Detection of choline transporter-like 1 protein CTL1 in neuroblastoma × glioma cells and in the CNS, and its role in choline uptake

Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma × glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.     

Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

Synthesis of acetylcholine (ACh) by non‐neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na⁺‐dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non‐neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non‐neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter‐like proteins, a five gene family choline‐transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na⁺‐independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non‐neuronal cell lines and presents a mechanism to target non‐neuronal ACh synthesis without affecting neuronal ACh synthesis.     

Regulation of endothelin-converting enzyme-1 expression in human neuroblastoma cells

In recent years endothelin-converting enzyme (ECE-1) has been suggested to play an important role in amyloid-beta peptide metabolism as one of the amyloid-degrading enzymes. In this connection, the analysis of the levels of expression and distribution of ECE-1 in the brain under normal and pathologic conditions could be important in neurodegeneration and pathogenesis of Alzheimer disease. In our previous studies, we have demonstrated that expression of ECE-1 was significantly reduced in the cortex of adult rats after 15 mins of global ischemia. It was also significantly reduced in the striatum of rats subjected to prenatal hypoxia. In the present study, we analyzed effects of hypoxia and oxidative stress on ECE-1 in human neuroblastoma NB7 cells and effects of the cholinergic agonist carbachol and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). We have found that chronic (24 hrs) hypoxia and oxidative stress resulted in 30% and 20% decrease in expression of ECE-1 at the protein level, respectively, although at the level of ECE-1 mRNA there were no statistically significant changes. Serum withdrawal from the incubation medium as well as addition of carbachol or PMA for 24 hrs also led to a significant reduction of the levels of ECE-1 protein in NB7 cells. Further study of the downstream signaling cascades involved in downregulation of ECE expression in NB7 cells and primary neuronal cells might provide us with new insights into possible therapeutic strategies for prevention or treatment of Alzheimer disease in elderly patients and those who suffer from stroke or cerebrovascular disorders.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V_max values from 1,973 ± 118 to 380 ± 18 nmol·mg^–1·min^–1, when the binding affinity did not change significantly (K_m values 56 ± 8 and 53 ± 6 µM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages. protein trafficking     

Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells.

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex consisting of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2. To explore possible pathophysiological roles of adrenomedullin and its receptor component RAMP2 in hypoxic tissues, we studied effects of hypoxia on expression of adrenomedullin and RAMP2 in two human neuroblastoma cell lines, IMR-32 and NB69, by radioimmunoassay and Northern blot analysis. Expression levels of adrenomedullin were increased by hypoxia in both cell lines. Treatment with cobalt chloride or desferrioxamine mesylate also increased expression levels of adrenomedullin mRNA. On the other hand, expression levels of RAMP2 mRNA were decreased in IMR-32 cells and were not changed in NB69 cells by hypoxia. Treatment with cobalt chloride or desferrioxamine mesylate decreased expression levels of RAMP2 mRNA in both IMR-32 and NB69 cells. These findings indicate that adrenomedullin expression is induced during hypoxia in IMR-32 and NB69 neuroblastoma cells, but RAMP2 expression is rather suppressed under the same conditions. The decreased expression of RAMP2 and the ADM expression induction under hypoxia may constitute one mechanism of cellular adaptation to hypoxic stress.     

Functional regulation of FEN1 nuclease and its link to cancer

Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5'-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochemical and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be critical to regulate protein-protein interactions and cellular localizations of FEN1. Lastly, we also review the biological significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.     

In vitro protein synthesis activity of poly(A) RNA from neuroblastoma cells

Protein synthesis activity of poly(A) RNA from M1 neuroblastoma cells was studied in a reticulocyte cell-free system. The activity of poly(A) RNA from M1 cells differentiated morphologically by bromodeoxyuridine was compared with that of proliferating cells. Poly(A) RNA from differentiated cells was about 10% more active than the corresponding RNA from proliferating cells. The products synthesized in vitro were fractionated by polyacrylamide gradient gel electrophoresis. A 420,000-dalton band was present in the translation products programmed by differentiated cell poly(A) RNA; it was absent in the translation products programmed by proliferating cell poly(A) RNA.This paper is part of thesis Doctorat d'Etat of A.D.     

Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.     

Radiofrequency radiation (900 MHz) induces Egr-1 gene expression and affects cell-cycle control in human neuroblastoma cells

Many environmental signals, including ionizing radiation and UV rays, induce activation of Egr-1 gene, thus affecting cell growth and apoptosis. The paucity and the controversial knowledge about the effect of electromagnetic fields (EMF) exposure of nerve cells prompted us to investigate the bioeffects of radiofrequency (RF) radiation on SH-SY5Y neuroblastoma cells. The effect of a modulated RF field of 900 MHz, generated by a wire patch cell (WPC) antenna exposure system on Egr-1 gene expression, was studied as a function of time. Short-term exposures induced a transient increase in Egr-1 mRNA level paralleled with activation of the MAPK subtypes ERK1/2 and SAPK/JNK. The effects of RF radiations on cell growth rate and apoptosis were also studied. Exposure to RF radiation had an anti-proliferative activity in SH-SY5Y cells with a significant effect observed at 24 h. RF radiation impaired cell cycle progression, reaching a significant G2-M arrest. In addition, the appearance of the sub-G1 peak, a hallmark of apoptosis, was highlighted after a 24-h exposure, together with a significant decrease in mRNA levels of Bcl-2 and survivin genes, both interfering with signaling between G2-M arrest and apoptosis. Our results provide evidence that exposure to a 900 MHz-modulated RF radiation affect both Egr-1 gene expression and cell regulatory functions, involving apoptosis inhibitors like Bcl-2 and survivin, thus providing important insights into a potentially broad mechanism for controlling in vitro cell viability.     

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

Oscillation of human period 1 (hPER1) reporter gene activity in human neuroblastoma cells in vivo

The mammalian period (Per) genes, which are components of the circadian clock, are mainly regulated via an autoregulatory feedback loop. Here we provide evidence that human Per1 (hPER1) reporter gene activity shows circadian rhythmicity in a human neuroblastoma, but not in a astrocytoma or a hepatoma cell line. Medium change and various pharmacological stimuli differentially induce this behavior. This circadian oscillation was strongly dampened and could be followed over maximally three cycles. It was even possible to phase-shift the course of this oscillation by repeated application of stimuli.     

Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells

Background  

Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).     

Regulation of insulin-like growth factor-II expression and its role in autocrine growth of human neuroblastoma cells

Insulin-like growth factor-II (IGF-II) is highly expressed in fetal tissues and may act as an autocrine growth factor during early embryogenesis. The SH-SY5Y human neuroblastoma cell line also expresses IGF-II and its receptors and responds to exogenous IGF-II with increased DNA synthesis, cell division, and neuritic outgrowth. For this study, we tested the hypothesis that IGF-II mediates autocrine growth of SH-SY5Y cells in serum-free media. SH-SY5Y cells plated at high densities proliferated in serum-free media, whereas sparsely plated cells did not. IGF-II mRNA levels increased within 24 hours of serum deprivation and were associated with increased immunoreactive IGF-II protein. Exogenous addition of IGF-II increased ³H-TdR incorporation and cell number in a dose- and time-dependent fashion. By nuclear labelling experiments using 5-Bromo-2′ deoxyuridine (BrdU), we detected a twofold higher percentage of S phase nuclei after a 24-hour incubation in IGF-II. Treatment of SH-SY5Y cells with anti-IGF-II antibodies in serum-free media inhibited cell proliferation, and this inhibition was partially overcome by the addition of increasing concentrations of IGF-II. Collectively, our results indicate that IGF-II mediates an autocrine growth mechanism in SH-SY5Y cells that is associated with increased IGF-II expression. © 1993 Wiley-Liss, Inc.     

Absence of MGST1 mRNA and protein expression in human neuroblastoma cell lines and primary tissue

A recent study identified a haplotype on a small region of chromosome 12, between markers D12S1725 and D12S1596, shared by all patients with familial neuroblastoma (NB). We previously localized the human MGST1 gene, whose gene product protects against oxidative stress, to this very same chromosomal region (12p112.1–p13.33). Owing to the chromosomal location of MGST1; its roles in tumorigenesis, drug resistance, and oxidative stress; and the known sensitivity of NB cell lines to oxidative stress, we considered a role for MGST1 in NB development. Surprisingly there was no detectable MGST1 mRNA or protein in either NB cell lines or NB primary tumor tissue, although all other human tissues, cell lines, and primary tumor tissue examined to date express MGST1 at high levels. The mechanism behind the failure of NB cells and tissue to express MGST1 mRNA is unknown and involves the failure of MGST1 pre-mRNA expression, but does not involve chromosomal rearrangement or nucleotide variation in the promoter, exons, or 3' untranslated region of MGST1. MGST1 provides significant protection against oxidative stress and constitutes 4 to 6% of all protein in the outer membrane of the mitochondria. As NB cells are extremely sensitive to oxidative stress, and often used as a model system to investigate mitochondrial response to endogenous and exogenous stress, these findings may be due to the lack of expression MGST1 protein in NB. The significance of this finding to the development of neuroblastoma (familial or otherwise), however, is unknown and may even be incidental. Although our studies provide a molecular basis for previous work on the sensitivity of NB cells to oxidative stress, and possibly marked variations in NB mitochondrial homeostasis, they also imply that the results of these earlier studies using NB cells are not transferable to other tumor and cell types that express MGST1 at high concentrations.