration in 4-6 h, and the effects were sustained for up to 72 h. TNCA was effective after oral as well as sc administration. Unlike calcitonin which produced only a transient reduction in serum calcium followed by escape, the effects of TNCA (0.6 mmol/kg) were sustained for 1 week and were accompanied by a decrease in mortality in tumor-bearing animals. We conclude that TNCA is a potent hypocalcemic action which has a rapid and prolonged effect without evidence of escape. This and related compounds should be tested further for use in treatment of hypercalcemia and other states characterized by excessive bone resorption.

Administration, Oral↗
PubMed1984

Tensile forces enhance prostaglandin E synthesis in osteoblastic cells grown on collagen ribbons.

An experimental system has been developed to examine the prostaglandin (PG) production induced by tensile mechanical forces in bone cells cultured on collagen ribbons. Fetal rat calvaria cells (osteoblast-enriched) were grown on collagen ribbons. The collagen ribbons were stretched under culture conditions in a machine that recorded force and displacement. Repeated stretching of the collagen ribbons (8 times, 5-10%, over 2 hours) increased the rate of prostaglandin synthesis approximately 3.5-fold over that of cells on nonstretched ribbons. This system should provide a suitable method to quantitatively study the effect of mechanical forces on various parameters of PG synthesis.

Animals↗
PubMed1984

The effect of dexamethasone on parathyroid hormone stimulation of adenylate cyclase in ROS 17/2.8 cells.

Treatment of ROS 17/2.8 osteosarcoma-derived cells with dexamethasone potentiates the PTH stimulation of adenylate cyclase in these cells, yielding a detectable response to as little as 10 pM PTH. Isoproterenol stimulation was also enhanced. The dexamethasone effect is first apparent at 12 h and increases with time of treatment. The apparent EC50 for dexamethasone is 3 nM. Hydrocortisone and corticosterone act similarly to dexamethasone, but require 30-fold higher concentrations. Dexamethasone treatment produces no change in high affinity phosphodiesterase activity. Glucocorticoid-potentiating effects are much more pronounced in whole cells than in broken cells and do not influence forskolin stimulation. Particulate fractions of dexamethasone-treated cells have higher adenylate cyclase specific activity, but are stimulated by guanyl-5'-yl imidodiphosphate to the same extent as control cells. These findings suggest that the glucocorticoids potentiate hormone responsiveness through promotion of hormone receptor-adenylate cyclase coupling by a mechanism dependent on cellular integrity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoblastic cells.

The culture media of three cell lines, a human prostate carcinoma (PC3), a rat Leydig cell tumor (Rice-500), and a rat carcinosarcoma (WRC-256), that were derived from tumors associated with humoral hypercalcemia of malignancy (HHM), were examined for stimulation of adenylate cyclase in ROS 17/2.8 osteoblastic cells and for bone resorptive activity in culture. Cells from a nonhypercalcemic variant of the WRC256 tumor served as control. Extracts from three solid human tumors, a lung adenocarcinoma from a patient with HHM and two adenocarcinoma from normocalcemic patients (lung and colon), were also examined for adenylate cyclase stimulation. We found excellent correlation between stimulation of cyclic AMP accumulation in ROS 17/2.8 cells and bone resorbing activity in culture, or production of HHM in vivo. Stimulation of adenylate cyclase by HHM factors was inhibited by the parathyroid hormone competitive inhibitor, [8norleucyl, 18norleucyl, 34tyrosinyl] bovine parathyroid hormone (3-34) amide.

Adenylyl Cyclases↗
PubMed1982

The effect of 1,25(OH)2D3 on alkaline phosphatase in osteoblastic osteosarcoma cells.

The effects of 1,25-dihydroxyvitamin D3 on growth, cell protein, and alkaline phosphatase were examined in cultures of a clonal osteoblast-like rat osteosarcoma cell line at different stages of phenotypic maturation, defined by the level of alkaline phosphatase activity. In "immature" cultures with low alkaline phosphatase specific activity, 1,25-dihydroxyvitamin D3 at 10(-8)-10(-7) M inhibited cell growth and elevated alkaline phosphatase activity; lower hormone concentrations had no effect. In cultures with higher alkaline phosphatase activity, the hormone had biphasic effects: at concentrations of 10(-8)-10(-7) M, 1,25-dihydroxyvitamin D3 inhibited growth and elevated alkaline phosphatase and total cell protein; however, lower hormone concentrations (10(-10) and 10(-9) M) reduced alkaline phosphatase activity. This effect was more pronounced in more "mature" cultures where half-maximum inhibition of alkaline phosphatase activity occurred near 10(-11) M. At this stage, the hormone caused a small increase in cell number. The data suggest that 1,25-dihydroxyvitamin D3 exerts differential effects on skeletal cells depending on their state of growth or maturation. This finding may offer new insights into the effects of 1,25-dihydroxyvitamin D3 on bone and may help resolve apparently conflicting experimental findings on the effects of the hormone on osteoblast-like cells in culture.

Alkaline Phosphatase↗
PubMed1982

Alkaline phosphatase inhibition by parathyroid hormone and isoproterenol in a clonal rat osteosarcoma cell line. Possible mediation by cyclic AMP.

The effect of parathyroid hormone (PTH 1-34 bovine) on alkaline phosphatase activity was investigated in an osteoblast-like clonal cell line derived from rat osteosarcoma (ROS 17/2). ROS 17/2 alkaline phosphatase resembled the bone enzyme in levamisole sensitivity and electrophoretic mobility but differed in heat stability. The specific activity of ROS 17/2 alkaline phosphatase increased with time in culture. This increase was inhibited by PTH (1-34) and (-)-isoproterenol in a dose-dependent manner starting at near-physiological hormone concentrations. The ID50 values were 0.02 nM for PTH (1-34) and 1.7 nM for isoproterenol. The two hormones stimulated ROS 17/2 adenylate cyclase, albeit at higher concentrations: Km values were 13 nM for PTH (1-34) and 16 nM for isoproterenol. The rise in alkaline phosphatase was also inhibited by dibutyryl cyclic AMP and 8-bromocyclic AMP (0.1 mM). These findings further document the osteoblastic properties of the ROS 17/2 osteosarcoma cell line, suggest that PTH inhibition of alkaline phosphatase represents a physiological response to the hormone in these cells, and implicate cyclic AMP as a mediator of this PTH effect.

Alkaline Phosphatase↗
PubMed1982

Unidirectional migration of osteosarcoma cells with osteoblast characteristics in response to products of bone resorption.

To investigate the mechanisms by which bone-forming cells are attracted to areas of bone resorption during bone remodeling, we have used in vitro methods to look for signals released by resorbing bone, which may be chemotactic for cultured bone cells. We have found that cultured rat osteosarcoma cells, which have characteristics associated with the osteoblastic phenotype, migrate in a unidirectional manner in response to a signal released by resorbing bones. These cells also migrated unidirectionally in response to Type I collagen, which comprises 95% of the bone matrix. This phenomenon of chemotaxis of bone-forming cells to sites of previous resorption may be an important component of the process of bone remodeling and the coupling of bone formation to bone resorption.

Animals↗