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)

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-cab11 against the amino acids 172-186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M(r) of about 48,000 and human intestine cytosol a broad band (50-63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5-6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.
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PMID:Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies. 147 57

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT2 and DHT3. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT2) was demonstrated in human plasma after administration of oral DHT2. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT3 in vitro showed that 25-hydroxy-DHT3 was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT3-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT3. The osteosarcoma cell line metabolized 25-OH-DHT3 in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1 alpha,25-dihydroxy-DHT3. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1 alpha,25-dihydroxy-DHT whereas compound Hb is possibly 1 beta,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1 alpha,25-dihydroxy-DHT is very effective in displacing radiolabelled 1 alpha,25-dihydroxyvitamin-D3 and is thus most likely to be the calcaemic metabolite of DHT.
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PMID:The metabolism of dihydrotachysterols: renal side chain and non-renal nuclear hydroxylations in vivo and in vitro. 156 63

The 1,25-dihydroxyvitamin D3 (vitamin D) receptor (VDR) is a key trans-activating protein that mediates calcium regulation as well as cellular proliferation and differentiation. Phosphorylation of the VDR contributes significantly to its functional activity, but the specific mechanisms that mediate this regulation are not well understood. Phosphorylation may influence DNA binding, ligand binding, and protein-protein interactions, including heterodimerization and/or transactivation functions. We used a protein kinase C inhibitor, staurosporine (ST), and an inhibitor of serine-threonine phosphatases, okadaic acid (OA), to elucidate the contribution of VDR phosphorylation to vitamin D-mediated transcription of the osteocalcin (OC) gene. Vitamin D-induced transcription was assayed in transfected ROS 17/2.8 osteosarcoma cells using chloraminphenicol acetyltransferase constructs containing the vitamin D-responsive element (VDRE) at its native locus in the rat OC promoter as well as fused to a heterologous promoter. Both ST and OA inhibit VDRE-mediated and vitamin D-dependent enhancement of OC gene transcription as well as OC biosynthesis, as assessed by RIAs. Results from gel mobility shift and Western blot analyses using nuclear proteins from ROS 17/2.8 cells show that binding of the VDR-retinoid-X receptor heterodimer complex to the OC VDRE is not inhibited in the presence of ST. In contrast, OA does inhibit the formation of complexes interacting with both the OC and osteopontin VDREs; immunoprecipitation studies using 32P-labeled ROS 17/2.8 cells reveal that OA treatment result in ligand-independent hyperphosphorylation of the VDR. Our results suggest that two distinct phosphorylation events modulate rat VDR function. One event is related to transactivation, and the other is also critical to the VDRE-binding activity of VDR-retinoid X receptor-DNA complexes with consequential effects on transactivation.
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PMID:Control of 1,25-dihydroxyvitamin D3 receptor-mediated enhancement of osteocalcin gene transcription: effects of perturbing phosphorylation pathways by okadaic acid and staurosporine. 758 24

A peptide of 27 amino acids, VDR(102-76), representing residues 76-102 immediately C-terminal to the second Zn finger of human vitamin D receptor (hVDR) was conjugated to fluorescein-labelled IgG using a bifunctional coupling reagent, m-maleimidobenzoyl n-hydroxysuccinimide. Upon microinjection into the cytoplasm of human osteosarcoma MG-63 cells, the chimeras accumulated in the nuclei. This transport was arrested by chilling or energy depletion. Two other peptides, VDR(80-67), spanning the N-terminal part of VDR(102-76), and VDR(108-97), spanning the C-terminal part of VDR(102-76), were not able to target the linked proteins to the nuclei. SV40(135-112), a peptide containing a well-characterized nuclear localization sequence (amino acids 112-135) of simian virus 40 (SV40) large T-antigen, caused complete nuclear accumulation under the same conditions. Wheat germ agglutinin, which inhibits SV40(135-112) transport, also inhibited the nuclear accumulation of VDR(102-76) as did energy depletion.
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PMID:A peptide C-terminal to the second Zn finger of human vitamin D receptor is able to specify nuclear localization. 805 6

Vitamin D responsive transcription of the bone-specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post-proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D-dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA-directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half-elements, whereas the V2 complex appears to recognize the proximal half-element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype.
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PMID:Variations in vitamin D receptor transcription factor complexes associated with the osteocalcin gene vitamin D responsive element in osteoblasts and osteosarcoma cells. 808 97

Although several studies have been performed on the biological activities of analogs of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) at the whole animal and cellular levels, little work has been done to analyze their transcriptional activation properties. A highly inducible 1,25-(OH)2 D3-responsive promoter composed of three copies of the mouse osteopontin vitamin D3 response element (VDRE3) inserted upstream of a herpes simplex virus thymidine kinase promoter has been constructed, and its transcriptional properties have been analyzed by transient transfection into the monkey kidney cell line COS-7 and the rat osteoblast-like osteosarcoma line ROS 17/2.8. We have studied systematically transcriptional activation by a number of 1,25-(OH)2 D3 analogs, particularly those substituted at positions 16, 23, 26, and 27, sites that are targets for metabolism. Strikingly, except for derivatives that bind the 1,25-(OH)2 D3 receptor (VDR) very weakly, we find no parallel between the potency of action of a derivative as a transcriptional inducer and its affinity for the VDR. Derivatives substituted by multiple bonds at positions 16 and/or 23, although having varying affinities for the VDR, all stimulate transcription more potently than D3, in some cases at 100-fold lower concentrations. The peak transcriptional activity observed varies by only approximately 20% among different active analogs, indicating little difference in the activity of the VDR once bound to ligand. Gel retardation assays with ROS 17/2.8 nuclear extracts suggest that the VDR binds to the mouse osteopontin VDRE predominantly as a heterodimer with retinoid X receptor(s) (RXR(s)). We find that 9-cis-retinoic acid, the cognate ligand for RXRs, does not have a significant effect on the response of the VDRE3 promoter to 1,25-(OH)2 D3 or a number of its derivatives in ROS 17/2.8 or in COS-7 cells, under conditions in which promoters containing retinoid X response elements are activated. This suggests that 9-cis-retinoic acid may not act on the response to 1,25-(OH)2 D3 or its derivatives by directly influencing the transcriptional activity of VDR/RXR heterodimers. This promoter/reporter system should be useful for analyzing the tissue-specific transcriptional activity of 1,25-(OH)2 D3 and its derivatives in any cell type amenable to transient transfection.
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PMID:Highly potent transcriptional activation by 16-ene derivatives of 1,25-dihydroxyvitamin D3. Lack of modulation by 9-cis-retinoic acid of response to 1,25-dihydroxyvitamin D3 or its derivatives. 830 Jun 29

We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10(-8) M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P]orthophosphate results in a rapid (< or = 30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25(OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro. 839 28

Genomic actions of the calciotropic hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)2D3 to 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[1-14C]bromoacetate(1,25(OH)2D3-[14C]BE) binding-site directed analogs of 1,25(OH)2D3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)2[3H]D3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1,25(OH)2D3-[14C]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)2D3. These results strongly suggested that 1,25(OH)2[3H]D3-BE and 1,25(OH)2D3-[14C]BE covalently modified the 1,25(OH)2D3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.
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PMID:Affinity labeling of the 1 alpha,25-dihydroxyvitamin D3 receptor. 856 52

It is well known that 17 beta-estradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) have important roles in bone metabolism. This study was undertaken to examine E2 regulation of 1,25(OH)2D3 receptor (VDR) expression and the biological action of 1,25(OH)2D3 in human osteoblast-like cells. When human osteosarcoma-derived osteoblast-like cells were treated with varying concentrations of E2, the VDR levels increased by up to 100% in a dose-dependent manner. VDR levels significantly increased at 10 nM E2 and this increase plateaued at 100 nM E2. E2-dependent increase of VDR was time dependent, plateauing at 24 hours and was maintained for at least 48 hours in human osteoblast-like cells. Scatchard analysis showed that E2 increased the number of VDR (12.3 +/- 0.4 versus 26.5 +/- 0.3 fmol/mg protein; mean +/- SE of three independent experiments) rather than the Kd (0.15 +/- 0.02 versus 0.16 +/- 0.01 nM; mean +/- SE of three independent experiments). Tamoxifen (50 nM), a specific competitor with E2, completely abolished the E2-induced increase of VDR. The levels of VDR mRNA (4.5 kb) from the cells increased in a dose-dependent manner after E2 treatment. With regard to the biological effects, E2 increased by 10-25% the inhibitory effect of 1,25(OH)2D3 on cell growth. However, E2 did not increase the stimulation of alkaline phosphatase activity by 1,25(OH)2D3. The present study suggests that E2 modulates the biological action of 1,25(OH)2D3 through VDR levels in bone cells.
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PMID:17 beta-estradiol increases the receptor number and modulates the action of 1,25-dihydroxyvitamin D3 in human osteosarcoma-derived osteoblast-like cells. 858 75

We have shown earlier that 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] induces cell growth suppression and cell differentiation of a human megakaryoblastic leukemia cell line, HIMeg. However, the molecular mechanism of 1,25(OH)2 D3 action is still unknown. Prompted by this, we have searched here for the presence of 1,25(OH)2 D3 receptor (VDR) expression in HIMeg cells by reverse transcription-polymerase chain reaction (RT-PCR). The amplified product showed an identical size to the product amplified from the control human VDR cDNA and hybridized specifically with the digoxigenin-labeled human VDR cDNA fragment. As expected, VDR mRNA is also expressed in HOS-8603, a human osteosarcoma cell line. These results represent the first reported evidence that VDR mRNA is expressed in megakaryoblastic cells. In addition, the regulation of VDR mRNA expression in HIMeg cells was studied by quantitative RT-PCR. It was found that [correction of the] VDR mRNA expression in HIMeg cells could be down-regulated rapidly by 1,25(OH)2 D3 (10 nM) in a time-dependent manner, reaching a maximal reduction to about 15% of control. However, VDR mRNA expression in HOS-8603 cells was not regulated by 1,25(OH)2 D3 at any time-point tested. Treatment of HIMeg cells with forskolin (1 microM), an activator of adenylate cyclase, caused an increase in VDR mRNA levels. Similarly, VDR mRNA expression in HOS-8603 cells was also up-regulated by forskolin. Consistent with the functionality of the VDR in other target cells, we found that the up-regulation of VDR expression in HIMeg cells by forskolin was accompanied by an increased responsiveness of HIMeg cells to 1,25(OH)2 D3 even though forskolin alone had no effects. Exposure to 1,25(OH)2 D3 in combination with forskolin resulted in a much more significant inhibition of cell proliferation than to 1,25(OH)2 D3 alone. Similarly, forskolin could also augment the differentiation induced by 1,25(OH)2 D3 reflected by a more evident morphological change and a higher percentage of development of cells with multilobular nuclei. These alterations were accompanied by a loss of clonogenic capacity and a decrease in the number of cells in the S phase. These data establish that HIMeg cells express functional VDR, which served to mediate actions of its ligand on the proliferation and differentiation of these cells.
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PMID:Demonstration of vitamin D receptor expression in a human megakaryoblastic leukemia cell line: regulation of vitamin D receptor mRNA expression and responsiveness by forskolin. 863 62

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