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)

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

The human ABCA2 transporter gene encodes a member of a large family of ATP-binding proteins that transport a variety of macromolecules across biological membranes. We have performed luciferase reporter gene assays with promoter constructs comprising the 5'-flanking region to identify cis-regulatory DNA elements and have mapped the minimal promoter region to 321 bp upstream of the translation start site. We have discovered a functional role for two GC-boxes located in the proximal promoter of the ABCA2 gene that contain overlapping sites for the EGR-1 and Sp1 transcription factors. We observed that oligonucleotides containing overlapping EGR-1/Sp1 sites bind the Sp1, Sp3 and Sp4 transcription factors. When BE(2)-M17 cells were treated with phorbol 12-myristate 13-acetate, we observed inducible expression and binding of the EGR-1 transcription factor to the two GC-boxes. Transfection of Sp1, Sp3 or Sp4 expression constructs into Drosophila S2 induced a dose-dependent increase in transcriptional activation of the ABCA2 promoter, but transfection of EGR-1 alone failed to activate transcription. When increasing amounts of EGR-1 were transfected into the BE(2)-M17 neuroblastoma cells we observed a dose-dependent decrease in expression of the ABCA2 promoter, although expression of the endogenous ABCA2 gene increased following transfection of EGR-1.
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PMID:Reciprocal regulation of expression of the human adenosine 5'-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors. 1256 May 8

The vesicular GABA transporter (VGAT) loads GABA from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons that contain GABA and/or glycine. To elucidate the molecular mechanisms of mouse VGAT (mVGAT) gene expression, we have isolated and characterized the mVGAT gene. The mVGAT gene was found to be 4.7 kilobases in size and to contain three exons and two introns by comparison of the cloned genomic DNA with the cDNA (termed mVGATa) sequence reported by Sagne et al. [FEBS Lett. 417 (1997) 177]. Analysis of transcripts and genomic DNA revealed an alternatively spliced mVGAT isoform (termed mVGATb) that retains intron 2 of mVGATa as an exon. This alternative transcript specifies 514 amino acid residues identical to VGATa followed by a unique C-terminal sequence of 11 amino acids encoded by intron 2. Fluorescent in situ hybridization studies showed that the mVGAT gene is localized on chromosome 2. One major transcription start site of the mVGAT gene is an A residue 209 bp upstream from the translational initiation site, as shown using the 5'-RACE method. RT-PCR analysis revealed that the mVGAT gene was expressed at a high level in retinoic acid-treated P19 embryonal carcinoma cells, at a very low level in non-treated P19 cells, and not detectably expressed in Neuro-2a neuroblastoma cells. Sequence analysis of the 5'-flanking region revealed a number of putative regulatory elements including Sp1, Egr-1 and Pitx binding sites. In transient transfection assays, 2 kilobases of the mVGAT 5'-flanking region generated similar levels of luciferase reporter activity in three kinds of cultured cells. Deletion analysis and gel mobility shift assays demonstrated that the region -161 to +155 contained the basal promoter activity of the mVGAT gene and that an activating region from -49 to -27 bound an Sp1-like protein. These results suggest a possible mechanism for regulation of the expression of the mVGAT gene.
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PMID:Mouse vesicular GABA transporter gene: genomic organization, transcriptional regulation and chromosomal localization. 1257 41

Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 bp region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA-protein interactions of the promoter, while mutations flanking this motif did neither. On super-shift, this region was shown to recruit Sp1 and Sp3 transcription factor proteins, while a functionally significant CT-box mutation resulted in their replacement by NF-1 transcription factor. Lysates from Drosophila S2 cells expressing exogenous Sp1, Sp3, and NF-1 proteins were able to partially mimic gel shift complexes seen with neuroblastoma nuclear extract and either wild type or mutant probes. Transient transfections of S2 cells showed that both individually and together, Sp1 and Sp3 were able to trans-activate a wild type CT-box-driven luciferase reporter construct in a dose-dependent manner. Transfection of the wild type but not mutant CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critically functional role, and in the basal state, allows for N-myc trans-activation by Sp1 and Sp3. Moreover when mutated, the CT-box may still function as a binding motif for alternate transcription factors such as NF-1 that can allow persistent N-myc expression.
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PMID:N-myc oncogene expression in neuroblastoma is driven by Sp1 and Sp3. 1456 77

Amplification of the MYCN gene, resulting in overexpression of MYCN, distinguishes a subset of neuroblastomas with poor prognosis. We recently identified MYCN as a target gene of the E2F transcription factors. Here we show that Sp1 and Sp3 cooperate with E2F-1 to activate the MYCN promoter. However, in a neuroblastoma cell line that does not express MYCN, overexpression of E2F-1 was not sufficient to activate the MYCN promoter even in the presence of trichostatin A and 5-aza-cytidine. This was because of a failure of E2F-1 to bind to the MYCN promoter in these cells, although access of E2F-1 to the inactive MYCN promoter was not blocked by a nucleosome. Differences in nucleosomal organization of the MYCN promoter in different cell lines did not correlate with gene activation per se but with the switch from basal to activated transcription. Binding of E2F and Sp1/Sp3 to the MYCN promoter in vivo correlated with acetylation of histones H3 and H4 and recruitment of RNA polymerase II and the protein acetyltransferase Tip60 but not with nucleosome remodeling. Our results define distinct chromatin states of the MYCN promoter, indicate that factors in addition to E2F and Sp1/Sp3 are required to activate MYCN in neuroblastomas, and provide evidence for a novel mechanism of controlling access of E2F to selected target genes.
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PMID:E2F and Sp1/Sp3 Synergize but are not sufficient to activate the MYCN gene in neuroblastomas. 1464 38

Chemoattractant-like receptor 1 (CMKLR1) is a functionally unknown ("orphan") G-protein coupled receptor. It has been implicated in osseous and cartilage development, and it also has a pathophysiological role as one of the minor coreceptors involved in human immunodeficiency virus type I (HIV-1)/simian immunodeficiency virus (SIV) infection of CD4(+) immune cells. Here we report the cloning of the mouse cmklr1 gene, the characterization of its genomic structure for comparison with the human gene, and the mapping and functional analysis of its 5' flanking sequence. The gene was found to contain three exons intercepted by one larger and one smaller intron. The overall structure resembles the human orthologue. The promoter lacks classical TATA and CCAAT boxes but contains several GC-rich regions as well as AP-4 elements, C/EBP motifs, and GATA-1 and GATA-2 binding sites. Promoter function was analyzed in mouse neuroblastoma (NB4 1A3) cells, endogenously expressing CMKLR1, as well as in mouse embryonic fibroblastic (3T3 clone A31) cells not expressing CMKLR1. 5' Deletion analysis and luciferase reporter gene assays of the promoter indicated that a 280-bp sequence adjacent to the transcription start site (established through 5'-RACE) is essential for initiating transcription. Within this region it was possible to identify four potential Sp1-binding sites that may be active in the transcription of the gene. Thus, we show that the mcmklr1 gene has several conserved features in common with its human counterpart, which suggests that they are regulated in a similar manner. The promoter does not seem to be tissue specific but other elements or enhancers may be missing. The results provide a necessary basis for further studies of the gene regulation and function of this chemoattractant-like receptor and will be useful when manipulating the gene in the development of transgenic animal models.
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PMID:Genomic organization and promoter analysis of the gene encoding the mouse chemoattractant-like receptor, CMKLR1. 1501 96

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthetic pathway of galactocerebroside (GalC), the most abundant glycolipid in myelin. Using a GalC expressing cell line, human oligodendroglioma (HOG), one which does not express GalC, human neuroblastoma (LAN-5), we previously demonstrated that the human CGT (hCGT) gene promoter functions in a cell-specific manner. Because the proximal (-292/-256) and distal (-747/-688) positive domains were shown to be critically involved in regulating the expression of several myelin-specific genes, we further investigated the functional roles of these two motifs in hCGT expression. Mutation analysis confirmed that a GC-box (-267/-259) and a CRE (-697/-690) were critical for hCGT expression. Electrophoretic mobility shift assay (EMSA) demonstrated that these motifs specifically bound to nuclear extracts from both cell lines. Using antibodies to Sp1, Sp3, pCREB-1, and ATF-1, these proteins were shown to be components of the EMSA complexes. However, the only difference between the HOG and LAN-5 cells was found in the EMSA profile of the CRE complexes. This difference may account for the differential transcription of the hCGT gene in the two cell types. Furthermore, the expression levels of ATF-1 detected were much higher in HOG cells than in LAN-5 cells. Thus, our data suggest that the GC-box and CRE function cooperatively, and that the CRE regulates the cell-specific expression of the hCGT gene.
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PMID:Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression: functional role of GC-box and CRE. 1522 98

Beta-1,4-galactosyltransferase (beta-1,4-GalT) V is a constitutively expressed enzyme that can effectively galactosylate the GlcNAcbeta1-->6Man group of the highly branched N-glycans that are characteristic of tumor cells. Upon malignant transformation of cells, the expression of the beta-1,4-GalT V gene increases in accordance with the increase in the amounts of highly branched N-glycans. Lectin blot analysis showed that the galactosylation of highly branched N-glycans is inhibited significantly in SH-SY5Y human neuroblastoma cells by the transfection of the antisense beta-1,4-GalT V cDNA, indicating the biological importance of the beta-1,4-GalT V for the functions of highly branched N-glycans. We cloned the 2.3-kb 5'-flanking region of the human beta-1,4-GalT V gene, and we identified the region -116/-18 relative to the transcription start site as that having promoter activity. The region was found to contain several putative binding sites for transcription factors, including AP2, AP4, N-Myc, Sp1, and upstream stimulatory factor. Electrophoretic mobility shift assay showed that Sp1 binds to nucleotide positions -81/-69 of the promoter region. Mutations induced in the Sp1-binding site showed that the promoter activity of the beta-1,4-GalT V gene is impaired completely in cancer cells. In contrast, the promoter activity increased significantly by the transfection of the Sp1 cDNA into A549 human lung carcinoma cells. Mithramycin A, which inhibits the binding of Sp1 to its binding site, reduced the promoter activation and expression of the beta-1,4-GalT V gene in A549 cells. These results indicate that Sp1 plays an essential role in the transcriptional activity of the beta-1,4-GalT V gene in cancer cells.
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PMID:Transcriptional regulation of the human beta-1,4-galactosyltransferase V gene in cancer cells: essential role of transcription factor Sp1. 1526 12

Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters. A deficiency in monoamine oxidase A results in aggressive behavior in both humans and mice. Studies on the regulation of monoamine oxidase A gene expression have shown that the Sp1 family is important for monoamine oxidase A expression. To search for novel transcription factors, the sequences of three Sp1 sites in the monoamine oxidase A core promoter were used in the yeast one-hybrid system to screen a human cDNA library. A novel repressor, R1 (RAM2), has been cloned. The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5'-end. The transfection of R1 in a human neuroblastoma cell line, SK-N-BE (2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity. The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression. R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment. A gel-shift assay indicated that the endogenous R1 protein in SK-N-BE (2)-C cells interacted with the R1 binding sequence. R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay. Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus, which suggested a role for R1 in transcriptional regulation. Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues. Taken together, this study shows that R1 is a novel repressor that inhibits monoamine oxidase A gene expression.
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PMID:R1, a novel repressor of the human monoamine oxidase A. 1565 81

The dopamine transporter is a plasma membrane protein that controls extracellular concentrations of the neurotransmitter dopamine. The physiological importance of the DAT provides the impetus for studies aimed at understanding the molecular mechanisms underlying regulation of the DAT gene. In this study, we identified a DAT-expressing neuroblastoma cell line (SK-N-AS) and employed it to investigate the transcriptional regulation of the human DAT gene. Two GC boxes (located at -130 and -60, respectively, relative to the transcriptional start site) were identified as important cis-acting elements mediating DAT promoter activity in dopaminergic SK-N-AS cells. Utilizing Sp-deficient Drosophila Schneider line (SL-2) cells, we showed that both Sp1 and Sp3 are strong activators of DAT transcriptional activity. Differential binding of Sp1 and Sp3 to the two GC boxes was demonstrated by electrophoretic mobility shift assays and super-shift assays. Our results indicate that the Sp1 family of proteins plays an important role in controlling the expression of the dopamine transporter gene within dopaminergic neurons.
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PMID:Sp1 and Sp3 activate transcription of the human dopamine transporter gene. 1581 70


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