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)

Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5'-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs (bp) upstream from the initiating ATG, separated from a 460-493 bp noncoding exon by a 4.8-kilobase intron. DNA sequence analysis shows that the non-coding exon is GC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elements including five Sp-1 binding sites, one AP-2 site, one AP-3 binding site and two E-boxes within the proximal 600 bp. A reporter construct consisting of 1440 bp of flanking DNA and 80 bp of the first exon cloned into a luciferase reporter plasmid, drove cell specific expression in transient transfection assays. Removal of 1088 bp of the 5' end of this construct resulted in expression in non-m4 expressing cell lines suggesting there is a repressor element in this region.
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PMID:Structure of the m4 cholinergic muscarinic receptor gene and its promoter. 853 49

Secretogranin II (SgII) is a member of the granin family of secretory proteins, which are selectively expressed in neuroendocrine cells. As a first step in understanding the molecular basis for cell type-specific expression of SgII, we isolated a 12-kb clone from a rat genomic library that contained the entire rat SgII coding region, the transcription initiation site, and approximately 3 kb of 5'-flanking region. Within 75 bp of the transcription start site (+1) we located a TATA box and a consensus cAMP responsive element. Within the 5'-flanking region, a number of potential cis-acting elements were identified, including 2 Pit-1 binding sites, 15 E box motifs, and near-perfect matches for AP-1 and AP-2 sites. To demonstrate cell type-specific expression the rat SgII gene, a plasmid containing 2.6 kb of the 5'-flanking region of the SgII gene fused to the luciferase reporter gene (p2774Luc) was transfected into rat pheochromocytoma PC-12 cells, rat pituitary GH4C1 (GH) cells, human BE(2)-M17 (M17) neuroblastoma cells, and mouse fibroblast NIH/3T3 cells. The promoter activity was 6- to 36-fold higher in neuroendocrine cells than in NIH/ 3T3 cells. Progressive deletions in the 5'-flanking region to 61 bp upstream of the start site (p223Luc) had no effect on promoter activity in PC-12 cells. On the other hand, a 5'-deletion in the SgII promoter to -1032 increased promoter activity 3.8-fold in GH cells. This level of expression was maintained when the SgII promoter was further truncated to -189, whereas truncation to -61 resulted in a 2.6-fold reduction in promoter activity. These results suggest that the sequence between -61 and +162 bp is sufficient for SgII promoter activity in PC-12 cells. However, other elements in the 5'-flanking region contribute to both positive and negative regulation of the rat SgII gene in GH cells.
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PMID:Cell-specific expression of the rat secretogranin II promoter. 875 52

The gene for amyloid precursor protein (APP) is expressed almost ubiquitously, with high levels of mRNA being detected in brain. The basal expression level of the APP gene can be modulated by physiological stimuli, and in this report we demonstrate that the second messenger cyclic AMP can regulate APP mRNA through transcriptional mechanisms. Northern blot analysis showed a 1.8-fold increase in steady-state levels of APP mRNA when the neuroblastoma x glioma hybrid cell line NG108-15 was treated with dibutyryl cyclic AMP. Although the upstream sequences of the APP gene do not contain a canonical cyclic AMP response element, transient transfection assays in NG108-15 cells using different portions of the APP promoter showed an increase in reporter gene activity mediated by sequences located between -303 to -204 and -488 to -2991. Cotransfection assays carried out in HepG2 cells with AP-2, a cyclic AMP-regulated transcription factor, failed to activate the APP promoter through the AP-2 consensus sequence (GCCNNNCGG) located at position -205. Electrophoretic mobility shift analysis revealed that the AP-2 binding activity present in HeLa nuclear extracts fails to recognize the APP AP-2 consensus sequence. We conclude that increases in cyclic AMP levels can lead to an up-regulation of APP gene transcription through at least two different regions of the APP promoter. This increase does not involve the AP-2 consensus sequence present in the APP promoter located at position -205, and, moreover, this putative site is not recognized by the transcription factor AP-2.
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PMID:Enhanced expression of amyloid precursor protein in response to dibutyryl cyclic AMP is not mediated by the transcription factor AP-2. 904 35

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the rat NSE gene 5' flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenous NSE. These data suggest that cis-acting elements responsible for the spatial and temporal pattern of NSE gene expression are located within the proximal 1.8 kb of the 5' flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to the cat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5' end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5' flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5' sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating that cis-acting elements controlling the regulation of NSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5' sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5' sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basal NSE promoter. Our results show that the 5' flanking region of the NSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.
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PMID:Sequences in the proximal 5' flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression. 906 16

The gene for choline acetyltransferase, synthesizing acetylcholine, is induced by several neurotrophic factors. A role for AP-2 in enhancing this transcription and limiting it to neural cells is strongly suggested. Previous studies demonstrated that base pairs +465-727 within the untranslated exon 1 of the porcine gene enhanced the expression of a reporter gene transfected into PC-12 cells. Deletion and mutation experiments indicate that base pairs +465-472 (CCGCGGGG) in the porcine gene, or +307-314 (CCTCGGGG) in the human sequence, were necessary and sufficient for increased gene expression in cholinergic or adrenergic but not liver cells. Constructs containing active sequences, but not inactive mutated sequences, specifically bind nuclear proteins from neuroblastoma cells, but not liver cells, in gel shift experiments. The human and porcine sequences are in agreement with an AP-2 consensus binding sequence, a nuclear transcription factor expressed only in cells derived from the neural crest. Gel shift experiments using recombinant AP-2 confirm this identification. AP-2 antibody further retarded the mobility of these DNA-nuclear extract or DNA-AP-2 complexes. These results support the importance of this AP-2 binding sequence in enhancing and limiting choline acetyltransferase expression in neural cells.
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PMID:An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells. 913 54

The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.
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PMID:Structure of the m1 muscarinic acetylcholine receptor gene and its promoter. 920 29

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.
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PMID:Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter. 920 30

Myc family proteins play an important role in cellular processes such as proliferation, differentiation, apoptosis and transformation. A number of interaction partners of Myc have been identified, such as Max, p107, TBP, YY1, Miz-1, AP-2 and Nmi. Both Max and Nmi also bind to MycN. In contrast to the well defined binding of Max to Myc family proteins the interaction of Nmi with Myc or MycN is only poorly characterized. By employing the yeast two-hybrid system we have mapped the regions of MycN and Myc responsible for binding to Nmi. For MycN exclusively a central region mediates binding to Nmi. In contrast, for Myc a C-terminal portion of the protein, and possibly also a central part, is involved in Nmi interaction. Nmi does not interact with Max and has no transactivation capabilities in yeast, suggesting that Nmi alone is not a transcriptional activator in mammalian cells. Immunofluorescence demonstrates that both in 293 embryonic kidney cells and in Kelly neuroblastoma cells all detectable ectopically expressed Nmi is localized in the cytoplasm, in part in a punctate, granular pattern. MycN, which is highly expressed in Kelly cells consequent to amplification, appears to be localized exclusively in the nuclei. This directly demonstrates that in the same cell at least the major proportion of MycN and Nmi is localized in different cellular compartments. This result is confirmed by the finding that endogenous Nmi, which is expressed in Kelly cells only after stimulation with interferon gamma, is detected exclusively in the cytoplasm of these cells. Therefore only a very small amount of MycN and Nmi is likely to be involved in MycN/Nmi interaction in vivo.
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PMID:Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN. 1059 90

NTAK (neural- and thymus-derived activator for the ErbB kinase, neuregulin-2) is a novel member of the epidermal growth factor (EGF) family. We have isolated and characterized the human NTAK gene, comprising 12 exons spanning in excess of 55 kilobases (kb). The 7. 0kb long mRNA of the human NTAK gene was expressed in the human neuroblastoma SK-N-SH cell line with two alternative isoforms detected. Furthermore, six isoforms have been identified from rat brain and PC-12 cells. Although the alpha isoform of the NTAK gene was found to be expressed in all tissues including brain, the beta isoform was expressed only in rat brain tissues. Potential regulatory regions included consensus binding sites for AP-2, TF-IIIA, Sp-1, and YY-1 located in the 5'-flanking region of the NTAK gene.
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PMID:Characterization of the human NTAK gene structure and distribution of the isoforms for rat NTAK mRNA. 1097 60

The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction.
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PMID:Induction of G protein-coupled receptor kinases 2 and 3 contributes to the cross-talk between mu and ORL1 receptors following prolonged agonist exposure. 1242 67


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