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:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed some functional aspects of the promoter of the human dopamine beta-hydroxylase (DBH) gene. A fragment of 1,247 bp directly 5' to the transcriptional start was progressively shortened, placed in front of a reporter gene, and tested in a human neuroblastoma cell line expressing DBH (SK-N-SH-TFM) and in a monkey kidney cell line (CV-1). A remarkably short region (267 bp), directly upstream from the transcription start, was sufficient to confer activity and tissue-specific expression. Furthermore, the expression of the DBH gene was shown to be inducible by cyclic AMP in SK-N-SH-TFM cells. This effect was demonstrated to occur at the transcriptional level, as shown by run-on assays, and was due to the presence of a near-consensus cyclic AMP-responsive element located in the untranscribed 5' regulatory region of the gene.
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PMID:Analysis of the human dopamine beta-hydroxylase promoter: transcriptional induction by cyclic AMP. 838 Jan 96

Human dopamine beta-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The Km of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.
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PMID:Expression of human dopamine beta-hydroxylase in Drosophila Schneider 2 cells. 854 10

Immunotoxins have been used to study the targeting of biologically active substances at neurons in vivo and to make experimental neural lesions. OX7-saporin, directed against Thy 1, destroys any neuron. 192 IgG-saporin, directed against the 'low affinity' neurotrophin receptor (p 75NTR), selectively destroys neurons expressing this receptor (sympathetic, sensory, cholinergic basal forebrain, cerebellar Purkinje). Anti-D beta H-saporin, directed against dopamine beta-hydroxylase, the enzyme that converts dopamine to norepinephrine, selectively destroys noradrenergic neurons (sympathetic, CNS). These agents show that several types of neural antigens may prove useful in treating pain, and anti-D beta H-saporin may be active against pheochromocytoma or neuroblastoma.
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PMID:Targeting toxins to neural antigens and receptors. 874 May 62

The dopamine beta-hydroxylase (DBH) gene is expressed selectively in noradrenergic and adrenergic neurons and neuroendocrine cells in the nervous system. A cAMP response element (CRE) residing at -181 to -174 bp from the transcription start site of the human DBH gene seems to be essential for DBH transcription. Potential cis-regulatory motifs such as AP1 and YY1 occur proximal to and overlap this CRE, endowing the area with a composite promoter structure. Using the DBH-expressing human neuroblastoma SK-N-BE(2)C and DBH-negative HeLa cell lines as model systems, we report here that this CRE/YY1/AP1 area interacts with multiple nuclear proteins, including CRE-binding protein (CREB) and transcription factor YY1 in a cell-specific manner. In support of the notion that multiple proteins bind to the CRE/YY1/AP1 area, DNase I foot-printing analysis has demonstrated that nuclear extracts protect an extended region (from -186 to -150 bp) relative to that protected by the purified CREB (from -186 to -171 bp). Site-directed mutational analysis has revealed differential roles of potential cis-regulatory motifs in regulation of DBH transcription. Strikingly, the YY1 element positively regulated basal DBH transcription while simultaneously regulating cAMP-mediated induction negatively, which is a novel mechanism of promoter function. Furthermore, three additional DNA-binding sites have been identified by DNase I footprint analysis in the upstream 260 bp promotor region of the human DBH gene, of which two sites are cell-specific. These results support a model whereby multiple proteins bind to the 5'-proximal area in a cell-specific manner and coordinately regulate the cell type-specific transcriptional activation of the DBH gene.
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PMID:Multiple protein factors interact with the cis-regulatory elements of the proximal promoter in a cell-specific manner and regulate transcription of the dopamine beta-hydroxylase gene. 875 72

Protein kinase C (PKC) activation after treatment of human neuroblastoma SK-N-BE(2)C cells with phorbol 12-myristate 13-acetate (PMA) was found to enhance transcription of the human dopamine beta-hydroxylase (DBH) in those cells. To identify which cis-acting element is responsive to the PMA treatment during DBH gene expression, we employed transient transfection assays with serially deleted constructs of the human DBH gene's 5' upstream region fused to the chloramphenicol acetyltransferase (CAT) gene. Treatment of transfected cells with PMA resulted in an approximate threefold increase in CAT expression for all deletion constructs ranging from -978 bp to -262 bp, while the enhancement did not occur with a construct shortened to -114 bp. The region between -262 and -114 bp from the initiation site of transcription contains several cis-regulatory elements including a cyclic AMP response element (CRE) and putative AP1 and YY1 sequences. Site-directed mutagenesis of those cis-acting elements were performed to identify which of the elements mediated the PMA-induced transcriptional enhancement. Substitution of bases in the putative AP1 site containing in part a putative YY1 sequence did not effect the PMA inducibility. However, specific mutations in the CRE sequence abolished the PMA-inducible effect. Changing the CRE sequence into an authentic AP1 sequence (TGACGTCC --> TGACTCA) did not affect the PMA inducibility, suggesting that AP1 factors might interact with the new AP1 site upon PKC activation. A specific PKC inhibitor, GF109203X, completely inhibited the stimulatory effect of PMA on the expression of the human DBH gene. PMA induced an increase in the DBH mRNA level as detected by Northern blot analysis. Gel retardation showed that the binding of nuclear factors to CRE, putative YY1, and AP1 was sequence specific. Our data suggest that the enhancement of the human DBH gene expression by PMA treatment is mediated by the CRE motif in the 5' upstream region of the gene, and occurs via a PKC-dependent pathway.
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PMID:A protein kinase C-activating phorbol ester enhances transcription of the human DBH gene through a cyclic AMP response element in SK-N-BE(2)C cells. 942 17

Prostaglandin E2 (PGE2) enhances transcription of the human dopamine beta-hydroxylase (DBH) gene in human neuroblastoma SK-N-BE(2)C cells. To identify a PGE2-responsive cis-acting element in the human DBH gene, serial deletion constructs of the human DBH 5'-upstream region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into SK-N-BE(2)C cells. Treatment of the transformed cells with PGE2 increased CAT expression two- to threefold in all constructs except where the promoter region was shortened beyond position -114 bp. There are several cis-regulatory elements in the region between -262 and -114 bp from the transcription initiation site that include a cyclic AMP response element (CRE) and a putative AP1 sequence. We presupposed that the CRE and AP1 might be candidates for PGE2 stimulation, and therefore, used site-directed mutagenesis to change the CRE and AP1 motives and test which of the two elements mediated the transcriptional enhancement. Only a specific mutation within the CRE sequence abolished the PGE2 effect. In addition, cotransfection with an expression vector expressing PKA inhibitor resulted in the specific blockage of the PGE2 effect on DBH gene expression. Northern blot analysis revealed that the increase in DBH gene transcription caused by PGE2 results in elevated DBH mRNA levels. Gel-retardation and competition assays confirmed that the binding of nuclear factors to the CRE site is sequence specific. Our data, therefore, indicate that PGE2 enhances the transcription of the human DBH gene. The effect is mediated by the CRE motif through activation of PKA.
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PMID:Stimulation of human DBH gene expression by prostaglandin E2 in human neuroblastoma SK-N-BE(2)C cells. 948 16

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators within the network of the general transcription machinery.
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PMID:The homeodomain protein Arix promotes protein kinase A-dependent activation of the dopamine beta-hydroxylase promoter through multiple elements and interaction with the coactivator cAMP-response element-binding protein-binding protein. 1064 60

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.
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PMID:Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression. 1109

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.
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PMID:Potentiation of PGE(2)-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells. 1167 58

We previously showed that ethanol regulates dopamine beta-hydroxylase (DBH) mRNA and protein levels in human neuroblastoma cells (Thibault, C., Lai, C., Wilke, N., Duong, B., Olive, M. F., Rahman, S., Dong, H., Hodge, C. W., Lockhart, D. J., and Miles, M. F. (2000) Mol. Pharmacol. 58, 1593-1600). DBH catalyzes norepinephrine synthesis, and several studies have suggested a role for norepinephrine in ethanol-mediated behaviors. Here, we performed a detailed analysis of mechanism(s) underlying ethanol regulation of DBH expression in SH-SY5Y cells. Transient transfection analysis showed that ethanol (25-200 mM) caused concentration- and time-dependent increases in DBH gene transcription. Progressive deletions identified ethanol-responsive sequences in the -262 to -142 bp region of the DBH gene promoter. Mutagenesis of cAMP-response element (CRE) sequences in this region abolished ethanol responsiveness while maintaining responsiveness to phorbol esters. Coexpression of dominant-negative CRE-binding protein greatly reduced ethanol induction of DBH. Inhibitors of protein kinase A, casein kinase II, and MAPK reduced ethanol induction of DBH promoter activity. Pharmacogenomic studies with microarrays showed that protein kinase A, MEK, and casein kinase II inhibitors blocked induction of DBH and a large subset of ethanol-responsive genes. These genes had diverse functional groupings, including multiple members of the MAPK and phosphatidylinositol signaling cascades. Real-time PCR analysis validated select microarray results. Taken together, these results suggest that ethanol regulation of DBH requires a functional CRE and its binding protein and may require interaction of multiple kinase pathways. This mechanism may also mediate ethanol responsiveness of a complex subset of genes in neural cells. These studies may have implications for behavioral responses to ethanol or mechanisms underlying ethanol-related neurological disease.
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PMID:Pharmacogenomic analysis of mechanisms mediating ethanol regulation of dopamine beta-hydroxylase. 1284 74


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