UMLS:C0027819 - FACTA Search

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

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.
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PMID:Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. 133 64

We have identified a positive regulatory cis-acting element of the adhesion molecule on glia (AMOG)/Na,K-ATPase beta 2 subunit gene as GAGGCGGGG at position -87 to -79 by transient transfection assay using B103 cells (rat neuroblastoma cell line). The newly identified AMOG regulatory element (AMRE) enhanced the promoter activity in a mutually compensating manner with the Sp1 element at position -147 to -142. AMRE acts as a positive regulatory element not only in B103 cells but also in 3Y1 (rat embryo cell line) cells to roughly the same extent and in MDCK (canine kidney cell line) cells to a lesser extent. AMRE also enhances other gene promoters, such as myelin basic protein (MBP) and herpes simplex virus (HSV) thymidine kinase (TK) gene promoters. The element is not a typical enhancer element because when it is introduced downstream of the HSV TK promoter, it has no enhancing activity.
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PMID:Identification and characterization of the cis-elements regulating the rat AMOG (adhesion molecule on glia)/Na,K-ATPase beta 2 subunit gene. 137 71

To investigate the regulatory processes involved in the expression of the D2 dopamine receptor gene, a rat genomic clone was isolated using a 21-mer oligonucleotide probe made of exon 1 sequences. A 1.3-kb region including all of exon 1, its 5'-flanking region, and part of intron 1 was sequenced. S1 nuclease analysis indicated three consecutive nucleotides as the main transcription start sites; several weaker sites were also noted between 321 and 363 nucleotides upstream from the 3' end of exon 1. The promoter region lacks TATA and CAAT boxes and is rich in G+C content with several putative Sp1 binding sites. Transient expression assays using chimeric constructs of D2 promoter deletion mutants-chloramphenicol acetyl-transferase gene in the neuroblastoma cell line NB41A3 which expresses D2 binding sites indicated strong transcription enhancing activity between nucleotides -75 and -30 and silencing activity between nucleotides -217 and -76. DNase I footprinting studies using nuclear extract from NB41A3 suggested Sp1 binding to its consensus sequence at nucleotide -48 but inhibition of Sp1 binding at nucleotide -86 by the extract. The D2 promoter could not induce transcription of the heterologous CAT gene in C6 glioma, embryonal NIH 3T3, or hepatic Hep G2 cells. It is concluded that the rat D2 gene shares with the human D1A dopamine receptor gene several features typical of "housekeeping" genes but they are both tissue-specific, regulated genes. Unlike the D1A gene, however, the D2 gene has a strong preference for transcription initiation to three consecutive nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of the promoter region of the rat D2 dopamine receptor gene. 139 Jun 23

Genomic and cDNA clones encoding the rat D1 receptor were isolated and sequenced. Comparison of the D1 receptor cDNA and genomic sequences revealed that the rat D1 receptor gene is organized into two exons separated by a small intron in the 5' untranslated region of its mRNA. The transcription start site is located 864 bp upstream from the translational initiation site. The 5'-flanking sequences of the D1 receptor gene do not contain TATA and CAAT canonical sequences, but have a high G+C content, potential cyclic AMP and glucocorticoid response element sequences, and binding sites for transcription factors such as Sp1, Ap1, and Ap2. Transfection studies using the D1 5'-flanking sequence and CAT gene fusion constructs have demonstrated that (1) the D1 promoter is active in D1-expressing neuroblastoma NS20Y cells, but inactive in D1-deficient glioma C6 and kidney 293 cells, (2) the information contained within 735 bp of 5'-flanking sequence of the D1 gene appears to be sufficient to confer its cell-specific expression, and (3) the D1 gene promoter responds to cyclic AMP induction, suggesting the existence of an auto-regulation mechanism by which the stimulation of D1 receptor exerts a positive feedback on its own gene expression.
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PMID:Characterization of gene organization and promoter region of the rat dopamine D1 receptor gene. 140 30

We cloned, sequenced and characterized a promoter region of the mouse homologue of the Alzheimer's disease amyloid precursor protein (APP)-encoding gene. The promoter region is highly homologous to that of the human APP (hAPP) gene. It has a high G+C content, lacks typical 'TATA' and 'CAAT' boxes, and contains possible binding sites for AP-1, heat-shock factor, Sp1 and AP-4. The promoter region was fused with the cat reporter gene, and the fusion genes were transfected to both the NB41A3 (mouse neuroblastoma) and L-cell lines. The promoter activity was monitored by chloramphenicol acetyltransferase (CAT) activity in a transient expression assay. The promoter was equally active in both cell lines. The deletion analysis revealed that there existed a negative regulatory element(s) between -153 and -100 bp and a positive element(s) between -100 and -37 bp. The negative element was shown to suppress the transcriptional activity of heterologous simian virus 40 promoter. DNase I footprinting experiments revealed that three nuclear protein-binding sites existed in the regulatory region, one in the negative and two in the positive regulatory regions. Gel retardation assay showed that Sp1 was one of the factors binding to the positive regulatory region. A nuclear factor binding to the negative regulatory region seemed to be missing in brain.
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PMID:Positive and negative regulatory elements for the expression of the Alzheimer's disease amyloid precursor-encoding gene in mouse. 155 68

Galanin (GAL) is a biologically active neuropeptide that has been suggested to play a role in stress-induced inhibition of insulin secretion, in dementia of the Alzheimer's type, and in the regulation of growth hormone secretion. We report here the isolation of a bovine genomic clone containing more than 5-kb 5'-flanking sequences. Partial sequence analysis of the genomic clone revealed an atypical TATA-box in the promoter (ATAAATA) and several consensus sequences that typically bind transcription factors, including those that bind NF kappa B, Sp1, and AP-2. Primer extension and RNase protection analyses revealed that transcription is initiated at two sites, 28 and 31 bp, respectively, downstream from the TATA-box. To locate functionally active regulatory elements on the GAL gene, we first identified a neural crest-derived human neuroblastoma cell line, SK-N-SH subclone SH-SY5Y, that expressed easily detectable levels of endogenous GAL mRNA. We then constructed plasmids containing various lengths of bovine GAL 5'-flanking sequences and the first exon fused to a reporter plasmid encoding luciferase. Transfection of these plasmids into the SH-SY5Y cells and analysis by transient expression indicated that 131 bp of 5' gene sequence was sufficient to obtain maximal basal expression. Further, expression was suppressed 16-fold when 5 kb were included, suggesting the presence of a distal repressor element(s). In another set of experiments, we found that GAL mRNA levels could be induced more than 10-fold by 20-hr treatment with phorbol 12-myristate 13-acetate (PMA). In cells transfected with the same plasmids, luciferase activity was also induced by PMA, but the degree of induction did not significantly differ among the deletion constructions (varying from six- to eight-fold), suggesting that elements conferring PMA induction and/or RNA stabilization may be located within 131 bp of the transcriptional start site, in the first exon, or on gene sequences not studied here.
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PMID:Primary sequence and functional analysis of the bovine galanin gene promoter in human neuroblastoma cells. 752 Jul 3

We report the first evidence that differential transcriptional regulation of human chromogranin A (CHGA) gene expression occurs during in vitro treatment of tumorigenic neuroblastoma (NB) cells with retinoic acid (5 microM) and/or dibutyryl-cAMP (1 mM). The CHGA gene encodes a tissue specific protein restricted to cells of the diffuse neuroendocrine system, but also widely expressed among NB tumours. We previously reported that CHGA as well as other neuroendocrine markers are modulated during NB differentiation in vitro. To investigate, at the molecular level, the mechanisms leading to NB tumour cell differentiation during the treatment with biologically active compounds, we sequenced and functionally characterised 2169 bp of a genomic DNA clone encompassing the 5' flanking region of the human CHGA gene. Computer-assisted analysis of the sequence revealed the presence of a cAMP responsive element at positions -56 to -49, and Sp1 binding sites at positions -181 to -176 and -216 to -210. Two novel 9 bp motifs, located at position -462 to -454 and -91 to -83 of the CHGA promoter were identified in the regulatory regions of two other neuroendocrine genes encoding for tyrosine hydroxylase and neuropeptide Y. In addition, in the first 1000 bp of the untranslated 5' region, we found the presence of several putative DNA binding sites of bHLH molecules, a protein family regulating tissue specific differentiation. Transient transfection experiments of chloramphenicol acetyltransferase (CAT) deletion constructs, showed the presence of an active promoter within the first 455 bp upstream from the start site. This region conferred tissue specific expression to a CAT reporter gene. In addition, the transcriptional activity of this fragment was modulated during the induction of differentiation of NB cells treated by retinoic acid and/or dibutyryl-cAMP. These observations provide preliminary data regarding CHGA transcriptional regulation in NB cells, and indicate that retinoic acid and cAMP activate distinct, apparently competitive, transcriptional pathways during NB cell differentiation. The molecular characterisation of the mechanisms regulating CHGA expression in tumour and normal neuroendocrine tissue could lead to the identification of novel molecules potentially relevant for future gene therapy of NB tumours.
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PMID:Retinoic acid and cAMP differentially regulate human chromogranin A promoter activity during differentiation of neuroblastoma cells. 757 43

The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes and has been shown to be extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, it is important to characterize its promoter. In this report, the 5' end of the human 5-HT2AR gene was cloned and characterized. Anchored PCR mapped multiple transcription initiation sites at nucleotides -1157, -1137, -1127, and -496. Transfection of chimeric growth hormone plasmids containing various DNA fragments into 5-HT2AR-positive human cell lines (SHSY-5Y, neuroblastoma; HeLa, cervix carcinoma) showed that the 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites between -1157 and -1127 and 5' of the downstream initiation site (at -496), exhibited significant promoter activity. This promoter activity was not affected by the sequence upstream of the 0.74 kb fragment. The sequence downstream (the 0.45 kb PvuII/SmaI fragment) strongly repressed this promoter activity, suggesting the presence of a silencer. Sequence analysis combined with gel retardation and Dnase 1 footprinting assay identified multiple cis and trans elements for this fragment, including Sp1, PEA3, cyclic AMP response element (CRE)-like sequence, and E-boxes. Two novel transcription factors have been detected by gel retardation and DNase 1 footprinting assay; one of them may be specific for human. The transcription factors and promoter activities were low in the negative cell line NCI-H460 (human lung large cell carcinoma). Interestingly, the 0.39 kb fragment, isolated from the 3' end of the 0.74 kb fragment, exhibited the highest promoter activity. The possibility that this 0.39 kb fragment may be an alternative promoter is discussed. These new data are essential for further study of the regulation of 5-HT2AR gene expression.
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PMID:Characterization of the human 5-HT2A receptor gene promoter. 762 19

We analyzed cis-acting elements regulating the expression of the gene encoding adhesion molecule on glia (AMOG) in primary cultured astrocytes from newborn rat cerebrum and cerebellum. The relative promoter activities among the series of 5' sequential deletion mutants are similar to those observed in B103 (rat neuroblastoma cell line) cells. The previously identified AMRE (AMOG regulatory element) of the GAGGCGGGG sequence functions as a positive regulatory element, not only in B103 cells, but also in astrocytes. Binding factors to the element were identified as Sp1 based on the following observations using nuclear extracts from the astrocytes and B103 cells: (1) The interaction of the factors with AMRE analyzed by DNase I footprinting and methylation interference analyses was similar to that of Sp1; (2) The binding of the factors to AMRE competed with an oligonucleotide containing the authentic Sp1 consensus sequence; (3) Sp1-specific antibody interfered with the formation of the AMRE gel retardation complexes. The functional implications of the factors in AMOG gene regulation are discussed.
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PMID:Sp1 binds to the adhesion-molecule-on-glia regulatory element that functions as a positive transcription regulatory element in astrocytes. 768 84

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.
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PMID:Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions. 774 11

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