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 regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in N1E-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA. Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation. These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.
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PMID:Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels. 787 Jan 90

Different portions of the 5'-upstream region of the mouse proliferating cell-nuclear-antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides -1231 and -624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5'-terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities. Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752-bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation-independent and position-independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long-terminal repeat and the mouse DNA polymerase beta gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase beta gene.
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PMID:Nucleotide sequence and promoter-specific effect of a negative regulatory region located upstream of the mouse proliferating cell nuclear antigen gene. 790 77

The structure of the mouse neurotrophin-3 (NT-3) gene has been analysed using genomic cloning and the rapid amplification of cDNA ends (RACE) method. The gene consists of two small upstream exons (exons IA and IB) and a larger downstream exon (exon II) that encodes the mature protein. Two classes of NT-3 transcripts, termed transcripts A and B, are generated by alternative splicing of exon IA or exon IB to the common exon II. The NT-3 gene also contains several transcription start sites in both upstream exons, and three different polyadenylation sites in exon II, as shown by RNase protection assays and by RACE, giving rise to multiple NT-3 mRNA variants of slightly different lengths. Cerebellar granule neurons express both classes of NT-3 transcripts, but only transcript B is regulated by tri-iodothyronine (T3) in these neurons. The effect of T3 on NT-3 mRNA is primarily due to transcription enhancement, as shown in nuclear run-on experiments. The levels of NT-3 mRNA are much lower in cultured mouse astrocytes and are undetectable in the human neuroblastoma cell line IMR 32. A TATA box is present in the upstream region of exon IB but not in that of exon IA. Promoter analysis using the chloramphenicol acetyltransferase reporter gene fused to different NT-3 upstream regions showed the presence of two active NT-3 promoters in cerebellar granule neurons. However, in IMR 32 cells, NT-3 promoter activity decreased dramatically with increasing length of the 5' flanking region. This suggests that expression of the NT-3 gene is regulated both by positive influences, such as T3, and by negative silencing elements present in the upstream regions of the NT-3 promoter.
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PMID:Two promoters direct transcription of the mouse NT-3 gene. 795 96

Three alpha 1-adrenergic receptors (ARs) have been cloned, i.e., the alpha 1B-, alpha 1C-, and alpha 1D-ARs. Compared with the alpha 1B subtype, the alpha 1A subtype in tissue is described as being insensitive to chloroethylclonidine and sensitive to SZL-49 and having a 10-100-fold higher affinity for a number of agonists and antagonists. The alpha 1A subtype is also expressed in a variety of rat tissues (as assessed by pharmacology), with greatest abundance in the cerebral cortex, hippocampus, vas deferens, and submaxillary gland. The cloned bovine alpha 1C-AR, though having an alpha 1A-AR pharmacology, was first reported as not being expressed in any rat tissue (as determined by Northern analysis) and was therefore designated as a new subtype. We report the cloning, expression, and characterization of the rat homolog of the bovine alpha 1C-AR. Using a human alpha 1C-AR probe obtained by polymerase chain reaction screening of a neuroblastoma cell line (SK-N-MC), both exon 1 and exon 2 of the rat alpha 1C-AR gene were cloned from a rat genomic library. These two exons were spliced together and cloned into the expression vector pMT2'. Transfection into COS-1 cells and analysis of the ligand-binding profile of the expressed protein receptor using 125I-HEAT revealed a 10-100-fold higher affinity for the alpha 1-AR antagonists 5-methylurapidil, (+)-niguldipine, WB-4101, and phentolamine and the agonists oxymetazoline and methoxamine, compared with the alpha 1B-AR. This ligand-binding profile is similar to that for endogenously expressed tissue alpha 1A-ARs. In addition, the rat alpha 1C-AR was the least sensitive of the three cloned subtypes to the alkylating effects of chloroethylclonidine but was the most sensitive to the alkylating prazosin analog SZL-49, properties also observed for the tissue alpha 1A subtype. Furthermore, by three different techniques, i.e., RNase protection assays, reverse transcription-polymerase chain reaction Northern blotting, and in situ hybridization histochemistry, the rat alpha 1C-AR mRNA was localized to alpha 1A-AR-rich tissues, such as rat vas deferens, hippocampus, aorta, and submaxillary gland. Taken together, these data suggest that this receptor may actually represent the alpha 1A subtype.
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PMID:Cloning, expression, and tissue distribution of the rat homolog of the bovine alpha 1C-adrenergic receptor provide evidence for its classification as the alpha 1A subtype. 796 68

We have isolated genomic clones which encode the promoter and flanking region of human nonmuscle myosin heavy chain (MHC)-A. The sequence of this region shows many features typical of a housekeeping gene; there is no TATA element and no functional CAAT box. The GC content is high, having an average GC content of 74% in the 600 base pairs (bp) surrounding the transcriptional start sites, and multiple GC boxes (putative Sp1 binding sites) are present. A number of nucleotide sites are utilized for the initiation of transcription. Promoter activity was monitored using luciferase as a reporter following transient transfection into NIH 3T3 cells. Analysis of 5' and 3' deletion mutants in the promoter region defines the core promoter as extending from nucleotide -112 to +61, where +1 is a major transcriptional start site. An essential sequence for core promoter activity resides in the 36-bp region from -77 to -112 which includes a single potential AP-2 binding site and a single potential Sp1 binding site. The region just downstream from the transcriptional start site (between +62 and +257) was found to be involved in cell type-specific activation of nonmuscle MHC-A gene expression. The increase in luciferase activity due to this proximal downstream region is approximately 15-fold in NIH 3T3 cells, but no increase was observed in C2C12 myotubes and neuroblastoma cells. This 196-bp region, which consists of 100 bp from exon 1 and 96 bp from intron 1, functions in a position- and orientation-dependent manner. Quantitation of luciferase mRNA content driven by the MHC-A promoter, using both competitive polymerase chain reaction and RNase protection assays, revealed that the increase seen in luciferase mRNA due to the 196-bp fragment is approximately 5-fold in NIH 3T3 cells. This only accounts for about one-third of the total increase seen in luciferase activity (protein amounts). Thus, this proximal downstream region appears to activate gene expression in NIH 3T3 cells via both pretranslational (transcription and/or mRNA stability) and translational mechanisms.
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PMID:Evidence for an internal regulatory region in a human nonmuscle myosin heavy chain gene. 819 47

The sequence of a high molecular weight (HMW) tau cDNA cloned from a neuroblastoma N115 library contains, in addition to the C- and N-terminal and middle regions present in the low molecular weight mouse brain tau proteins, a 711-bp nonhomologous domain (exon 4a) and a region of 198 bp corresponding to exon 6 of the tau gene. Protein immunoblot analysis, performed with antibodies specific either for a sequence present in the N-terminal region of all the tau variants or for exon 4a revealed several bands suggesting that more than one tau form is expressed in this cell line. Northern blot experiments performed with a number of cDNA probes spanning domains common and uncommon to low molecular weight and HMW tau allowed the identification of four tau transcripts differing in the size of their coding and noncoding regions. All these transcripts contain the sequence encoded by exon 6, but two of them lack exon 4a. As shown by RNase protection assays, the N-terminal region of these transcripts is also variable and contains either exon 1, or exons 1 and 2, or exons 1-3. Yet all these HMW tau forms contain four homologous repeats in their C-terminal domain both in the differentiated and nondifferentiated cells, i.e., have adult characteristics. In conclusion, the data reported in this article demonstrate that several HMW tau variants are expressed in neuroblastoma N115 cells and that the transition between immature to mature tau forms occurring during brain development is not required for neurite outgrowth during morphological differentiation of this cell line.
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PMID:Heterogeneity of the high molecular weight tau proteins in N115 neuroblastoma cells. 836 Jun 88

The promoter region of the alpha-subunit of the calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) gene was inserted into a beta-galactosidase (beta-gal) reporter plasmid, and beta-gal activities were examined in neuroblastoma (NB2a) and pheochromocytoma (PC12) cells after transient or stable transfections. The alpha-CaMKII promoter was 12- to 45-fold more active in NB2a compared with PC12 cells after transient or stable transfections. All-trans retinoic acid (RA) stimulated reporter gene expression at both protein and mRNA levels in transfected PC12 cells. RA increased the level of endogenous alpha-CaMKII mRNA in untransfected PC12 cells by 4.4-fold. The transcription initiation site(s) (TIS) of the alpha-CaMKII gene in PC12 cells and rat brain was examined by RNase protection assays (RPA) and reverse transcriptase PCRs. The TIS for the alpha-CaMKII/beta-gal reporter gene in transfected PC12 cells was indistinguishable from the TIS+1 in rat hippocampus. In contrast, the only detectable TIS for the alpha-CaMKII gene in untransfected PC12 cells was located near the ATG translation start codon, 147 nucleotides 3' to TIS+1 in hippocampus. This unusual TIS was also the predominant TIS in rat cerebellum. These results suggest that the alpha-CaMKII promoter may contain sequences that respond directly or indirectly to RA. In addition, the unusual TIS of the alpha-CaMKII gene in PC12 cells and rat cerebellum may contribute to the very low expression of this gene compared with that in hippocampus.
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PMID:Retinoic acid stimulates alpha-CAMKII gene expression in PC12 cells at a distinct transcription initiation site. 879 26

The occurrence of multidrug resistance (MDR) is one of the main obstacles in the successful chemotherapeutic treatment of cancer. MDR cell lines are resistant to the so-called naturally occurring anti-cancer drugs, such as anthracyclines, Vinca alkaloids and epipodophyllotoxins, but are not cross-resistant to alkylating agents, antimetabolites and cisplatin. So far, three separate forms of MDR have been characterized in more detail: classical MDR, non-Pgp MDR and atypical MDR. Although all three MDR phenotypes have much in common with respect to cross-resistance patterns, the underlying mechanisms certainly differ. Atypical MDR is associated with quantitative and qualitative alterations in topoisomerase II alpha, a nuclear enzyme that actively participates in the lethal action of cytotoxic drugs. Atypical MDR cells do not overexpress P-glycoprotein, and are unaltered in their ability to accumulate drugs. In this review we will focus on classical and non-Pgp MDR. The molecular mechanism of classical and non-Pgp MDR is transcriptional activation of membrane-bound transport proteins. These transport proteins belong to the ATP-binding cassette (ABC) superfamily of transport systems. The classical MDR phenotype is characterized by a reduced ability to accumulate drugs, due to activity of an energy-dependent uni-directional, membrane-bound, drug-efflux pump with broad substrate specificity. The classical MDR drug pump is composed of a transmembrane glycoprotein (P-glyco-protein-Pgp) with a molecular weight of 170 kD, and is, in man, encoded by the so-called multidrug resistance (MDR1) gene. Typically, non-Pgp MDR has no P-gly-coprotein expression, yet has about the same cross-resistance pattern as classical MDR. This non-Pgp MDR phenotype is caused by overexpression of the multidrug resistance-associated protein (MRP) gene, which encodes a 190 kD membrane-bound glycoprotein (MRP). MRP probably works by direct extrusion of cytotoxic drugs from the cell and/or by mediating sequestration of the drugs into intracellular compartments, both leading to a reduction in effective intracellular drug concentrations. For the classical MDR phenotype, evidence is accumulating that it plays a role indeed, in clinical drug resistance, especially in some hematological malignancies (acute myeloid leukemia, multiple myeloma and non-Hodgkin's lymphoma) and solid tumors (soft tissue sarcomas and neuroblastoma). The association of MRP with clinical drug resistance has not been elaborated, yet, and studies on MRP expression in human cancer have just begun. We found that overexpression of MRP, as determined by RNase protection assay as well as by immunohistochemistry, occurs in several human cancers, among which are cancer of the lung, esophagus, breast and ovary, and leukemias. Further studies are indicated to establish whether elevated MRP expression at diagnosis is an unfavorable prognostic factor for clinical outcome of chemotherapy.
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PMID:Molecular mechanisms of multidrug resistance in cancer chemotherapy. 888 Aug 78

As an initial approach to define the regulatory elements and transcriptional factors that account for cell-restricted expression of the alpha2c-adrenergic receptor (AR) gene, we isolated and characterized the receptor gene and identified regions of the gene conferring cell-specific expression. A 4300-nucleotide (nt) fragment of the 5'-flanking region of the rat alpha2c-AR gene was isolated from a genomic library. The genomic sequence contained the uninterrupted sequence of the 5'-untranslated region of a previously isolated alpha2c-AR cDNA clone indicating the lack of introns in the 5' gene segment. RNase protection assays and/or RNA blot analysis indicated the expression of alpha2c-AR mRNA in rat brain but not in kidney or liver, which is consistent with the major expression of this gene in neuronal tissue. The 5' gene segment was used to identify sites of transcriptional initiation and promoter activity by RNase protection assays and transient transfection of reporter gene constructs. With the use of RNA probes progressively upstream of the translational start site, RNase protection assays with rat brain total RNA indicated multiple sites of transcriptional initiation within a approximately 70-nt span (-660 to -730 nt 5' to the translational start codon). The zone of transcriptional initiation was part of a larger GC-rich area of the 5' gene segment that is a characteristic of genes initiating transcripts at multiple sites. The promoter activity of this zone of transcriptional initiation and the influence of gene segments 5' to this area were addressed using chloramphenicol acetyl transferase reporter gene constructs. Transient transfection of reporter gene constructs indicated that a 96-nt gene fragment (-699/-603 relative to the translational start codon) was sufficient to direct transcription in the neuroblastoma X glioma hybrid cell line NG108-15, a cell line expressing the endogenous alpha2c-AR. Promoter activity was not observed in constructs lacking the zone of transcriptional initiation. The promoter segment was inactive when introduced into the rat glioma cell line C6B4, the rat submandibular cell line RSMT-A5, and the rat pancreatic beta cell line RIN-5AH, all of which do not express the endogenous alpha2c-AR gene. Upon incubation with nuclear extracts, a 129-nt fragment encompassing the promoter exhibited a gel mobility shift pattern that was specific for cells expressing the receptor protein and involved a nuclear protein that recognized a Sp1 oligonucleotide. These data indicate that a 96-nt gene promoter segment of the alpha2c-AR gene functions in a cell-type-specific manner.
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PMID:Analysis of the alpha2C-adrenergic receptor gene promoter and its cell-type-specific activity. 896 63

We have used SH-SY5Y neuroblastoma cells as a model for differentiating neurons to examine the mechanisms that regulate responses to the neuropoietic cytokine ciliary neurotrophic factor (CNTF). Retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) each induced differentiation of SH-SY5Y cells. Cells treated for 24 h with retinoic acid (10 microM) showed a threefold increase in 125I-CNTF binding sites and were up to five times more sensitive to CNTF than untreated cells in stimulating the tyrosine phosphorylation of the transcription factor STAT3. TPA (10 nM) induced a transient 42% decrease in 125I-CNTF binding sites after 4 h of treatment that recovered to near control levels after 7 h of continuous exposure. TPA-treated cells showed a decreased sensitivity to CNTF and a sevenfold decrease in levels of STAT3. The retinoic acid-induced increase in 125I-CNTF binding could be prevented by administration of either cycloheximide or actinomycin D, whereas neither agent altered the TPA-induced decrease in 125I-CNTF binding. In addition, levels of mRNA for both the CNTF receptor alpha and gp130 subunits increased twofold as measured by RNase protection after treatment with retinoic acid for 30 h. The increase in CNTF receptor alpha subunit mRNA was not due to a decrease in its turnover rate, and therefore, was likely due to an increase in gene expression. Thus, retinoic acid and TPA regulate CNTF receptors on neuroblastoma cells differently, and the results demonstrate the importance of transcriptional control of CNTF receptors and also implicate translational and post-translational mechanisms in the regulation of cytokine receptors and responses on neurons.
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PMID:Opposing regulation of ciliary neurotrophic factor receptors on neuroblastoma cells by distinct differentiating agents. 898 65

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