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 report a flexible strategy for the high level expression of a recombinant human monoclonal antibody (mAb) in Chinese hamster ovary (CHO) cells, initially using COS monkey kidney cell transfections to evaluate rapidly modifications to immunoglobulin (Ig) DNA constructs. Using sequential transfections with two amplifiable markers, we generated CHO cell lines and clones that secrete 80-110 micrograms/10(6) cells/24 hours of a mouse-human chimeric IgG1 kappa mAb. This cellular productivity is considerably greater than most murine hybridomas and transfected myelomas. Our data also demonstrate that genomic kappa sequences can improve mAb expression in COS and CHO cells. As a paradigm, we focused our expression studies on a human chimeric form of 3F8, a murine mAb that binds to ganglioside GD2 on neuroblastoma and melanoma tumor cells.
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PMID:High level expression on a chimeric anti-ganglioside GD2 antibody: genomic kappa sequences improve expression in COS and CHO cells. 138 57

Using test plasmids containing the SV40 origin, we found a wide spectrum of permissiveness to their replication in different human cell lines. N-myc overexpressing neuroblastoma cells were highly permissive. LA-N-1 neuroblastoma cells were the most permissive of all the cell lines that we tested including the homologous CV-1 or COS-1 monkey kidney cells. Other human cell lines expressing various amounts of c-myc, and the 293 cell line expressing adenovirus E1A and E1B exhibited intermediate levels of permissiveness. T24 and EJ bladder carcinoma cells, which do not express the myc genes, were nonpermissive. Transient expression of c-myc or N-myc from plasmid vectors resulted in a modest stimulation of replication. Replication of test plasmids containing different configurations of the SV40 origin region was activated by the myc proteins. The high efficiency of replication in LA-N-1 cells is due to a combination of reasons including the overproduction of N-myc, high efficiency of expression of the SV40 replication initiator protein large T antigen from a cotransfected expression plasmid (containing the T antigen gene under the RSV LTR control), and other unknown host cell replication stimulatory factors. Replication of test plasmids was not detected in N-myc or c-myc overexpressing cells when the T antigen expression plasmid was not provided, showing that the myc proteins cannot substitute for T antigen in SV40 DNA replication.
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PMID:Efficient replication of plasmids containing the SV40 origin in N-myc overexpressing human neuroblastoma cells. 165 Apr 40

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotide-binding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the previously cloned catecholamine receptors. Northern blot analysis of various neural tissues revealed a transcript of approximately 4 kb that was predominantly located in the striatum with lesser amounts in the cortex and retina. In contrast, no mRNA was detected in the cerebellum, hippocampus, olfactory bulb, mesencephalon, or pituitary. In situ hybridization analysis also revealed a high abundance of mRNA in the striatum as well as in the olfactory tubercle. To establish the identity of this cDNA, we performed transient expression experiments in COS-7 cells. [3H]SCH-23390, a D1-selective radioligand, exhibited specific, saturable binding only in cells that were transfected with this cDNA. Competition binding analysis with a variety of dopaminergic ligands demonstrated a D1 dopaminergic pharmacology. In addition, dopamine as well as other D1-selective agonists stimulated cAMP accumulation in transfected COS-7 cells. We conclude that we have cloned a cDNA encoding the D1 dopamine receptor linked to the activation of adenylyl cyclase activity.
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PMID:Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation. 216 56

Thy-1 is a cell surface differentiation marker which shows distinct patterns of tissue-specific expression in different species. In man, the Thy-1 antigen is encoded by chromosome 11. We have examined the regulatory signals determining human Thy-1 expression through serologic analysis of rodent-human somatic cell hybrids retaining human chromosome 11 in which the fusion partners belong to distinct differentiation lineages. Cell surface expression of human Thy-1 was determined by mixed hemadsorption assays with two monoclonal antibodies (mAb), K117 and L127, shown to detect authentic human Thy-1 through analysis of COS-7 monkey kidney cells transfected with a cloned human Thy-1 gene. Three different patterns of human Thy-1 expression were observed when hybrid cells, constructed with different human and rodent cell types, were tested with mAb K117 and L127. Hybrids formed between Thy-1+ human neuroblastoma cells and Thy-1- mouse neuroblastoma cells, or hybrids between Thy-1+ human fibroblasts and the Thy-1- mouse kidney carcinoma, RAG, retain human Thy-1 expression. In contrast, hybrids formed between either Thy-1+ human neuroblastoma cells or Thy-1+ human fibroblasts and Thy-1- mouse L cells lose expression of human Thy-1 even though chromosome 11 is retained. Finally, hybrids formed between Thy-1- human peripheral lymphocytes or a Thy-1- lymphoblastoid B cell line and Thy-1- Chinese hamster fibroblasts begin to express human Thy-1. These studies suggest that both positive and negative trans-acting signals may play a role in the tissue-specific regulation of the human Thy-1 gene.
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PMID:Differential expression of the human Thy-1 gene in rodent-human somatic cell hybrids [corrected]. 288 61

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.
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PMID:Cloning and expression of cDNA encoding mouse tyrosinase. 300 90

Murine N1E-115 neuroblastoma cells are shown to express a single class of angiotensin II (Ang II) receptors that display all the pharmacological properties defining the Ang II receptor subtype 2 (AT2): high affinity for 125I-labelled AT2-selective agonist CGP 42112 (Kd 91 +/- 19 pM); expected rank order of potency (CGP 42112 = (Sar1,Ile8)Ang II > or = Ang II > PD 123319 >> DUP 753) for several Ang II analogues; increased binding in the presence of the reducing reagent dithiothreitol (DTT); and insensitivity to analogues of GTP. Molecular cloning of cDNA encoding AT2 receptors from N1E-115 cells reveals nucleotide sequence identity with the AT2 subtype expressed in fetal tissue. Murine AT2 receptors transiently expressed in COS cells display the same pharmacological profile as endogenous Ang II receptors of N1E-115 cells. Taken together, these data reveal the exclusive presence of the AT2 receptor subtype in N1E-115 cells. Incubation of N1E-115 cells with Ang II leads to a marked decrease in the level of tyrosine phosphorylation of several proteins with apparent molecular masses of 80, 97, 120, 150 and 180 kDa respectively. Tyrosine dephosphorylation of the same set of proteins is observed after treatment with the AT2-specific agonist CGP 42112. The response to both effectors is rapid and transient, showing a maximum between 5 and 10 min, and returning to basal levels after 20-30 min. In both cases, tyrosine dephosphorylation can be prevented by co-incubation with an excess of the antagonist Sarile. These data thus establish that AT2 receptor activation leads to protein tyrosine dephosphorylation in N1E-115 cells, and support a possible role for AT2 receptors in the negative regulation of cell proliferation.
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PMID:Angiotensin II AT2 receptors are functionally coupled to protein tyrosine dephosphorylation in N1E-115 neuroblastoma cells. 753 1

The 5-hydroxytryptamine3 receptor 5-HT3R has been implicated in gut and cardiac motility and in behavioral disorders. Characteristics of 5-HT3Rs appear to be heterogeneous among species, but human 5-HT3R cDNA has not been identified. We isolated a cDNA encoding 5-HT3R from human hippocampus. The mouse 5-HT3R gene has been reported to generate two alternative splicing isoforms that differ by six amino acids. All of our isolated human clones corresponded to the shorter isoform. Amino acid identities with mouse neuroblastoma N1E-115 and rat brain 5-HT3Rs were 84% for each. Southern blot analysis of human genomic DNA suggested that our cloned transcript encoded a human counterpart for the rodent 5-HT3Rs. This gene was assigned to chromosome 11 using polymerase chain reaction analysis of a human/rodent somatic cell hybrid panel. With the use of Northern blot analysis, 5-HT3R transcripts were identified in human small intestine, colon, and brain regions including hippocampus, amygdala, and striatum. In human heart, 5-HT3R expression was not detectable even with reverse transcriptase-polymerase chain reaction analysis, although it was detectable in mouse heart. Transfection of COS-1 with human 5-HT3R cDNA induced specific binding of the 5-HT3R-selective radioligand [3H]YM060. Human 5-HT3R showed typical characteristics of the 5-HT3R, but its affinity for the 5-HT3R agonist m-chlorophenylbiguanide was much lower than that of rat 5-HT3R. When injected with human 5-HT3R cRNA, the oocytes responded to 5-HT3R agonists with a rapidly developing inward current. The potency of the agonists to induce inward current paralleled that to compete with the radioligand binding, and 2-methyl-5-hydroxytryptamine, a partial agonist for mouse 5-HT3R, was a full agonist for human 5-HT3R. Our data revealed that the 5-HT3R molecule has interspecies differences in both tissue distribution and functional profile.
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PMID:Molecular cloning of human 5-hydroxytryptamine3 receptor: heterogeneity in distribution and function among species. 756 20

We have examined the 5'-flanking region (944 bp) of the human choline acetyltransferase (hChAT) gene for sequences that modulate its transcriptional activity and identified a sequence 5'-TGACCCA-3' which confers c-Jun/c-Fos (AP-1) inducibility of homologous and heterologous promoters. Using transient transfections in neuroblastoma NE-1-115 and COS-1 cells, we show that ligand-activated estrogen receptor (HEGo) represses the transcriptional activation by c-Fos/c-Jun. Testing HEGo mutants in transfection assays reveals that the ligand-binding domain is crucial for this repression, whereas the N-terminal (A/B) region and the DNA-binding domain are not essential. Gel retardation assays show that the hChAT AP-1 recognition sequence binds in vitro baculovirus-produced c-Jun/c-Fos proteins. This binding is inhibited by addition of baculovirus-produced HEGo. In contrast to HEGo, ligand-activated glucocorticoid, androgen, and retinoic acid receptors (RARs) enhance the transcription activation induced by c-Jun/c-Fos. All three types of RARs--RAR alpha, beta, gamma--and RXR alpha are able to stimulate AP-1 activity on the proximal hChAT promoter. Several mechanism possibilities involving protein-protein interaction are discussed to explain the phenomena.
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PMID:Positive and negative effects of nuclear receptors on transcription activation by AP-1 of the human choline acetyltransferase proximal promoter. 774 8

We have used the mouse delta-opioid receptor (mDOR) cDNA to isolate the mDOR gene and its human homologue. In both species the coding region is interrupted by two introns with conserved exon-intron boundaries located after transmembrane domains 1 and 4. Using the polymerase chain reaction and primers based on the sequence of the cloned human delta-opioid receptor (hDOR) gene, we have obtained a full length cDNA encoding the hDOR from SH-SY5Y neuroblastoma cells. The cDNA sequence is 100% identical to the cloned human genomic sequence and 94% identical to the mouse sequence at the protein level. When expressed in COS cells, hDOR displays nanomolar affinities for delta-selective ligands, whereas the affinities for mu- and kappa-selective ligands are in the micromolar range. The delta agonists [D-Ala2, D-Leu5]enkephalin, cyclic [D-penicillamine2,D-penicillamine5]enkephalin, and BW373U86 efficiently decrease forskolin-induced cAMP levels in hDOR-expressing COS cells, indicating functional coupling of the receptor. The distribution of hDOR mRNA in human brain was investigated using delta-selective reverse transcription-polymerase chain reaction amplification, followed by Southern hybridization with a delta-specific probe. The transcript is found in cortical areas, including olfactory bulb, hippocampus, and amygdala, as well as in basal ganglia and hypothalamus. No expression is detected in internal globus pallidus, thalamus, any investigated brainstem structure, or pituitary gland. Taken together, our results indicate similar structural, pharmacological, functional, and anatomical properties for the hDOR and the mDOR and therefore support the use of rodent models for the study of these receptors in opioid function.
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PMID:The human delta-opioid receptor: genomic organization, cDNA cloning, functional expression, and distribution in human brain. 780 19

RC3 encodes a thyroid hormone-dependent, calmodulin-binding, protein kinase C substrate (neurogranin, p17) present in the dendritic spines of discrete neuronal populations in the forebrain. Its physiological role could be related to synaptic plasticity, memory, and other processes. In the present work we have isolated and sequenced 2.4 kbp of genomic DNA upstream from the origin of transcription and determined its nucleotide sequence. The major features of the RC3 promoter are the absence of TATA and CAAT boxes and the presence of an Initiator sequence surrounding the cap site. By sequence analysis we identified several cis-acting regulatory elements, among them response elements for retinoic acid and steroid (glucocorticoids/progesterone) hormone receptors. An oligonucleotide containing the retinoic acid responsive element bound to retinoic acid receptors specifically in vitro and conferred retinoic acid regulation to a heterologous promoter after transfection in COS-7 cells. Retinoic acid and dexamethasone, respectively, increased activity of the RC3 promoter in neuroblastoma cells when a deletion construct containing the retinoic acid and the glucocorticoid responsive elements was cotransfected with retinoic acid receptor or glucocorticoid receptor expression vectors. When added together all-trans retinoic acid and dexamethasone had additive effects. Despite the fact that RC3 expression in vivo is thyroid hormone-dependent, no evidence for the presence of a thyroid hormone responsive element was found within the 2.4 kbp flanking region analyzed and thyroid hormone did not increase reporter activity after cotransfection of suitable constructs with thyroid hormone receptor expression vectors. Our results suggest that the expression of RC3 in vivo could be subject to complex physiological signals, including retinoids and steroid hormones in addition to thyroid hormones.
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PMID:Characterization of the promoter region and flanking sequences of the neuron-specific gene RC3 (neurogranin). 789 4


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