CHAT

Gene Summary

Gene:CHAT; choline O-acetyltransferase
Aliases: CMS6, CMS1A, CMS1A2, CHOACTASE
Location:10q11.2
Summary:This gene encodes an enzyme which catalyzes the biosynthesis of the neurotransmitter acetylcholine. This gene product is a characteristic feature of cholinergic neurons, and changes in these neurons may explain some of the symptoms of Alzheimer's disease. Polymorphisms in this gene have been associated with Alzheimer's disease and mild cognitive impairment. Mutations in this gene are associated with congenital myasthenic syndrome associated with episodic apnea. Multiple transcript variants encoding different isoforms have been found for this gene, and some of these variants have been shown to encode more than one isoform. [provided by RefSeq, May 2010]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:choline O-acetyltransferase
HPRD
Source:NCBIAccessed: 06 August, 2015

Ontology:

What does this gene/protein do?
Show (28)
Pathways:What pathways are this gene/protein implicaed in?
Show (1)

Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 06 August 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Nerve Growth Factors
  • Cell Line
  • Chromosomes, Human, 13-15
  • Neuroblastoma
  • Base Sequence
  • Gene Expression Regulation
  • Breast Cancer
  • Childhood Cancer
  • Chromosome Aberrations
  • Choline O-Acetyltransferase
  • Xeroderma Pigmentosum
  • Chromosomes, Human, 21-22 and Y
  • Transcription
  • Messenger RNA
  • Neurons
  • Transfection
  • Newborns
  • Translocation
  • Microscopy, Electron
  • Cri-du-Chat Syndrome
  • Cell Differentiation
  • Cervical Cancer
  • Chromosome Disorders
  • Klinefelter Syndrome
  • Down Syndrome
  • Turner Syndrome
  • Chromosomes, Human, 6-12 and X
  • Tyrosine 3-Monooxygenase
  • Infant
  • Chromosome 10
  • Karyotyping
  • Trisomy
  • Mutation
  • Acetylcholine
  • Signal Transduction
  • Cancer Gene Expression Regulation
  • rac GTP-Binding Proteins
  • Cell Division
  • Chromosomes, Human, 16-18
  • Molecular Sequence Data
Tag cloud generated 06 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CHAT (cancer-related)

Zhang L, Ramchandren R, Papenhausen P, et al.
Transformed aggressive γδ-variant T-cell large granular lymphocytic leukemia with acquired copy neutral loss of heterozygosity at 17q11.2q25.3 and additional aberrations.
Eur J Haematol. 2014; 93(3):260-4 [PubMed] Related Publications
T-cell large granular lymphocytic leukemia (T-LGLL) is a rare indolent lymphoproliferative disorder characterized by cytopenias, splenomegaly, and various degrees of T-cell lymphocytosis, due to a clonal expansion of CD8-positive cytotoxic T-cells. Phenotypic variants of T-LGLL include CD4(+) /CD8(-) T-cells, with dual CD4(-) /CD8(-) /γδ(+) T-cells being even rarer. Cytogenetic abnormalities in T-LGLL have rarely been reported, and there is scientific debate regarding the existence of aggressive or transformed variants of T-LGLL. We report a patient with T-LGLL, γδ variant, with nearly 20-year-long duration of cytopenias before transformation to an unusual clinical scenario, manifesting with marked lymphocytosis >100 × 10(9) /L and infiltration of lymph nodes, tonsils, and subcutaneous tissue. Single-nucleotide polymorphism assays revealed acquired copy neutral loss of heterozygosity at 17q and deletion of 3p21.31, in addition to trisomy 5, monosomy X, and monosomy 21. These genetic abnormalities provided a better understanding of the molecular nature and the potentiality of disease transformation.

Inazu M, Yamada T, Kubota N, Yamanaka T
Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy.
Pharmacol Res. 2013; 76:119-31 [PubMed] Related Publications
Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

Andres D, Keyser BM, Petrali J, et al.
Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.
BMC Neurosci. 2013; 14:49 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells.
RESULTS: We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG.
CONCLUSION: Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Parnell EA, Calleja-Macias IE, Kalantari M, et al.
Muscarinic cholinergic signaling in cervical cancer cells affects cell motility via ERK1/2 signaling.
Life Sci. 2012; 91(21-22):1093-8 [PubMed] Related Publications
AIMS: The etiology of cervical cancer depends primarily on infection with human papillomaviruses, but tobacco smoking is the most important behavioral risk factor for this cancer. Therefore, we have previously confirmed involvement of nicotinic acetylcholine receptors (nAChRs) in cervical cancer biology. In order to comprehensively evaluate the role of cholinergic signaling in cervical cells, we have addressed additional participation of muscarinic acetylcholine receptors (mAChRs).
MAIN METHODS: We have studied the expression of mAChRs and cholinergic system components by reverse transcription PCR and Western blots, the motility of cervical cancer cells in cell culture, and the signaling from mAChRs via the ERK1/2 signaling pathway.
KEY FINDINGS: The cervical cancer cells HeLa, SiHa and CaSki express four of the five mAChRs, M1, M3, M4, and M5, and the acetylcholine (ACh) synthesizing and degrading enzymes choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), and vesicular ACh transporter (VAChT). mAChR-dependent signaling induces cervical cell motility, which requires ERK1/2 activation, and could be abrogated by mAChR antagonists.
SIGNIFICANCE: The epidemiological finding that tobacco smoke raises the prevalence of cervical cancer has led to analysis of the cholinergic signaling in cervical biology and carcinogenesis. Cervical cancer cells express several nAChRs and mAChRs, whose activation leads to changes of cellular properties such as increased motility and proliferation that favor a carcinogenic phenotype. The signaling involves intracellular phosphorylation cascades including ERK1/2.

Hughes L, Phelps C
"The bigger the network the bigger the bowl of cherries...": exploring the acceptability of, and preferences for, an ongoing support network for known BRCA 1 and BRCA 2 mutation carriers.
J Genet Couns. 2010; 19(5):487-96 [PubMed] Related Publications
There is increasing evidence to suggest that the ongoing information and support needs of BRCA gene mutation carriers are not being met. This qualitative study investigated preferences for an on-going support network for mutation carriers in Wales, UK. Seventeen female BRCA1/2 mutation carriers participated in focus groups which explored their current and on-going information and psychological support needs. The interviews were transcribed and thematically analysed. The results reflected a diversity of experiences and support needs. The majority of participants felt they and their families would benefit from an on-going 'support network' which should incorporate information-provision alongside elements of a traditional support group alongside, internet-based support such as web-based chat forums, matching schemes and professionally led workshops. Some degree of professional input into any such initiative was believed to be important. This study has informed the development of an appropriate support network based on a hub and spoke model to help carriers and their families adapt to living and coping with their genetic risk.

Wu X, Rauch TA, Zhong X, et al.
CpG island hypermethylation in human astrocytomas.
Cancer Res. 2010; 70(7):2718-27 [PubMed] Free Access to Full Article Related Publications
Astrocytomas are common and lethal human brain tumors. We have analyzed the methylation status of over 28,000 CpG islands and 18,000 promoters in normal human brain and in astrocytomas of various grades using the methylated CpG island recovery assay. We identified 6,000 to 7,000 methylated CpG islands in normal human brain. Approximately 5% of the promoter-associated CpG islands in the normal brain are methylated. Promoter CpG island methylation is inversely correlated whereas intragenic methylation is directly correlated with gene expression levels in brain tissue. In astrocytomas, several hundred CpG islands undergo specific hypermethylation relative to normal brain with 428 methylation peaks common to more than 25% of the tumors. Genes involved in brain development and neuronal differentiation, such as BMP4, POU4F3, GDNF, OTX2, NEFM, CNTN4, OTP, SIM1, FYN, EN1, CHAT, GSX2, NKX6-1, PAX6, RAX, and DLX2, were strongly enriched among genes frequently methylated in tumors. There was an overrepresentation of homeobox genes and 31% of the most commonly methylated genes represent targets of the Polycomb complex. We identified several chromosomal loci in which many (sometimes more than 20) consecutive CpG islands were hypermethylated in tumors. Seven such loci were near homeobox genes, including the HOXC and HOXD clusters, and the BARHL2, DLX1, and PITX2 genes. Two other clusters of hypermethylated islands were at sequences of recent gene duplication events. Our analysis offers mechanistic insights into brain neoplasia suggesting that methylation of the genes involved in neuronal differentiation, in cooperation with other oncogenic events, may shift the balance from regulated differentiation towards gliomagenesis.

Tong M, de la Monte SM
Mechanisms of ceramide-mediated neurodegeneration.
J Alzheimers Dis. 2009; 16(4):705-14 [PubMed] Related Publications
Obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic steatohepatitis (NASH) can be associated with cognitive impairment or early neurodegeneration. Previously, we showed that diet-induced obesity with T2DM and NASH results in mild neurodegeneration with some features of AD, including brain insulin resistance. In a companion study, we correlated obesity/T2DM/NASH-associated central nervous system (CNS) abnormalities with increased pro-ceramide gene expression in liver. Since ceramides are neurotoxic and cause insulin resistance, we directly investigated the role of ceramides as mediators of neurodegeneration using an in vitro culture model. We treated PNET2 human CNS neuronal cells with D-erythro-Ceramide analogs (C2Cer:N-acetylsphinganine and C6Cer:N-hexanoylsphinganine), or the inactive dihydroceramide analog (C2DCer) for 48 h, and probed for changes in genes and proteins that are critical to insulin/IGF signaling, and associated with neurodegeneration. Exposure to C6Cer>C2Cer impaired energy metabolism, viability, and insulin and insulin-like growth factor signaling mechanisms, and resulted in increased levels of AbetaPP-Abeta and pTau, whereas C2D had no significant effect on these parameters. CNS exposure to neurotoxic ceramides from exogenous sources, including liver, can cause neurodegeneration with impairments in insulin and IGF signaling mechanisms, similar to the findings in experimental models of obesity/T2DM, and NASH.

Kim HS, Park H, Lim JH, et al.
Morphometric evaluation of oesophageal wall in patients with nutcracker oesophagus and ineffective oesophageal motility.
Neurogastroenterol Motil. 2008; 20(8):869-76 [PubMed] Related Publications
The pathogenesis of nutcracker oesophagus (NE) and ineffective oesophageal motility (IEM) is unclear. Damage to the enteric nervous system or smooth muscle can cause oesophageal dysmotility. We tested the hypothesis that NE and IEM are associated with abnormal muscular or neural constituents of the oesophageal wall. Oesophageal manometry was performed in patients prior to total gastrectomy for gastric cancer. The oesophageal manometries were categorized as normal (n = 7), NE (n = 13), or IEM (n = 5). Histologic examination of oesophageal tissue obtained during surgery was performed after haematoxylin and eosin (H&E) and trichrome staining. Oesophageal innervation was examined after immunostaining for protein gene product-9.5 (PGP-9.5), choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). There were no significant differences in inner circular smooth muscle thickness or degree of fibrosis among the three groups. Severe muscle fibre loss was found in four of five patients with IEM. The density of PGP-9.5-reactive neural structures was not different among the three groups. The density of ChAT immunostaining in the myenteric plexus (MP) was significantly greater in patients with NE (P < 0.05) and the density of nNOS immunostaining in the circular muscle (CM) was significantly greater in IEM patients (P < 0.05). The ChAT/nNOS ratio in both MP and CM was significantly greater in NE patients. NE may result from an imbalance between the excitatory and inhibitory innervation of the oesophagus, because more than normal numbers of ChAT-positive myenteric neurones are seen in NE. Myopathy and/or increased number of nNOS neurones may contribute to the hypocontractile motor activity of IEM.

Near RI, Zhang Y, Makkinje A, et al.
AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation and altered breast cancer cell morphology.
J Cell Physiol. 2007; 212(3):655-65 [PubMed] Free Access to Full Article Related Publications
Over-expression of AND-34/BCAR3/NSP2 (BCAR3) or its binding-partner p130Cas/BCAR1 generates anti-estrogen resistance in human breast cancer lines. Here, we have compared BCAR3 to two related homologs, NSP1 and NSP3/CHAT/SHEP, with regards to expression, anti-estrogen resistance, and signaling. BCAR3 is expressed at higher levels in ERalpha-negative, mesenchymal, than in ERalpha-positive, epithelial, breast cancer cell lines. Characterization of "intermediate" epithelial-like cell lines with variable ER-alpha expression reveals that BCAR3 expression correlates with both mesenchymal and ERalpha-negative phenotypes. Levels of the BCAR3/p130Cas complex correlate more strongly with the ERalpha-negative, mesenchymal phenotype than levels of either protein alone. NSP1 and NSP3 are expressed at lower levels than BCAR3 and without correlation to ERalpha/mesenchymal status. Among NSP-transfectants, only BCAR3 transfectants induce anti-estrogen resistance and augment transcription of cyclin D1 promoter constructs. Over-expression of all homologs results in activation of Rac, Cdc42 and Akt, suggesting that these signals are insufficient to induce anti-estrogen resistance. BCAR3 but not NSP1 nor NSP3 transfectants show altered morphology, transitioning from polygonal cell groups to rounded, single cells with numerous blebs. Whereas stable over-expression of BCAR3 in MCF-7 cells does not lead to classic epithelial-to-mesenchymal transition, it does result in down-regulation of cadherin-mediated adhesion and augmentation of fibronectin expression. These studies suggest that BCAR's ability to induce anti-estrogen resistance is greater than that of other NSP homologs and may result from altered interaction of breast cancer cells with each other and the extracellular matrix.

Tsai LK, Tsai MS, Shyue SK, et al.
Adenoviral interneuronal transportation after retrograde gene transfer in mice.
Brain Res Mol Brain Res. 2005; 142(2):151-5 [PubMed] Related Publications
Although retrograde gene transfer from infected muscles to neurons by viral vectors has been known for years, it is still unknown whether interneuronal gene transportation of viral vectors occurs after retrograde gene transfer. To determine this, we injected adenoviral vectors carrying eGFP gene with or without a neural tracer into the right gastrocnemius muscles of mice. After 7 days, some spinal motor neurons were detected with green fluorescence but without the signal of neural tracer. In addition, nerves with green fluorescence could be noted in the right lumbosacral paraspinal muscles of viral-injected mice. The green fluorescence in the right lumbosacral paraspinal muscles might have resulted from retrograde gene transportation from the viral-injected gastrocnemius muscles to the spinal neurons, followed by interneuronal transfer and anterograde expression of eGFP in the axons belonging to neurons innervating the paraspinal muscles. This phenomenon of interneuronal transportation raises the possibility that we could treat motoneuron diseases by injection of viral vectors containing therapeutic genes into a few muscles resulting in widespread beneficial effects.

Traiffort E, Ruat M, O'Regan S, Meunier FM
Molecular characterization of the family of choline transporter-like proteins and their splice variants.
J Neurochem. 2005; 92(5):1116-25 [PubMed] Related Publications
We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

Mason PJ, Bessler M
Heterozygous telomerase deficiency in mouse and man: when less is definitely not more.
Cell Cycle. 2004; 3(9):1127-9 [PubMed] Related Publications
Telomerase, whose core components are a reverse transcriptase (TERT) and an integral RNA (TERC) maintains telomere ends. In somatic cells in the absence of telomerase telomeres get shorter leading to replicative cell senescence. In cancer cells abundant telomerase is present and cells do not senesce. Hence levels of telomerase may be crucial in regulating senescence and the transition to the neoplastic state. Heterozygous TERC mutations in man have been shown to underlie the rare inherited skin and bone marrow failure condition dyskeratosis congenita and a number of patients initially classified as idiopathic aplastic anemia have also been found to be mutated in one allele of the TERC gene. Families in which TERC mutations are segregating show disease anticipation, the severity of the disease increasing in successive generations due to decreasing telomere length. These data, along with biochemical analysis of mutated Terc and studies of Terc deficient mice show that in man and mouse haploinsufficiency for TERC leads to inability to correctly maintain telomeres, and highlights the importance of finely controlled telomerase levels in striking a balance between the processes of aging and cancer. Here we review several scenarios in which telomerase levels are disturbed, in human diseases or following genetic manipulation in mice.

Hashemi SH, Li JY, Ahlman H, Dahlström A
SSR2(a) receptor expression and adrenergic/cholinergic characteristics in differentiated SH-SY5Y cells.
Neurochem Res. 2003; 28(3-4):449-60 [PubMed] Related Publications
Somatostatin (SS) is an inhibitory regulator of secretory and proliferative responses that activates a group of receptors in the plasma membrane termed SSR1-5. SSR2 is one of the most abundant SSR, which also is expressed in high numbers in many neuroendocrine tumor types. Here, we describe a study of the presence and intracellular localization of the spliced variant SSR2(a) and its endogenous ligand SS in the cultured human neuroblastoma (NB) cell line, SH-SY5Y, by immunohistochemistry and confocal laser scanning. The integral neuronal synaptic vesicle membrane proteins synaptophysin (p38) and SV2 were studied, as well as the IR of catecholaminergic and cholinergic markers. RA treatment was used as an inducer of neuronal-like differentiation in our SH-SY5Y cell line. After the treatment, the presence of catecholaminergic markers (including NPY) decreased while the cholinergic markers (including VIP) increased. p38 and SV2 as well as VIP were shifted into the rather long neuritic processes, indicating efficient intracellular transport. The SSR2(a) protein was significantly increased by RA treatment, but only minor increases in mRNA for this receptor protein could be seen. No subcellular co-localization between p38/SV2 and the cytoplasmic granular receptor material was demonstrated. The SSR2(a) receptor ligand SS was found to be present not only in the cytoplasm but also in the nucleus, and more strongly so after RA treatment. The possible reason for this may be that this peptide, like other small peptides, may serve as transcription factor, or cofactor.

Nagai A, Suzuki Y, Baek SY, et al.
Generation and characterization of human hybrid neurons produced between embryonic CNS neurons and neuroblastoma cells.
Neurobiol Dis. 2002; 11(1):184-98 [PubMed] Related Publications
A human hybrid neuronal cell line A1 has been generated by somatic fusion between a human fetal cerebral neuron and a human neuroblastoma cell, and RT-PCR, immunochemical, and electrophysiological studies of the hybrid cells indicated that the cells express faithfully of morphological, immunochemical, physiological, and genetic features of human cerebral neurons. A1 hybrid neurons express neuron-specific markers such as neurofilament-L (NF-L), NF-M, NF-H, MAP-2, and beta tubulin III. A1 human hybrid neurons express messages for various cytokines and cytokine receptors which are similar to parental human CNS neurons and different from the other parental cell line, SK-SH-SY5Y neuroblastoma. A1 hybrid neurons also express messages for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD), indicating that they could differentiate into various subsets of neuronal types. Whole-cell patch clamp experiments showed that A1 hybrid neurons expressed Na+ currents, which were completely blocked by tetrodotoxin. In addition, depolarizing and hyperpolarizing voltage clamp steps evoked respective outward and inward K+ currents in these cells. When A1 hybrid neurons were exposed to beta amyloid for 72 hr, there was three-fold increase in TUNEL positive cells over controls, indicating that beta amyloid is neurotoxic to A1 hybrid neurons. The present study indicates that the A1 human hybrid neuronal cell line should serve as a valuable in vitro model for studies of biology, physiology, and pathology of human neurons in health and disease.

Robert I, Quirin-Stricker C
A novel untranslated 'exon H' of the human choline acetyltransferase gene in placenta.
J Neurochem. 2001; 79(1):9-16 [PubMed] Related Publications
To investigate the existence of 5'-region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5'-untranslated regions (UTR) in human neuronal and non-neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG-3 and neuroblastoma CHP126 and MC-IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT-PCR). We used a sense primer located in the 5'-flanking region, in the previously defined intronic sequence and an anti-sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG-3 cells whereas it was absent in spinal cord, CHP126 and MC-IXC cells. It was designated 'H-type' of ChAT mRNA. Whereas CHP126 produced the R- and N-type of ChAT mRNAs, no transcript of the N-and R-type was detected in JEG-3 and human placenta. In addition, CHP126 and JEG-3 cells and placenta showed the expression of the M-type of ChAT mRNA. The identity of the amplified 567 bp product (H-type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H-type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M-type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.

Lochhead PA, Coghlan M, Rice SQ, Sutherland C
Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression.
Diabetes. 2001; 50(5):937-46 [PubMed] Related Publications
A major action of insulin is to regulate the transcription rate of specific genes. The expression of these genes is dramatically altered in type 2 diabetes. For example, the expression of two hepatic genes, glucose-6-phosphatase and PEPCK, is normally inhibited by insulin, but in type 2 diabetes, their expression is insensitive to insulin. An agent that mimics the effect of insulin on the expression of these genes would reduce gluconeogenesis and hepatic glucose output, even in the presence of insulin resistance. The repressive actions of insulin on these genes are dependent on phosphatidylinositol (PI) 3-kinase. However, the molecules that lie between this lipid kinase and the two gene promoters are unknown. Glycogen synthase kinase-3 (GSK-3) is inhibited following activation of PI 3-kinase and protein kinase B. In hepatoma cells, we find that selectively reducing GSK-3 activity strongly reduces the expression of both gluconeogenic genes. The effect is at the level of transcription and is observed with induced or basal gene expression. In addition, GSK-3 inhibition does not result in the subsequent activation of protein kinase B or inhibition of the transcription factor FKHR, which are candidate regulatory molecules for these promoters. Thus, GSK-3 activity is required for basal activity of each promoter. Inhibitors of GSK-3 should therefore reduce hepatic glucose output, as well as increase the synthesis of glycogen from L-glucose. These findings indicate that GSK-3 inhibitors may have greater therapeutic potential for lowering blood glucose levels and treating type 2 diabetes than previously realized.

Lee TJ, Kim SJ, Park JH
Influence of the sequence variations of the HLA-DR promoters derived from human melanoma cell lines on nuclear protein binding and promoter activity.
Yonsei Med J. 2000; 41(5):593-9 [PubMed] Related Publications
In previous studies we reported that the expression of HLA-DR on melanoma cell lines was differentially modulated by IFN- gamma and that the transcription rate was responsible for this differential modulation. We have also reported the nucleotide sequence variations in the promoter region of HLA-DR genes, and proposed that differences in the promoter activity by the sequence variations of the HLA-DR promoters might contribute to such a differential transcriptional regulation at the promoter level. In this study, in order to assess whether the sequence variations of the HLA-DR promoters affect the factor binding and exert influence on the promoter activity, nuclear factor binding to our previous six HLA-DRA and fourteen HLA-DRB promoter clones was evaluated with the nuclear protein extracted from a B-lymphoblastoid cell line (BLCL), BH, together with the chloramphenicol acetyltransferase (CAT) reporter assay. In the HLA-DRA promoters, clone #35 containing one bp nucleotide sequence variation at the octamer binding site (OCT) (GATTTGC to GATCTGC) showed relatively weak factor binding. In the HLA-DRB promoters, clusters I, III, and IV of our previous HLA-DRB promoter homologues, containing one bp nucleotide sequence variation (GATTCG) in their Y boxes exhibited weak factor binding and CAT activity compared to other clusters (GATTGG) that showed strong factor binding and CAT activity. This data suggests chat the binding patterns of transcription factors influenced by the nucleotide sequence variations of the HLA-DR promoter could affect the promoter activity and the DNA sequence elements in the HLA-DR promoter could mediate transcriptional regulation.

Huang RP, Fan Y, deBelle I, et al.
Egr-1 inhibits apoptosis during the UV response: correlation of cell survival with Egr-1 phosphorylation.
Cell Death Differ. 1998; 5(1):96-106 [PubMed] Related Publications
UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1.

Pancrazio JJ, Ma W, Grant GM, et al.
A role for inwardly rectifying K+ channels in differentiation of NG108-15 neuroblastoma x glioma cells.
J Neurobiol. 1999; 38(4):466-74 [PubMed] Related Publications
The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.

Tanaka H, Shimojo M, Wu D, Hersh LB
Regulation of the cholinergic gene locus.
J Physiol Paris. 1998; 92(2):149-52 [PubMed] Related Publications
DNase I hypersensitive site mapping of the human cholinergic gene locus has been used to detect cholinergic specific potential regulatory sites. Analysis of mutant PC12 cell lines provides evidence that protein kinase A II is required and coordinately regulates basal expression of both the ChAT and VAChT genes.

Pinkel D, Segraves R, Sudar D, et al.
High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.
Nat Genet. 1998; 20(2):207-11 [PubMed] Related Publications
Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

Twyman RM, Jones EA
Sequences in the proximal 5' flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression.
J Mol Neurosci. 1997; 8(1):63-73 [PubMed] Related Publications
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.

Rabinovsky ED, Ramchatesingh J, McManaman JL
Regulation of tyrosine hydroxylase gene expression in IMR-32 neuroblastoma cells by basic fibroblast growth factor and ciliary neurotrophic factor.
J Neurochem. 1995; 64(6):2404-12 [PubMed] Related Publications
The actions of basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) on tyrosine hydroxylase (TH) gene expression were studied using IMR-32 neuroblastoma cells. Treatment of these cells with bFGF for 3 days induced the expression of detectable levels of immunoreactive TH protein and TH mRNA. In contrast, CNTF did not affect TH expression unless bFGF was present. In the presence of saturating amounts of bFGF, CNTF increased TH protein and mRNA levels of TH two-to threefold over those found in bFGF-treated cultures. The effects of CNTF on TH expression diminished with increasing culture time, and after 6 days of incubation CNTF no longer enhanced TH levels. The requirement for bFGF as cofactor in the effects of CNTF on TH was specific, as CNTF did not affect TH when it was coadministered with 8-(4-chlorophenylthio)-cyclic AMP, another agent that stimulates TH development in this cell line, and bFGF was not required for CNTF to stimulate the development of choline acetyltransferase. Moreover, cotreatment with bFGF reduced the ability of CNTF to enhance choline acetyltransferase. These results demonstrate that bFGF and CNTF can enhance expression of TH and that bFGF can modify the effects of CNTF on neurotransmitter phenotype.

Li YP, Baskin F, Davis R, et al.
A cell type-specific silencer in the human choline acetyltransferase gene requiring two distinct and interactive E boxes.
Brain Res Mol Brain Res. 1995; 30(1):106-14 [PubMed] Related Publications
We have previously reported that cholinergic neuron-specific expression of the human choline acetyltransferase gene is mediated by two co-operative silencers. We have now localized the proximal silencer to the region from nucleotide -2195 to -2409, which contains two distinct E boxes (CACCTG and CATGTG). Deletion or mutation of either of these E boxes results in a loss of silencer activity. There are specific nuclear proteins in adrenergic cells which bind to each of the two E boxes. However, nuclear proteins from cholinergic cells only bind the 5' E box not the 3' E box. It is this interaction which appears to be the cause of the inactivity of this silencer in these cells.

Usami S, Matsubara A, Shinkawa H, et al.
Neuroactive substances in the human vestibular end organs.
Acta Otolaryngol Suppl. 1995; 520 Pt 1:160-3 [PubMed] Related Publications
In order to evaluate the involvement of neuroactive substances in the human vestibular periphery, the immunocytochemical distribution of substance P (SP), calcitonin gene-related peptide (CGRP), and choline acetyltransferase (ChAT) was examined. SP-like immunoreactivity (LI) was present around and beneath sensory hair cells, probably corresponding to their afferent nerve endings. SP-LI was found predominantly in subpopulations of the primary afferents distributed in the peripheral region of the end organs. ChAT-LI and CGRP-LI were found throughout as small puncta below the hair cell layer, probably corresponding to efferent endings. The present results indicate that these neuroactive substances, previously described in animals, are also distributed in the human vestibular periphery, and almost certainly contribute to human vestibular function.

Yamamoto H, Itoh F, Senota A, et al.
Expression of matrix metalloproteinase matrilysin (MMP-7) was induced by activated Ki-ras via AP-1 activation in SW1417 colon cancer cells.
J Clin Lab Anal. 1995; 9(5):297-301 [PubMed] Related Publications
The matrix metalloproteinase matrilysin (MMP-7) is a member of the matrix metalloproteinase gene family, which is believed to play an important role in tumor invasion and metastasis. We have previously found that matrilysin mRNA is specifically expressed in colorectal cancers and adenomas and that its message is localized in the tumor cells themselves. We examined the effects of activated Ki-ras oncogene on the expression of matrilysin in colon cancer cells. We showed that both mRNA and the enzymatic activity of matrilysin were induced by the introduction of activated Ki-ras into SW1417 colon cancer cells. To understand the mechanisms regulating this induction, we analyzed alterations of AP-1 activity induced by activated Ki-ras, using the chloramphenicol acetyltransferase assay. AP-1 activity in SW1417 cells expressing activated Ki-ras was higher than that in control cells. The gel-shift assay also showed higher levels of AP-1 binding protein in SW1417 cells expressing activated Ki-ras than those in control cells. Our results suggest that activated Ki-ras may play a role in inducing expression of matrilysin through an AP-1-dependent pathway in colon cancer cells.

Quirin-Stricker C, Nappey V, Simoni P, et al.
Trans-activation by thyroid hormone receptors of the 5' flanking region of the human ChAT gene.
Brain Res Mol Brain Res. 1994; 23(3):253-65 [PubMed] Related Publications
Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect. We localized several potential T3REs and characterized the most proximal T3RE (position 3280-3291) which contains two hexameric half-sites arranged as a direct repeat without a base pair spacer. An oligonucleotide containing this sequence confers T3 responsiveness to a heterologous promoter. The transcriptional response of this T3RE is markedly reduced after mutation of the first or second half-site indicating that both half-sites are required for a maximal T3 response. We have found that RAR alpha, RXR alpha and COUP-TF do not enhance T3 responsiveness and therefore they may not interact with T3R alpha in NG108-15 cells on this regulatory sequence. T3R monomer and dimer specific binding to the proximal T3RE is demonstrated by gel-retardation DNA binding assays and by methylation interference experiments. In COS-1 cells, T3R inhibits transcriptional activation by the transcription factor AP-1 whereas in NE1-115 cells T3R enhances AP-1 mediated activation in a T3 dependant fashion. It is likely that these effects involve protein-protein interactions. These results suggest that the T3 receptor can act as a positive transcriptional regulatory factor on the hChAT gene.

Inoue H, Baetge EE, Hersh LB
Enhancer containing unusual GC box-like sequences on the human choline acetyltransferase gene.
Brain Res Mol Brain Res. 1993; 20(4):299-304 [PubMed] Related Publications
The minimum requirement of an enhancer for the human choline acetyltransferase (ChAT) gene was analyzed by mutagenesis and protein-DNA interaction studies. A series of deletion and site-specific mutants were expressed transiently in a rat cholinergic neuroblastoma cell, NS20Y. The results revealed that the distal region between -970 and -941 base pairs (bp) from the transcription start site is essential for efficient transcription of the human ChAT gene. Two GC box-like sequences were located within this region, and they were shown to bind purified Sp1 transcription factor by footprinting and gel retardation analyses. This sequence, however, has an orientational effect and is located approximately 1000 bp from the transcription start site, unusual for the conventional GC box sequence. Furthermore, the sequence between these GC box-like sequences is shown to bind another novel trans-acting factor by gel retardation assay and to be necessary for efficient enhancer activity. On the other hand, gel retardation assays using a nuclear extract from Drosophila SL2 cells suggested that non-Sp1 trans-acting factors could also participate in the enhancing activity of this element. Taken together, the results demonstrate that two GC box-like sequences and another trans-acting factor-binding sequence located between -970 and -941 bp act coordinately and are essential for efficient transcription of this low expressed human ChAT gene.

Li YP, Baskin F, Davis R, Hersh LB
Cholinergic neuron-specific expression of the human choline acetyltransferase gene is controlled by silencer elements.
J Neurochem. 1993; 61(2):748-51 [PubMed] Related Publications
Choline acetyltransferase (ChAT) is specifically expressed in cholinergic neurons. To identify control mechanisms regulating the cell-specific expression of the gene encoding ChAT, transient expression of the luciferase gene driven by human ChAT gene 5'flanking sequences was compared in cholinergic and noncholinergic cell lines. Analysis of the gene indicated the presence of two regulatory elements with selective silencing activity. These elements, located between nucleotides -2043 to -3347 and nucleotides -3347 to -6550, act cooperatively to repress promoter activity > 10-fold in a human adrenergic neuroblastoma cell line, SHSY5Y, and a human osteosarcoma cell line, 143 TK-, while exhibiting less than a two-fold effect in cholinergic cell lines. Deletion of either nucleotides -2043 to -3347 or nucleotides -3348 to -6550 reduced cell-specific repression by approximately half. Such differential repression appears to be responsible for the selective expression of the ChAT component of the cholinergic phenotype.

Bausero P, Schmitt M, Toussaint JL, et al.
Identification and analysis of the human choline acetyltransferase gene promoter.
Neuroreport. 1993; 4(3):287-90 [PubMed] Related Publications
Choline acetyltransferase (ChAT) is the key enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is reduced in various central neurodegenerative diseases. From a previously selected 12.6 kb human choline acetyltransferase (hChAT) genomic clone, we have identified and characterized a promoter region of 895 bp. Sequence analysis revealed the presence of a TATA-like box, a CAAT box and several putative regulatory responsive elements. Three transcription initiation sites were determined by primer extension analysis. The Northern blot of poly(A)+ RNA, showed a single band of 2300 Nt in the human nucleus accumbens and facial nucleus. By using transient transfections into NE-1-115 and COS-1 cells of the 5' flanking region of the hChAT gene we identified a sequence of 66 bp upstream of the transcription start site which confers responsiveness to proto-oncogenes c-Fos/c-Jun. These data suggest that the hChAT gene may be a physiological target of c-Fos/c-Jun and therefore may play a role in neuronal responses to various stimuli.

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