Growth inhibition of clonal osteosarcoma cell-lines by low concentrations of glucocorticoid hormones

Eilam, Y.; Silbermann, Michael; Szydel, N.

doi:10.1016/0006-291x(80)91214-0

Cited by 9 publications

(4 citation statements)

References 11 publications

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“…5D shows that, after phosphatase treatment, there is an increased representation of the more rapidly migrating protein-DNA complex (VDR, lower arrowhead) and several additional high-mobility complexes with a corresponding decreased representation ofthe slower mobility complex (VDR, upper arrowhead). This suggests that phosphorylation of the vitamin D receptor and/or an accessory factor (37-39) is required for formation of the lower-mobility protein-DNA complex and is consistent with reports that phosphorylation of the receptor is required for both ligand binding (40) and for complex formation at the VDRE (41)(42)(43) (44,45) and in transformed osteosarcoma cells (46,47). The synergism observed in osteoblasts could reflect an effect on the state of differentiation (33) or an up-regulation of the vitamin D receptor (44) caused by dexamethasone.…”

Section: Resultssupporting

confidence: 70%

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Bortell

Owen

Shalhoub

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormonemediated transcriptional modulation is reflected by modifications in transcription factor binding at (s) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.Genes that support structural and metabolic properties of mature osteoblasts are expressed in actively proliferating transformed osteoblasts and osteosarcoma cells. This is in contrast to the predominant expression ofbone-related genes [e.g., osteocalcin (OC), osteopontin, and alkaline phosphatase] after completion ofproliferation (post-proliferatively) in normal diploid osteoblasts (1,2). It therefore appears that modified growth control in osteosarcoma cells (3) is accompanied by a loss of regulatory mechanisms that mediate the relationships between proliferation and differentiation associated with both progressive development and maintenance of the osteoblast phenotype. This is reflected by loss of the three-stage developmental sequence (4-6) that establishes a bone-tissue-like organization of multilayered cells in a mineralized extracellular matrix (4-10). Thus, in osteosarcoma cells, there is a striking deregulation of growth-related controls whereby proliferation supports initial stages of osteoblast phenotype development, whereas down-regulation of proliferation is required to signal induction of gene expression that is restricted to the post-proliferative period of osteoblast differentiation.To address molecular mechanisms associated with the altered proliferation-differentiation relationship in osteosarcoma cells, we have examined modifications in transcriptional control of cell growth and bone-...

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Section: Resultssupporting

confidence: 70%

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Bortell

Owen

Shalhoub

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…In the present studies, we focused on the influence of glucocorticoids on osteocalcin gene expression based on the well-documented effects of this steroid hormone on bone cell growth and differentiation, both in vivo as well as in vitro [Canalis, 1983;Chen et al, 1977;Eilam et al, 1980;Ekenstam et al, 1988;Lukert et al, 1986;Majeska et al, 1980Majeska et al, ,1985Morrison et al, 1989;Wong et al, 19901. Our results from studies carried out with two ROS 1 7 / 2 3 osteosarcoma cell sublines indicate that while dexamethasone does not significantly influence basal levels of osteocalcin gene expression as reflected by osteocalcin biosynthesis, cellular levels of osteocalcin mRNA, and osteocalcin gene transcription, dexamethasone reduces or abrogates the vitamin D upregulation of the osteocalcin gene.…”

Section: Discussionmentioning

confidence: 99%

Influence of dexamethasone on the vitamin D–mediated regulation of osteocalcin gene expression

Schepmoes

Breen

Owen

et al. 1991

J of Cellular Biochemistry

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The influence of dexamethasone on expression of the osteocalcin gene which encodes the most abundant non-collagenous and only reported bone-specific protein was examined in ROS 17/2.8 osteosarcoma cells which express a broad spectrum of genes related to bone formation. Consistent with previous reports, quantitation of cellular osteocalcin mRNA levels by Northern blot analysis, osteocalcin gene transcription by activity of the osteocalcin gene promoter fused to a chloramphenicol acetyl-transferase (CAT) mRNA coding sequence following transfection into ROS 17/2.8 cells, and osteocalcin biosynthesis by radioimmunoassay indicate that dexamethasone in a concentration range of 10(-6) to 10(-9) M only modestly modifies basal levels of osteocalcin gene expression. However, dexamethasone significantly inhibits these parameters of the vitamin D-induced upregulation of osteocalcin gene expression in both proliferating and in confluent ROS 17/2.8 cells. In this study, we observed that the extent to which abrogation of the vitamin D response occurs is dependent on basal levels of osteocalcin gene expression as reflected by a complete inhibition of the vitamin D-induced upregulation in a ROS 17/2.8K subline with low basal expression and only a partial reduction of the vitamin D stimulation in a ROS 17/2.8C subline with eightfold higher levels of basal expression. This effect of glucocorticoid appears to be at the transcriptional and post-transcriptional levels as demonstrated by a parallel decline in the cellular representation of osteocalcin mRNA, osteocalcin gene promoter activity, and osteocalcin biosynthesis. The complexity of the glucocorticoid effect on vitamin D-mediated transcriptional properties of the osteocalcin gene is indicated by persistence of sequence-specific protein-DNA interactions at two principal osteocalcin gene promoter regulatory elements, the osteocalcin (CCAAT) box which modulates basal level of transcription, and the vitamin D responsive element, where vitamin D-mediated enhancement of osteocalcin gene transcription is controlled.

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“…Thus bone cell responsiveness to GC is a function of both the culture system and upon the experimental conditions. Studies performed on different bone cell systems reveal that although GC generally repress cellular proliferation [Eilam et al, 1980;Canalis, 1983;Chen et al, 1983a;Chyun et al, 1984;Wonget al, 19901, there are reports of enhanced [Chen et al, 1983al andbiphasic effects [McCulloch andTenenbaum, 1986;Ng et al, 19891 on bone cell growth. Similarly, alkaline phosphatase also exhibits biphasic expression [Canalis, 19831 or inhibition [Chen and Feldman, 19791, but in most bone systems is increased by GC treatment [Hahn et al, 1984;Leboy et al, 1991;McCulloch and Tenenbaum, 1986;Wong et al, 1990;Majeska et al, 19851.…”

mentioning

confidence: 99%

Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

Shalhoub

Conlon

Tassinari

et al. 1992

J of Cellular Biochemistry

198

142

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To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.

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Growth inhibition of clonal osteosarcoma cell-lines by low concentrations of glucocorticoid hormones

Cited by 9 publications

References 11 publications

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Influence of dexamethasone on the vitamin D–mediated regulation of osteocalcin gene expression

Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

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