Recent progress in the molecular biology of the clonedN-acetylglucosaminyltransferases

Several genes which code for theN-acetylglucosaminyltransferases have been cloned and characterized. Physiological and pathophysiological roles of the genes still remain to be elucidated but accumulated evidence suggests that theN-acetylglucosaminyltransferase genes are implicated in differentiation, morphogenesis and cancer metastasis.     

Potentiation of PGE2-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.     

Heparin and dibutyryl cAMP modulate gene expression in stimulated human saphenous vein smooth muscle cells

Summary Increased expression of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A chain, and tissue plasminogen activator (tPA) by smooth muscle cells (SMC) has been postulated to mediate the progression of intimal hyperplasia. We tested whether heparin would suppress the expression of these genes in stimulated human saphenous vein SMC. Quiescent cultured human saphenous vein SMC were stimulated for 4 h with heat-inactivated fetal bovine serum (10% by vol) in the presence or absence of heparin (1 to 250μg/ml). Heparin (50μg/ml) attenuated the induction by serum of bFGF mRNA, tPA mRNA, and tPA secretion. Nonanticoagulant heparin also attenuated serum induction of bFGF and tPA mRNA levels. To further study the role of second messenger signaling, a more specific mode of SMC stimulation was used with thrombin (3 U/ml) in the presence or absence of dibutyryl cyclic AMP (Bu₂-cAMP; 0.5 mM). In contrast to heparin, which had no effect on PDGF expression, Bu₂-cAMP decreased the induction by thrombin of PDGF-A chain mRNA levels. In thrombin-stimulated SMC, Bu₂-cAMP significantly decreased secretion of PDGF-AA protein. Thrombin, however, caused an increase in bFGF mRNA levels which was potentiated by Bu₂-cAMP with associated potentiation by Bu₂-cAMP of intracellular bFGF protein levels. The induction of tPA mRNA and tPA secretion by thrombin was sharply blocked by Bu₂-cAMP. These results suggest that heparin reduces intimal hyperplasia at least partly via partial inhibition of SMC gene expression.     

Altered expression of genes regulating cell growth,proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture

An elevation of the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing approximately 6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4, 24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppressors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.     

MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.     

Application of bromodeoxyuridine (BrdU)-labelled Escherichia coli strains in studies on their adherence to human endothelial cells

Fimbriated and fimbria-less strains of Escherichia coli were isolated from urine of pyelonephritis patients, labelled with bromodeoxyuridine and their adhesion to human umbillical vein endothelial cells was studied employing ELISA and immunocytochemistry. No significant differences were noted in adhesion of the two types of strains.     

Embryonic cAMP and developmental potential in Drosophila melanogaster

Summary Measurements of cAMP in early embryos of Drosophila melanogaster demonstrate that the dunce gene plays a major role, and the rutabaga gene a secondary role, in maternal regulation of embryonic cAMP content. Studying the double mutant combination, we find that variability in elevated cAMP content between individual embryos is associated with a wide variability in developmental potential. Embryos with about five times the normal cAMP content define a threshold between apparently normal and abnormal development. Measurements of cAMP content in anterior and posterior halves of embryos indicate that the posterior embryonic region, which is developmentally more sensitive to the effects of elevated cAMP than the anterior region, does not contain more cAMP than the anterior region. The variety of developmental defects observed is discussed in relation to possible targets of cAMP action. Offprint requests to: J.A. Kiger, Jr     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.     

Cloning and expression of a porcine UDP-GalNAc: polypeptideN-acetylgalactosaminyl transferase

By employing a bovine UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyl transferase (O-GalNAc transferase) cDNA as a probe, we isolated four overlapping cDNAs from a porcine lung cDNA library. Both the nucleotide sequence of the porcine cDNA and the predicted primary structure of the protein (559 amino acids) proved to be very similar to those of the bovine enzyme (95% and 99% identity, respectively). Transient expression of the clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes a porcine O-GalNAc transferase. The intracellular O-GalNAc transferase activity was increased approximately 100-fold by transfecting cells with the porcine cDNA.Abbreviations O-GalNAc transferase UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyltransferase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - GnT-III UDP-N-acetylglucosamine: -mannoside -1,4N-acetylglucosaminyltransferase III     

Effects of NGF on acetylcholine, acetyl-CoA metabolism, and viability of differentiated and non-differentiated cholinergic neuroblastoma cells

Nerve growth factor (NGF) is a peptide displaying multiple cholinotropic activities. The aim of this work was to explain mechanisms of the positive and negative effects of NGF on phenotypic properties and viability of cholinergic cells. To discriminate these effects we used two p75NTR receptor-positive lines of cholinergic neuroblastoma cells, SN56 and T17 that are devoid of or express high affinity NGF (TrkA) receptors, respectively. cAMP and retinoic acid caused differentiation of both cell lines. In addition to the morphologic maturation, the increase of choline acetyltransferase activity, acetylcholine, Ca and cytoplasmic acetyl-CoA levels and decrease of mitochondrial acetyl-CoA and cell viability were observed. NGF caused similar effects in non-differentiated T17 cells but had no influence on non-differentiated SN56 cells. On the contrary, in both cAMP/all-trans-retinoic acid (RA) differentiated cell lines, NGF resulted in a similar suppression of cholinergic phenotype along with an increase of mitochondrial acetyl-CoA and cell susceptibility to nitric oxide and amyloid-beta25-35. These effects of NGF were prevented by an antibody against the p75NTR receptor. Data indicate that: (i) positive cholinotrophic effects of NGF required activation of both TrkA and p75NTR receptors; (ii) cAMP/RA-evoked differentiation inhibited NGF effects mediated by TrkA receptors and activated its p75NTR-dependent suppressing influences and (iii) a differentiation-evoked decrease of mitochondrial acetyl-CoA and an elevation of mitochondrial Ca could augment impairment of cholinergic neurons by neurotoxic signals.     

Effects of long-term in vitro exposure to aluminum,cadmium or lead on differentiation and cholinergic receptor expression in a human neuroblastoma cell line

Neurotoxicity of long-term exposure to lead, aluminum and cadmium has been studied in vitro on the human neuroblastoma cell line IMR32 by measuring cytotoxicity, and the effects on neuronal-specific characteristics such as nitrite outgrowth and expression of cholinergic receptors as parameters of toxicity. Cytotoxicity was highest with cadmium, intermediate with lead and lowest with aluminum exposure. Lead, but not cadmium and aluminum, interfered with neurite growth. The expression of a-bungarotoxin binding sites and muscarinic receptors was markedly increased by cadmium and not affected by aluminum exposure. Lead induced only an increase of toxin binding sites. These in vitro modifications are discussed in relation to the possible use of neuronal cell lines for detecting neurotoxic effects of heavy metals.Abbreviations ACh acetylcholine - -Bgtx -bungarotoxin - BrdUr 5'-bromodeoxyuridine - CNS central nervous system     

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.     

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.     

Effects of salicylhydroxamic acid on the proliferation of Atriplex and murine neuroblastoma cells, and on Drosophila egg laying and development

Salicylhydroxamic acid (SHAM) inhibits the proliferation of cultured plant (Atriplex halimus) and murine neuroblastoma cells with IC50 of 90 and 250 microM, respectively. After 2 h of application, SHAM induces an acceleration of the neuroblastoma cell cycle from G1/S to G2 phases and, after 6 h, it induces an accumulation of the cells in S phase and a cell swelling. Up to 300 microM, SHAM is not cytotoxic and does not induce electrophysiological differentiation of neuroblastoma cells. When Drosophila females are grown in media containing 0.6-1.25 mM SHAM, the rate and number of laid eggs are increased. Furthermore, SHAM stimulates the different development stages from embryo to adult. A general interpretation of the effects of SHAM on cell proliferation and differentiation is proposed.     

Effects of granulocytes on human neuroblastoma cells measured by chemiluminescence and chromium-51 release assay

We investigated whether polymorphonuclear leukocytes (PMN) are able to kill human neuroblastoma cells either directly or if coated with antibody MAb 14.18 that recognizes ganglioside GD₂ present on the cell surface of most neuroblastoma cells. Neuroblastoma cells could not be destroyed directly, whereas in the antibody-dependent reaction (ADCC-reaction) they were easily eliminated. In order to answer the question whether reactive oxygen intermediates are involved in this process, chemiluminescence measurements were performed. Compared to the signals that could be measured using opsonized zymosan as stimulus, only weak CL-signals could be registered during the ADCC reaction. Pretreatment of PMN with granulocyte-macrophage colony stimulating factor (GM-CSF) enhanced the CL-signals, catalase and SOD reduced it; however, cell killing was only slightly influenced in the presence of catalase and superoxide dismutase. These data suggested that reactive oxygen compounds do not play a prominent role in the killing process. Definitive evidence for this suggestion could be obtained using PMN from a patient with chronic granulomatous disease (CGD): MAb 14.18 coated neuroblastoma cells could be killed effectively, but no CL-signal could be registered, either in the ADCC-reaction or using opsonized zymosan as stimulus.