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)

Intracisternal A-type particle (IAP) transcripts are endogenous retrovirus-like sequences expressed during specific stages of normal development and in a variety of murine tumors. In this study, we have analyzed two cell lines derived originally from the SEWA murine osteosarcoma and grown either as ascites or as solid tumors, for proteins that might regulate IAP expression. We found that subline AA7-NA, originally derived from the ascites tumor, expressed about five times more IAP RNA than the AS12-AD subline, which was derived from a solid tumor. In view of this finding, we examined the binding of cellular proteins from the two cell lines to the 5' end of an IAP long terminal repeat sequence. Gel retardation assays of DNA-protein complexes and DNase I footprinting assays identified several DNA sequences within the long terminal repeat fragment that were protected by protein extracts from both SEWA sublines. Gel retardation assays using specific synthetic oligonucleotide sequences that correspond to two of these protected regions revealed different patterns of DNA-protein complexes with extracts from the two SEWA sublines. These data suggest that expression of IAP sequences is regulated by complex mechanisms involving several proteins that appear to differ between the two sublines.
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PMID:Interaction of SEWA sarcoma cell proteins with the intracisternal A-type particle long terminal repeat DNA sequence. 154 43

Potential cis-acting regulatory elements of the human platelet derived growth factor-B (PDGF-B) gene were identified by DNase I hypersensitive site mapping. The transcription unit was examined for the presence of hypersensitive sites in chromatin DNA isolated from human term placental cytotrophoblasts, human placental fibroblasts, the JEG-3 choriocarcinoma cell line and the U2-OS osteosarcoma cell line. A number of cell type-specific hypersensitive sites were identified, all within the 1st intron. Transient transfection of JEG-3 cells with CAT constructs containing regions of the c-sis 1st intron linked to the basal c-sis promoter identified a cell type-specific positive regulatory activity within the intron, composed of at least two distinct elements. One element appeared to be specific for JEG-3 cells, while the other was also active in U2-OS cells. The overall positive regulatory activity of the 1st intron region was specific for JEG-3 cells, but did not function as a classically defined enhancer, as it was orientation-dependent (unless stably integrated into chromatin DNA). In addition, the activator appears to require interaction with the c-sis promoter, as little or no activation was seen when either the SV40 or human beta-globin promoters were substituted for the c-sis promoter. The 1st intron also contained a negative regulatory element, which was specific for U2-OS cells and silenced an abnormally high basal c-sis promoter activity in these cells. The complexity of the transcriptional control of the PDGF-B gene is discussed.
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PMID:Expression of the human PDGF-B gene is regulated by both positively and negatively acting cell type-specific regulatory elements located in the first intron. 202 39

The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.
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PMID:Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter. 821 10

Nuclease hypersensitive sites were mapped in the proximal promoter of the osteocalcin gene, which is expressed only in bone cells exhibiting the mature osteoblast phenotype. Nuclei from proliferating and confluent rat osteosarcoma (ROS) 17/2.8 cells were subjected to DNase I digestion, and hypersensitivity was assayed by the indirect end-labeling method, using osteocalcin gene probes. Hypersensitive sites were detected in two promoter domains: -590 to -390, which spans the vitamin D responsive element, and -170 to -70, which spans the TATA box and the CCAAT-containing OC box domain. Together, these elements regulate basal and vitamin D enhanced osteocalcin gene transcription. We observed a parallel relationship between the intensity of bands representing the hypersensitive sites and the extent to which the osteocalcin gene is transcribed. Both in confluent cultures and in response to vitamin D, when osteocalcin transcription was upregulated, the hypersensitive bands were significantly intensified. Additionally, the bands were decreased under conditions that downregulate osteocalcin gene transcription. A functional relationship between the presence of hypersensitive sites and osteocalcin gene transcription is further supported by the absence of hypersensitivity in nonosseous cells that do not express osteocalcin, although these proliferating cells exhibited hypersensitivity in a cell cycle regulated histone gene promoter. Our results suggest the involvement of chromatin structure in transcriptional responsiveness of the osteocalcin gene to physiologic modulation.
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PMID:DNase I hypersensitive sites in promoter elements associated with basal and vitamin D dependent transcription of the bone-specific osteocalcin gene. 828 56

Equine infectious anemia virus (EIAV) is a lentivirus that causes a chronic disease of horses characterized by cyclic episodes of fever, anemia, and viremia. Although the genome and promoter of EIAV are much less complex than those of its relatives the primate immunodeficiency viruses, the cellular proteins that activate and regulate transcription of EIAV have not yet been identified. In this report, we show by electrophoretic mobility shift assays and DNase I footprinting that the EIAV promoter contains multiple binding sites for ubiquitous, cell type-specific, and inducible cellular proteins. Functional analysis by transient transfection of canine osteosarcoma (D17) and human epithelial carcinoma (HeLa) cells with EIAV promoters containing deletions or individually mutated DNA-binding sites demonstrated that these DNA-binding elements cooperatively regulate transcriptional activity. A methylated DNA-binding site (MDBP; also designated EF-C or EP) acts as either a positive or negative regulator of promoter activity, depending on the cell type or condition. Two PEA2 elements, an AP-1 site, and an ets/PEA3 motif confer a positive effect on promoter activity. The EIAV promoter is shown to be activated by treatment of HeLa cells with phorbol myristate acetate (PMA). DNA-binding activities were induced in PMA-treated HeLa cells and formed complexes on oligonucleotides that contain the EIAV AP-1 and ets/PEA3 elements. Functional analysis of mutated promoters indicated that the ets/PEA3 motif was the principal mediator of PMA activation.
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PMID:Physical and functional characterization of transcriptional control elements in the equine infectious anemia virus promoter. 838 28

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

Transcription of the osteocalcin gene, which encodes a 10 kDa bone-specific protein, is controlled by modularly organized basal regulatory sequences and hormone-responsive enhancer elements. We have previously shown that in the ROS 17/2.8 rat osteosarcoma cell line, which continuously expresses the osteocalcin gene, key regulatory elements reside in two DNase I hypersensitive sites that are fucntionally correlated with transcriptional activity. We now report that a specific nucleosomal organization supports this constitutive expression in ROS 17/2.8 cells, and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of the osteocalcin gene during differentiation of normal diploid rat osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNAse I hypersensitive sites (-170 to -70 and -600 to -400) and a selective nucleosome positioning over the OC gene promoter are directly associated with developmental stage-specific transcriptional activation in bone-derived cells.
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PMID:Changes in chromatin structure support constitutive and developmentally regulated transcription of the bone-specific osteocalcin gene in osteoblastic cells. 866 2

In order to investigate the subnuclear interactions of the WT1 gene product, nuclear fractionation analyses were performed with human osteosarcoma HOS and myelogenous leukemia K562 cells. The WT1 protein was tightly associated with the nucleus and was resistant to high-salt or detergent extraction and DNase I digestion. Both the expression level and stability of WT1 and its resistance to high salt and DNase I treatments remained constant during the cell cycle. In addition, human WT1 ectopically expressed in mouse NIH3T3 cells was also resistant to these treatments. These results suggest that WT1 functions in tight association with the nuclear matrix.
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PMID:The Wilms tumor protein is persistently associated with the nuclear matrix throughout the cell cycle. 920 4

Developmental control of bone tissue-specific genes requires positive and negative regulatory factors to accommodate physiological requirements for the expression or suppression of the encoded proteins. Osteocalcin (OC) gene transcription is restricted to the late stages of osteoblast differentiation. OC gene expression is suppressed in nonosseous cells and osteoprogenitor cells and during the early proliferative stages of bone cell differentiation. The rat OC promoter contains a homeodomain recognition motif within a highly conserved multipartite promoter element (OC box I) that contributes to tissue-specific transcription. In this study, we demonstrate that the CCAAT displacement protein (CDP), a transcription factor related to the cut homeodomain protein in Drosophila melanogaster, may regulate bone-specific gene transcription in immature proliferating osteoblasts. Using gel shift competition assays and DNase I footprinting, we show that CDP/cut recognizes two promoter elements (TATA and OC box I) of the bone-related rat OC gene. Overexpression of CDP/cut in ROS 17/2.8 osteosarcoma cells results in repression of OC promoter activity; this repression is abrogated by mutating OC box I. Gel shift immunoassays show that CDP/cut forms a proliferation-specific protein/DNA complex in conjunction with cyclin A and p107, a member of the retinoblastoma protein family of tumor suppressors. Our findings suggest that CDP/cut may represent an important component of a cell signaling mechanism that provides cross-talk between developmental and cell cycle-related transcriptional regulators to suppress bone tissue-specific genes during proliferative stages of osteoblast differentiation.
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PMID:The CCAAT displacement protein/cut homeodomain protein represses osteocalcin gene transcription and forms complexes with the retinoblastoma protein-related protein p107 and cyclin A. 1060 45

An ideal gene carrier requires both safety and transfection efficiency. Polyethylenimine (PEI) is a well-known cationic polymer, which has high transfection efficiency owing to its buffering capacity. But it has been reported that PEI is cytotoxic in many cell lines and non-degradable. In this study, we synthesized degradable PEI-alt-poly(ethylene glycol) (PEG) copolymers using Michael-type addition reactions as a new gene carrier and characterized them. These copolymers were complexed with plasmid DNA and the resulting complexes were characterized by dynamic light scattering, gel retardation and atomic force microscopy to determine particle sizes, complex formation and complex shape, respectively. Cytotoxicity and transfection efficiency of the copolymers were also checked in cultured HeLa human cervix epithelial carcinoma cells, HepG2 human hepatoblastoma cell line and MG63 human osteosarcoma cells. PEG to PEI ratio in the copolymers was near 1 and the molecular weight of the copolymer ranged from around 8000 to 12,900. These copolymers degraded rapidly at 37 degrees C in 0.1 M phosphate buffered saline (PBS, pH 7.4). The complete copolymer/DNA complex was formed at an N/P ratio of 12, producing a complex resistant to DNase I. Particle sizes decreased with increasing N/P ratio and PEG molecular weight, exhibiting a minimum value of 75 nm at an N/P ratio of 45 with PEI-alt-PEG (700). Cytotoxicity study showed that copolymers exhibited no cytotoxic effects on cells even at high copolymer concentration. Also, transfection efficiency was influenced by PEG molecular weight and, in case of PEI-alt-PEG (258), the transfection efficiency was higher than that for PEI 25 K in HepG2 and MG63, whereas it was lower than that for PEI 25K in HeLa cells.
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PMID:Degradable polyethylenimine-alt-poly(ethylene glycol) copolymers as novel gene carriers. 1593 8


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