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)

Osteocalcin (OC) is a bone-specific vitamin D- responsive protein that is developmentally expressed during osteoblast differentiation. In transient transfection assays, as little as approximately 0.1 kilobase (kb) of the OC proximal promoter is sufficient for basal expression. Because eukaryotic genes are packaged as nucleosomes that contribute to both chromatin organization and transcriptional control, we functionally examined the activity of OC promoter constructs within a chromatin context. ROS 17/2.8 osteosarcoma cells were stably transfected with a series of rat OC promoter-reporter constructs, containing progressive 5'-deletions. The results demonstrate that in contrast to transient transfection assays, the proximal 0.11-kb promoter is no longer active when integrated in the genome. Progressive gain of basal expression with 0.35-, 0.53-, and 0.72-kb promoters suggests that upstream sequences facilitate the formation of an appropriate higher order nuclear structure, thereby potentiating the activity of the chromosomally integrated proximal promoter elements. This is consistent with location of both deoxyribonuclease I (DNase I)-hypersensitive sites and nuclear matrix protein-DNA interaction sites in the osteocalcin promoter. Vitamin D responsiveness in the stably transfected cells is obtained with the inclusion of 0.53 kb or additional upstream promoter sequences. Therefore, these sequences satisfy the requirements for binding of basal and enhancer transcription factors as well as interactions between them within a chromatin context. Both maximal basal expression and maximal vitamin D responsiveness are obtained with cells carrying either the 0.72-kb or the 1.1-kb promoter fragment. Cells carrying the 1.1-kb promoter show DNase I hypersensitivity at both the basal promoter and the vitamin D response element-containing domains, locations that also exhibit DNase I hypersensitivity in the endogenous OC promoter. In addition, we have documented changes in the basal activity and vitamin D responsiveness of the stably integrated 1.1 kb promoter as a function of cell density-mediated growth inhibition, which is accompanied by up-regulation of bone phenotypic genes. Thus, important aspects of OC gene transcriptional regulation that cannot be investigated in transient transfection assays can be addressed using ROS 17/2.8 cells stably transfected with OC promoter-reporter constructs.
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PMID:Basal and vitamin D-responsive activity of the rat osteocalcin promoter in stably transfected osteosarcoma cells: requirement of upstream sequences for control by the proximal regulatory domain. 860 77

Ketoconazole (keto) or liarozole (liaro), inhibitors of the cytochrome P450 enzymes that mediate vitamin D and A hydroxylations, could potentiate the antiproliferative effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analogs. Proliferation of MCF-7 and T47-D human breast cancer cells, MG-63 human osteosarcoma cells and HL-60 human promyeloid leukemia cells was concentration dependently inhibited by 1alpha,25(OH)2D3. The vitamin D analogs KH 1060 [20-epi-22-oxa-24,26,27-trihomo-1alpha,25(OH)2D3], RO 23-6010 [16-ene-23-yne-26-trifluoro-1,25(OH)2D2D3], ZXY 835 [20-epi-23-yne-25,26-epoxy-1alpha(OH)D3], and CD 99 [11alpha-methyl-1alpha,25(OH)2D3] were 150-,58-,16- and 7-fold more potent than 1alpha,25(OH)2D3 in inhibiting the proliferation of MCF-7 cells, respectively. A similar rank order of potency was observed in other cell lines. The antiproliferative effects of the vitamin D hormone and analogs was enhanced in MCF-7 cells when coincubated with 1 microM keto (7-, 10-, 5-, 25- and 1.3-fold more potent than in the absence of keto), respectively. The antiproliferative effect was less enhanced when 1alpha,25(OH)2D3 or its analogs KH 1060, ZXY 835 and RO 23-6010 were combined with liaro (3-, 7-, 2- and 3-fold, respectively). Keto and liaro did not markedly potentiate the activity of 1alpha,25(OH)2D3 or its analogs in MG-63 or HL-60 cells. These results suggest that differences in cellular metabolism can at least partially explain the different potency of vitamin D analogs. Moreover, the metabolism of vitamin D analogs is cell-type specific.
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PMID:Enhancement of antiproliferative activity of 1alpha,25-dihydroxyvitamin D3 (analogs) by cytochrome P450 enzyme inhibitors is compound- and cell-type specific. 864 29

We have compared the actions of calcitriol and its three synthetic analogs, 20-epi-22-oxa-24a,26a,27a-trihomo-1 alpha,25-dihydroxyvitamin D3 (KH 1060), 1 alpha,24S-(OH)2-22-ene-26,27-cyclopropyl vitamin D3 (MC 903) and 20-epi-1 alpha,25-dihydroxyvitamin D3 (MC 1288), on the expression of two marker genes of differentiated osteoblasts, namely alkaline phosphatase and osteocalcin, using human MG-63 osteosarcoma cells. Calcitriol and the analogs had qualitatively similar stimulatory effects on target-gene activation. Quantitatively, MC 903 behaved in most experiments essentially as the parent compound calcitriol. In vitamin D receptor/DNA complex formation MC 903, however, was more potent than calcitriol. In contrast, the 20-epi analogs, KH 1060 and MC 1288, were much more potent even at lower concentrations, than calcitriol and MC 903 in stimulating alkaline phosphatase activity, osteocalcin mRNA synthesis and osteocalcin secretion. The stimulation occurred to a greater degree and for a longer period than with calcitriol. This effect was apparently mediated by stronger and longer lasting binding of the vitamin D receptor to the osteocalcin vitamin D responsive element by the 20-epi analogs. After a 6-h treatment and during subsequent culture without hormone, the effects of the 20-epi analogs were also stronger and lasted longer than those with calcitriol or MC 903. Collectively, at comparable and lower concentrations, the 20-epi analogs, KH 1060 and MC 1288, mediate much stronger and longer lasting stimulatory effects than calcitriol or its analog MC 903 on target-gene expression associated with the differentiated phenotype of the MG-63 human osteosarcoma cells.
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PMID:Synthetic 20-epi analogs of calcitriol are potent inducers of target-gene activation in osteoblastic cells. 866 57

Unlike estrogen and progesterone receptors that operate as homodimers on response elements, retinoid X receptors (RXRs) and vitamin D receptors (VDRs) can function as heterodimers. Studies concerning the significance of heterodimeric partnerships are usually performed utilizing mammalian or insect cells. These cells express endogenous nuclear receptors, making it impossible to assign a role for one receptor subtype over another while studying the function of transfected receptor(s). Yeast lacks endogenous VDRs and RXRs and their ligands and provides a unique cellular context to study nuclear receptor function. We examined the interaction between human VDR and human RXR alpha, mouse RXR beta 2, and mouse RXR gamma to identify physiologically important receptor interactions. DNA binding studies on consensus, osteocalcin, or the rat 24-hydroxylase vitamin D response elements (VDREs) indicated that although RXR complexes can form on the consensus DNA elements, RXR:VDR heterodimers preferentially interact with the natural VDREs. The interaction is RXR isotype-specific and affected by ligands. Transactivation studies using the rat 24-hydroxylase VDREs indicated that VDR preferentially associated with RXR alpha or RXR gamma to stimulate transcription, and the activity was potentiated by ligand. Although RXR beta 2:VDR bound tightly to DNA, the resulting heterodimer transactivated poorly. The regulation of the 24-hydroxylase promoter observed in yeast is similar with respect to transactivation potential of specific VDRE and fold activation observed in osteosarcoma cells. Ligand binding to both receptors in a RXR:VDR complex is required for maximal transcriptional activity, indicating that the isotype-specific RXR partner significantly contributes to the ability of RXR:VDR heterodimers to transactivate from target response elements in yeast.
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PMID:Retinoid X receptor isotype identity directs human vitamin D receptor heterodimer transactivation from the 24-hydroxylase vitamin D response elements in yeast. 872 85

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyl-transferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25-(OH)2D3 responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25-(OH)2D3 responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D3. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D3. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2,8 cells and from vitamin D receptor. (VDR)-enriched COS 1 cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25-(OH)2D3 is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.
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PMID:DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression. 877 27

Fibronectin (FN) is an important adhesive noncollagenous glycoprotein involved in maintenance of the extracellular matrix and cell adhesiveness, loss of which has been implicated in the metastatic potential of cells. Regulation of FN occurs at the transcriptional level by the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Transient transfection of homologous and heterologous promoter reporter constructs into ROS 17/2.8 (rat osteosarcoma), NIH 3T3 (mouse fibroblast), and MCF-7 (human mammary carcinoma) cell lines showed a consistent two- to threefold induction of transcription when stimulated with 1,25-(OH)2D3. These heterologous promoter transfection studies with gel shift analysis locate a third, natural DR6-type vitamin D responsive element (VDRE) at nucleotide positions -171 to -154 in the murine FN promoter. Interestingly, this VDRE is also present in rat and human FN promoters. This study shows that 1,25-(OH)2D3 induces FN transcription from an existing elevated basal transcriptional activity by acting through two putative hexameric core binding motifs which bind VDR homodimers. Furthermore, the FN VDRE is the first homodimer-type VDRE that is not overlaid by a DR3-type structure.
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PMID:Identification of a vitamin D3 response element in the fibronectin gene that is bound by a vitamin D3 receptor homodimer. 886 8

The actions of the hormonal form of vitamin D, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2 D3], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1 alpha, 25-(OH)2D3 in many cell types including osteoblastic cells. We have compared the effects of 1 alpha, 25-(OH)2D3 and a novel 1 alpha, 25-(OH)2D3 bromoester analog (1,25-(OH)2-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 x 10(-8) M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)2-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1 alpha, 25-(OH)2D3 and 1,25-(OH)2-BE intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 x 10(-8) M, both 1 alpha,25-(OH)2D3 and 1, 25-(OH)2-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)2-BE are similar to those of 1 alpha,25-(OH)2D3, and since 1,25-(OH)2-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors.
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PMID:Effects of the vitamin D3 analog 1 alpha, 25-dihydroxyvitamin D3-3 beta-bromoacetate on rat osteosarcoma cells: comparison with 1 alpha, 25-dihydroxyvitamin D3. 891 81

Osteocalcin (OC) is a bone matrix protein, synthesized by osteoblasts, which contains three residues of gammacarboxyglutamic acid (GLA). A fraction of circulating OC, which is not fully carboxylated and does not bind to hydroxyapatite, is called undercarboxylated OC (ucOC). In elderly institutionalized women, we have shown an increase of circulating ucOC level which may result not only from vitamin K deficiency but also from vitamin D deficiency (Szulc et al., J Clin Invest 91:1769; 1993). This intriguing finding prompted us to study the effect of vitamin D on the secretion of ucOC by osteoblastic cells in vitro in the presence of warfarin, an inhibitor of gammacarboxylation of GLA-containing proteins. The potential influence of retinoic acid (RA) was also studied, because its mechanism of action involves pathways that are similar to vitamin D. In the presence of warfarin (0.05 microg/mL), 1alpha,25(OH)2D (10(-8)-10(-6) mol/L) decreased dose dependently ucOC secretion by human osteosarcoma MG63 cells (from 3.87 +/- 0.96 to 2.12 +/- 0.13 ng/10(6) cells). When expressed as a fraction of total OC, secretion ucOC decreased from 47.4 +/- 1.4% to 24.8 +/- 3.2% in the MG63 cells. The secretion of total OC was stimulated by RA and by Ro 13-7410, which is a specific ligand of retinoic acid receptor (RAR), but not by 9-cis retinoic acid (9-cisRA), which is a physiologic ligand of retinoid X receptor (RXR). RA and Ro 13-7410 decreased ucOC secretion and ucOC% in warfarin-treated MG63 cells (RA: from 50.4 +/- 13.3% to 13.5 +/- 2.8%; Ro 13-7410: from 28.4 +/- 8.2% to 11.3 +/- 8.4%). 9-cisRA had no effect on OC gammacarboxylation. These results show that vitamin D, RA, and Ro 13-7410, but not 9-cisRA, may modify the gammacarboxylation of OC in human MG63 cells.
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PMID:Influence of vitamin D and retinoids on the gammacarboxylation of osteocalcin in human osteosarcoma MG63 cells. 896 28

1alpha,25(OH)2-16-ene-D3, a synthetic analog of the steroid hormone, 1alpha,25(OH)2D3, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1alpha,25(OH)2-16-ene-D3 expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1alpha,25(OH)2-16-ene-D3 and 1alpha,25(OH)2D3 are metabolized differently. 1alpha,25(OH)2-24-oxo-16-ene-D3, an intermediary metabolite of 1alpha,25(OH)2-16-ene-D3 formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1alpha,25(OH)2-24-oxo-D3, the corresponding intermediary metabolite of 1alpha,25(OH)2D3. In a subsequent in vivo study, we also reported that 1alpha,25(OH)2-24-oxo-16-ene-D3 exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1alpha,25(OH)2-24-oxo-16-ene-D3, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1alpha,25(OH)2-16-ene-D3 and 1alpha,25(OH)2D3 are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1alpha,25(OH)2-24-oxo-16-ene-D3 in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1alpha,25(OH)2-16-ene-D3 and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1alpha,25(OH)2D3. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1alpha,25(OH)2-16-ene-D3 and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1alpha,25(OH)2D3 itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1alpha,25(OH)2-24-oxo-16-ene-D3, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1alpha,25(OH)2-16-ene-D3.
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PMID:1alpha,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1alpha,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells. 901 Mar 46

Bone is an estradiol-responsive tissue. Estrogen withdrawal during the menopause causes loss of bone mass and clinically relevant osteoporosis in a third of all women. Sufficient or impaired local production, as well as degradation of estradiol in cells present in the bone microenvironment might be an important mechanism of rescue or might contribute to the development of osteoporosis, respectively. We therefore investigated aromatase and 17beta-hydroxysteroid dehydrogenase type IV (17beta-HSD IV) expression in osteoblast- and osteoclast-like cells. Aromatase mRNA was increasingly expressed in myeloid THP 1 cells differentiated along the monocyte/phagocyte pathway exploiting vitamin D and either granulocyte-macrophage-stimulating factor (GMCSF) or macrophage-stimulating factor (MCSF). In long-term cultures, when sequentially exposed to vitamin D (days 0-21) and GMCSF (days 5-10) and plated on collagen, the amount of expression of aromatase mRNA steadily increased along with the increasing expression of osteopontin mRNA, alpha(v) integrin mRNA, c-fms (MCSF-receptor) mRNA and multinucleated cells developing. The conversion of estradiol from testosterone (10(-7) M/l) in the supernatants of dishes mirrored changes in aromatase mRNA expression and by day 21 rose to 30,000 ng/10(7) cells/24 h. 17Beta-HSD IV mRNA expression was abundant in undifferentiated THP 1 cells and was decreased to approximately 50% by day 21. Unstimulated SV-40 immortalized fetal osteoblasts did not express aromatase mRNA, but the expression was stimulated by the addition of the phorbol ester phorbol myristate acetate (PMA). Unstimulated osteoblasts from primary cultures did not express aromatase mRNA. Osteoblast-like osteosarcoma cells MG 63 expressed faint levels of aromatase mRNA in contrast to the osteosarcoma cell line HOS 58. 17Beta-HSD IV mRNA was expressed in fetal osteoblasts as well as in osteoblasts from primary culture, MG 63 and HOS 58 cells. In summary, we can show the expression of estradiol metabolizing enzymes in cells which are present in the bone microenvironment. Impaired aromatase expression and/or enhanced expression of 17beta-HSD IV may contribute to the pathogenesis of osteoporosis.
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PMID:Local estradiol metabolism in osteoblast- and osteoclast-like cells. 936 87

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