Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0029463 (osteosarcoma)
 16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genes regulated by p53, as well as the factors modulating its function, need to be identified before the mechanism of action of p53 in control of cell growth can be adequately understood. Binding of the SV40 large T-antigen protein to an evolutionally conserved (conformational) domain of p53 inhibits p53's DNA-binding and transcription activation activities. Cellular proteins might also bind to this same region of p53 to regulate its function. A hybrid protein composed of protein A fused to the conformational domain (amino acids 115-295) of p53 was expressed in Escherichia coli and used as an affinity probe for binding proteins in detergent lysates of non-small cell lung carcinoma (NSCLC) cells. The wild-type p53 hybrid protein associated with several major proteins of molecular weights 45 K, 56 K, and 70 K, as well as other minor species ranging in molecular weight from 30 K to 90 K. These proteins bound specifically to the p53 sequence of the hybrid protein. Protein A did not associate with these proteins and the two p53 hybrid proteins containing missense mutations at codons 273 and 175 exhibited a 40-80% weaker association. In addition, T antigen competed with the cellular proteins for binding to the conformational domain. The conditions of cell growth had a profound effect on the expression of the p53 binding proteins. Considerably more p53 binding proteins were expressed in actively growing cells than in cultures maintained under conditions for slow growth. Quantitative differences in expression of p53-binding proteins were observed among different NSCLC cell lines. The expression of p53-binding proteins was not restricted to NSCLC cell lines; detergent extracts of an osteosarcoma cell line yielded similar p53-binding proteins.
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PMID:Binding of cellular proteins to a conformational domain of tumor suppressor protein p53. 824 46

The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give mutant NLS(NQ)] or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/delta 22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.
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PMID:A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity. 833 4

Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein. A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase. We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast. A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter. The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141). hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and [Tyr34]hPTHrP(1-34)amide. Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles.
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PMID:Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae. A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach. 838 31

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.
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PMID:Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization. 852 4

The gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is widely expressed in normal and neoplastic tissues. This study demonstrates that expression of the PTHrP gene has features of early response genes, including up-regulation after serum repletion of serum-starved ROS 17/2.8 (rat osteosarcoma) cells. The PTHrP messenger RNA (mRNA) levels were induced within 30 min and peaked at 4 h. Elevated mRNA levels were accompanied by an increase in secreted PTHrP. The serum effects on PTHrP mRNA levels were blocked by actinomycin D, suggesting a requirement for gene transcription. Nuclear run-on assays revealed a 3-fold increase in PTHrP gene transcription 4 h after exposure to serum. Deletions of the 5' flanking sequence of the rat PTHrP gene fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 cells showed that the serum-responsive region is located between -1.05 kb and -0.3 kb upstream of the transcription start site. PTHrP mRNA levels were also induced by cycloheximide, another feature common to early response genes. The PTHrP mRNA half-life in serum-starved cells was 56 min. Serum treatment prolonged the half-life 2.7-fold, suggesting serum-induced stabilization of the mRNA. Insulin and epidermal growth factor also induced PTHrP mRNA expression in a time-dependent manner analogous to serum, indicating that the effects of serum may be mediated, at least partially, through these agents. In summary, serum up-regulated PTHrP mRNA expression through both transcriptional and posttranscriptional mechanisms. This rapid stimulation by growth factors suggests that PTHrP may contribute to the early cellular response after growth factor stimulation.
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PMID:Serum stimulation of parathyroid hormone-related peptide gene expression in ROS 17/2.8 osteosarcoma cells through transcriptional and posttranscriptional mechanisms. 875 33

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

Recently the high-mobility group protein gene HMGIC has been found to be rearranged in a variety of benign mesenchymal tumors with 12q13-q15 aberrations, such as angiomyxoma, fibroadenomas of the breast, lipomas, pleomorphic salivary gland adenomas, polyps of the endometrium, pulmonary chondroid hamartomas, and uterine leiomyomas. Here we report on HMGIC aberrations in the osteosarcoma cell line OsA-CI. In Northern blot studies, aberrant HMGIC transcripts were detected. Analysis of cDNA sequence data obtained after 3' rapid amplification of cDNA ends, indicated these to consist of 5' HMGIC sequences encoding the three DNA binding domains fused to ectopic sequences apparently derived from part of the human lumican (keratan sulphate proteoglycan) gene (LUM), which we mapped by fluorescence in situ hybridization (FISH) to chromosome 12q22-q23. Moreover, Southern blot analysis revealed amplification of this fusion gene but not of the 3'HMGIC sequences. This observation was independently confirmed by FISH analysis using yeast artificial chromosome (YAC) and cosmid clones, which furthermore indicated that the amplified 5'HMGIC sequences were contained within an amplicon of about 200 kb. Our results indicate that aberrations in HMGIC might not be restricted to benign mesenchymal tumors.
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PMID:Amplification of a rearranged form of the high-mobility group protein gene HMGIC in OsA-CI osteosarcoma cells. 890 60

Bone sialoprotein (BSP) is a noncollagenous matrix glycoprotein localized predominantly in mineralized tissues but also detected in extraskeletal sites undergoing focal mineralization. We have previously characterized the human BSP gene and have shown that the upstream sequence contains inverted TATA and CCAAT motifs at the expected locations from the transcriptional start site (J. M. Kerr et al. [13]) and a potential YY1 binding motif located within the first 30 bp of intron 1 of the human gene. Deletion analyses of the human BSP promoter/exon 1 sequence fused to a CAT reporter gene indicate that CCAAT enhances basal transcription of BSP in transiently transfected rat UMR106-01 BSP osteosarcoma and rat skin fibroblasts. Though this enhancing activity was lost with inclusion of 68 bp of intron containing a YY1 motif in these constructs, reporter activity in the UMR106-01-BSP cells was elevated four- to seven-fold relative to that of rat fibroblasts. Gel electrophoretic mobility shift, UV-crosslinking, and south-western experiments indicate that YY1 is present only in the extracts of nuclei isolated from the UMR cells and may contribute to the elevated transcriptional activity of the human BSP promoter construct in UMR106-01-BSP.
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PMID:The human bone sialoprotein gene contains an NF-E1/YY1 cis-acting sequence with putative regulatory activity. 906 66

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.
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PMID:Parathyroid hormone (1-34)-mediated interleukin-6 induction. 932 32

Chromosomal translocations resulting in chimaeric transcription factors underlie specific malignancies, but few authentic target genes regulated by these fusion proteins have been identified. Desmoplastic small round-cell tumour (DSRT) is a multiphenotypic primitive tumour characterized by massive reactive fibrosis surrounding nests of tumour cells. The t(11;22)(p13;q12) chromosomal translocation that defines DSRT produces a chimaeric protein containing the potential transactivation domain of the Ewing-sarcoma protein (EWS) fused to zinc fingers 2-4 of the Wilms tumour suppressor and transcriptional repressor WT1 (refs 2,3). By analogy with other EWS fusion products, the EWS-WT1 chimaera may encode a transcriptional activator whose target genes overlap with those repressed by WT1 (ref. 4). To characterize its functional properties, we generated osteosarcoma cell lines with tightly regulated inducible expression of EWS-WT1. Expression of EWS-WT1 induced the expression of endogenous platelet-derived growth factor-A (PDGFA), a potent secreted mitogen and chemoattractant whose promoter contains the many potential WT1-binding sites. Native PDGFA was not regulated by wild-type WT1, indicating a difference in target gene specificity between this tumour suppressor and its oncogenic derivative. PDGFA was expressed within tumour cells in primary DSRT specimens, but it was absent in Wilms tumours expressing WT1 and Ewing sarcomas with an EWS-Fli translocation. We conclude that the oncogenic fusion of EWS to WT1 in DSRT results in the induction of PDGFA, a potent fibroblast growth factor that contributes to the characteristic reactive fibrosis associated with this unique tumour.
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PMID:The EWS-WT1 translocation product induces PDGFA in desmoplastic small round-cell tumour. 935 95


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