Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2-4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4-6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells.
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PMID:Regulation of connexin43 expression and function by prostaglandin E2 (PGE2) and parathyroid hormone (PTH) in osteoblastic cells. 940 10

A quantitative method for measuring 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was developed utilizing a luciferase reporter gene under the control of the highly inducible 25-hydroxyvitamin D3 24-hydroxylase promoter in a stably transfected cell line. Transient transfections with constructs containing the 24-hydroxylase gene promoter 5' to a luciferase reporter were first performed in cell lines with high levels of vitamin D receptor, i.e., the rat osteosarcoma (ROS 17/2.8) and human breast cancer (T-47D) cell lines. ROS 17/2.8 cells, stably transfected with the plasmid, gave a 60-fold stimulation with 10(-10) M 1,25-(OH)2D3. A standard curve was constructed showing a large range of response to 1,25-(OH)2D3 (1 pg to 1 ng). The assay was adapted to microtiter plates, which permits a large number of samples to be assayed simultaneously. Other metabolites of vitamin D and analogs such as 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 1 alpha-hydroxyvitamin D3 have negligible effects on the detection of 1,25-(OH)2D3, thus eliminating the need for purification of sample. The sensitivity of the method permitted the use of 100 microliters of serum with excellent results. Comparison of this method with a commercially available assay demonstrates that it gives higher sensitivity, simpler manipulations, and comparable results.
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PMID:A highly sensitive method for large-scale measurements of 1,25-dihydroxyvitamin D. 944 54

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n, n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 microM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30-80 mM phosphate, at pH 5.6-6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.
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PMID:Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA. 951 70

The ability of different picornavirus internal ribosome entry site (IRES) elements to direct initiation of protein synthesis has been assayed in different cell lines in the presence and absence of viral proteases that inhibit cap-dependent protein synthesis. Reporter plasmids that express dicistronic mRNAs, containing different IRES elements, with the general structure CAT/IRES/LUC, have been assayed. In each plasmid, the CAT sequence encodes chloramphenicol acetyl transferase and the LUC sequence encodes luciferase. The poliovirus (PV) 2A protease and the foot-and-mouth disease virus (FMDV) Lb protease induce the cleavage of the translation initiation factor elF4G and hence inhibit the activity of the cap-binding complex, elF4F. In human osteosarcoma (HTK-143) cells, each of the various IRES elements functioned efficiently. In these cells, the co-expression of the viral proteases severely inhibited the expression of CAT, but the proteases had little effect on the activities of the various IRES elements. In contrast, in baby hamster kidney (BHK) cells, the efficiencies of the different IRES elements varied significantly, whereas, in normal rat kidney (NRK) cells, each of the IRES elements was relatively inefficient. In both BHK and NRK cells, the activities of those IRES elements that functioned inefficiently were strongly stimulated by the co-expression of the PV 2A or FMDV Lb proteases. This stimulation was independent of the loss of cap-dependent protein synthesis and was not achieved by the co-expression of the C-terminal fragment of elF4G. The results suggest that the PV 2A and FMDV Lb proteases induce the cleavage of another cellular protein, in addition to elF4G, which influences IRES function.
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PMID:Recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins. 958 94

This study examines the cooperative effects of a human estrogen receptor-alpha (ERalpha) isoform on estrogen (E2)-mediated gene activation in U2-OS osteosarcoma cells. Delta5ERalpha, an alternatively spliced ERalpha variant lacking exon 5, is coexpressed with normal ERalpha in several E2-responsive neoplastic tissues. However, the potential interactions of delta5ERalpha with normal ERalpha have not been functionally characterized. Delta5ERalpha encodes the hormone-independent trans-activating function (AF-1), as well as the constitutive receptor dimerization and DNA-binding domains. It is generated by an alternate splice event that omits exon 5 and alters the reading frame of the resulting mRNA. The delta5ERalpha protein is prematurely truncated and lacks the majority of the hormone-binding and activating function-2 (AF-2) domains. When delta5ERalpha mammalian expression vector was transfected alone in human ERalpha/ERbeta-negative osteosarcoma U2-OS cells, it had no effect on either basal or E2-mediated EREtk81Luc reporter transcriptional activity, while transfected cells expressing control normal ERalpha increased EREtk81 Luc activity up to 20-fold in response to 10 nM E2. However, when delta5ERalpha was cotransfected with normal ERalpha, both basal and E2-stimulated EREtk81Luc reporter activation were increased approximately 500% over levels observed when cells were transfected with ERalpha alone. Similar effects of delta5ERalpha and normal ERa coexpression were observed using an E2-responsive human C3 promoter/luciferase reporter construct. The effects of delta5ERalpha on normal ERalpha were further assessed in U2-OS cells stably transfected with normal ERalpha. Transfection of increasing amounts of delta5ERalpha expression vector into [ERalpha+]OS cells resulted in potentiation of E2-stimulated ERELuc activity in a synergistic, dose-dependent manner. Moreover, coexpression of delta5ERalpha in [ERalpha+]OS cells improved E2 sensitivity 100-fold over cells expressing ERalpha alone. Proliferation rates of stable U2-OS cell lines expressing delta5ERalpha were significantly increased (P < 0.05), with cell doubling times reduced from 35 h in control parental U2-OS cells to 28 h in [delta5ERalpha]OS cells. However, growth rates were not affected by either E2 or tamoxifen treatment. Electromobility shift/supershift assays using nuclear extracts of U2-OS cells stably transfected with ERalpha and delta5ERalpha confirmed the constitutive binding of delta5ERalpha and ERalpha protein to estrogen-response element (ERE) sequence independent of E2 and also showed an increase in delta5ERalpha/ERalpha-ERE complexes with E2 treatment. These data are consistent with interactive effects of normal ERalpha and delta5ERalpha on transcription from classic ERE gene promoters. Delta5ERalpha appears to therefore act as a dominant positive receptor that increases both basal and E2-stimulated gene transactivation of normal ERalpha.
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PMID:A tumor-specific truncated estrogen receptor splice variant enhances estrogen-stimulated gene expression. 973 4

The mitogenic and regulatory effects of estrogen (E2) in adenohypophysial cells are known to be mediated through the nuclear estrogen receptor (ER alpha). Expression of ER alpha and several of its messenger ribonucleic acid (RNA) alternate splice variants has been shown to be restricted to prolactinomas and gonadotroph tumors. However, little is known about gene expression patterns of the novel nuclear hormone receptor ER beta in the neoplastic pituitary. ER beta has high homology to ER alpha in the DNA- and ligand-binding domains, but encodes a distinct transcriptional activating function-1 (AF-1) domain. Using RT-PCR analysis of total RNA from 38 human pituitary adenomas, we found that ER beta messenger RNA was coexpressed with ER alpha and its splice variants in 60% of prolactinomas, 100% of mixed GH/PRL tumors, and 29% of gonadotroph tumors. ER beta gene expression was not limited to ER alpha-positive tumor subtypes, however, and was also found in 100% of null cell tumors, 80% of somatotroph tumors, and 60% of corticotroph tumors. Because ER beta is coexpressed with ER alpha and its splice variants in prolactinomas and gonadotroph tumors, we functionally characterized the potential interactions between ER beta and ER alpha. We also examined the potential cooperative effects on ER beta-mediated gene expression of a tumor-specific truncated delta 5ER alpha splice variant that has been shown to be coexpressed in the majority of ER alpha-positive tumors. This exon 5 splice variant encodes the AF-1 domain as well as regions critical for DNA binding and nuclear localization, but lacks the ligand-binding and AF-2 domains. Mammalian expression vectors encoding ER alpha, delta 5ER alpha, and/or ER beta complementary DNAs were transiently transfected along with an E2 response element promoter-luciferase (ERELuc) reporter into human ER alpha/ER beta-negative osteosarcoma U2-OS cells. ER beta was less potent than ER alpha in activating E2-stimulated ERELuc activity (4-vs. 14-fold relative to basal control levels). However, when delta 5ER alpha was coexpressed with ER beta or ER alpha, E2-stimulated ERELuc activity was markedly increased to 8- and 57-fold, respectively, relative to basal control levels when each full-length isoform was expressed alone. Finally, coexpression of ER beta with ER alpha did not significantly alter the E2-stimulated ERELuc activity induced by ER alpha alone. Cotreatment with tamoxifen markedly inhibited all E2-stimulated ERELuc responses to baseline levels. Together, these data suggest that ER beta has a minor role in mediating E2 responses in ER alpha-positive tumors, but may be the main mediator of E2-stimulated gene expression when expressed alone in somatotroph, corticotroph, and null cell tumors. This low, but significant, level of ER beta trans-activation potential may be enhanced by coexpression of delta 5ER alpha in neoplastic pituitary. Therefore, E2-mediated gene expression in normal and neoplastic pituitary appears to be highly dependent on the expression of ER alpha and ER beta isoforms, which have varying transcriptional activities.
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PMID:Differential expression of estrogen receptor-beta (ER beta) in human pituitary tumors: functional interactions with ER alpha and a tumor-specific splice variant. 974 46

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-alpha gene (PDGFRalpha). Mice heterozygous for the PDGFRalpha-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRalpha and Pax1. Using the human PDGFRalpha promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRalpha gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln --> His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRalpha-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRalpha expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein-DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRalpha gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRalpha expression may be causally related to NTDs.
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PMID:Altered regulation of platelet-derived growth factor receptor-alpha gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins. 982 22

Recent studies show that the p53 tumor suppressor protein is overexpressed in rheumatoid arthritis (RA) synovium and that somatic mutations previously identified in human tumors are present in RA synovium (Firestein, G. S., Echeverri, F., Yeo, M., Zvaifler, N. J., and Green, D. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10895-10900; Firestein, G. S., Nguyen, K., Aupperle, K. R., Yeo, M., Boyle, D. L., and Zvaifler, N. J. (1996) Am. J. Pathol. 149, 2143-2151; Reme, T., Travaglio, A., Gueydon, E., Adla, L., Jorgensen, C., and Sany, J. (1998) Clin. Exp. Immunol. 111, 353-3581). We hypothesize that the abnormality of p53 seen in RA synovium may contribute to joint degeneration through the regulation of human matrix metalloproteinase-1 (hMMP-1, collagenase-1) gene expression. Transcription assays were performed with luciferase reporters driven by the promoter of the hMMP-1 gene or by a minimal promoter containing tandem repeats of the consensus binding sequence for activator protein-1, cotransfected with p53-expressing plasmids. The results revealed that (i) wild-type (wt) p53 down-regulated the promoter activity of hMMP-1 in a dose-dependent fashion; (ii) four of six p53 mutants (commonly found in human cancers) lost this repression activity; and (iii) this p53 repression activity was mediated at least in part by the activator protein-1 sites found in the hMMP-1 promoter. These findings were further confirmed by Northern analysis. The down-regulation of hMMP-1 gene expression by endogenous wt-p53 was shown by treatment of U2-OS cells, a wt-p53-containing osteogenic sarcoma line, and Saos-2 cells, a p53-negative osteogenic sarcoma line, with etoposide, a potent inducer of p53 expression. p53, activated by etoposide, appears to block hMMP-1 promoter activity induced by etoposide in U2-OS cells. In summary, we have shown for the first time that the hMMP-1 gene is a p53 target gene, subject to p53 repression. Because MMP-1 is principally responsible for the irreversible destruction of collagen in articular tissue in RA, abnormality of p53 may contribute to joint degeneration through the regulation of MMP-1 expression.
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PMID:p53 down-regulates human matrix metalloproteinase-1 (Collagenase-1) gene expression. 1020 59

Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors.
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PMID:Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase. 1031 53

We have synthesized several 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D] derivatives and evaluated their biological activity in terms of their binding affinity for the vitamin D receptor (VDR) and vitamin D-binding protein (DBP), antiproliferative or differentiation-inducing effects on human promyelocytic leukemic HL-60 cells, and transcriptional activity on a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), and human osteocalcin gene promoter, including a VDRE in transfected human osteosarcoma MG-63 cells. Furthermore, human VDR- or retinoic acid X receptor alpha (RXR alpha)-mediated luciferase activities of the derivatives were also measured by a one-hybrid system in human epitheloid carcinoma, cervix HeLa cells and African green monkey kidney CV-1 cells. Binding affinity for VDR, bone-resorbing activity, antiproliferative and cell-differentiating effects, transactivation potencies on target genes and VDR- or RXR alpha-mediated gene regulations of 1alpha,25(OH)2D2 and 1alpha,25(OH)2D4 were almost comparable to the effects of 1alpha,25(OH)2D3 while 24-epi-1alpha,25(OH)2D2 and 1alpha,25(OH)2D7 were much less active than 1alpha,25(OH)2D3 in these respects. This is the first report concerning biological assessment of 1alpha,25(OH)2D2, 1alpha,25(OH)2D3, 1alpha,25(OH)2D4, 24-epi-1alpha,25(OH)2D2 and 1alpha,25(OH)2D7 at the molecular level, especially with regards to the structural differences at the 24R- or 24S-methyl group and a double bond between carbons 22 and 23 in the side chain of 1alpha,25(OH)2D derivatives.
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PMID:Biological activity profiles of 1alpha,25-dihydroxyvitamin D2, D3, D4, D7, and 24-epi-1alpha,25-dihydroxyvitamin D2. 1032 56


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