ABSTRACT
Human neuron-specific enolase, NSE, (E.C.4.2.1.11), has been purified from the brain. The method includes a salt precipitation and column chromatography on DEAE-Sephacel, Sephacryl S-300, DEAE-Sepharose and Blue-Sepharose. The specific activity of the purified NSE was 51 units/mg protein and the yield was 28%. The molecular weight of human NSE was compared to the bovine form purified by the same procedure. On Sephadex G 150 both human and bovine NSE chromatographed, in the dimeric form, as 77,000 Dalton proteins, while the molecular weight of the monomer was 45,000 Daltons as determined by SDS poly-acrylamide gel electrophoresis. An antiserum, specific for NSE, was raised in rabbits and a radioimmunoassay developed. The contribution of lyzed blood cells to the serum NSE levels in eight healthy donors was determined and correlated with the haemoglobin content. Minimal haemolysis can add 5-10 ng NSE/ ml to the true serum level. For accurate serum NSE determinations, serum samples must be free from haemolysis.
Subject(s)
Brain/enzymology , Isoenzymes/isolation & purification , Phosphopyruvate Hydratase/isolation & purification , Chromatography, Agarose , Electrophoresis, Polyacrylamide Gel , Hemolysis , Humans , Molecular Weight , Phosphopyruvate Hydratase/blood , Phosphopyruvate Hydratase/immunology , RadioimmunoassayABSTRACT
SH-SY5Y human neuroblastoma cells differentiate morphologically and biochemically in the presence of 12-0-tetradecanoylphorbol-13-acetate (TPA). The degree of differentiation, as demonstrated by the appearance of cell surface projections, growth inhibition and increase in noradrenalin concentration, was dependent on the TPA concentration and had an optimum at 1.6 X 10(-8) M of TPA. At that concentration neuron specific enolase (NSE) increased to a maximum level after 10 days of culture with no further changes in the NSE level during additional culture for 10 days. In contrast, the noradrenalin concentration reached a maximum after 4 days of TPA treatment and decreased during longer exposures to TPA. Based on the facts that the phorbolester induced differentiation shows stereo specificity and was optimal at the same concentration range as common polypeptide hormones, a putative TPA-hormone receptor interaction is discussed. An opposite effect of TPA on the SH-SY5Y cells, antagonizing the differentiation effect, is further suggested to explain the decrease in differentiation observed at TPA concentrations higher than 1.6 X 10(-8) M.
Subject(s)
Caenorhabditis elegans Proteins , Cell Differentiation/drug effects , Neuroblastoma/pathology , Phorbols/pharmacology , Protein Kinase C , Receptors, Drug , Tetradecanoylphorbol Acetate/pharmacology , Carrier Proteins , Cell Line , Humans , Kinetics , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Norepinephrine/metabolism , Phosphopyruvate Hydratase/metabolism , Receptors, Cell Surface/drug effectsABSTRACT
Thirteen human neuroblastoma and six Wilms' tumor biopsies have been analyzed for neuron specific enolase (NSE). THe relative activity of NSE in the neuroblastomas (including ganglioneuroblastoma and ganglioneuroma) ranged from 28% to 62.5% of total enolase activity. The corresponding figures for the Wilms' tumors were 1% to 4.5%. It appears that NSE can serve as a biochemical marker for neuroblastoma and be useful in the differential diagnosis of neuroblastoma and Wilms' tumor.
Subject(s)
Kidney Neoplasms/analysis , Neuroblastoma/analysis , Neurons/enzymology , Phosphopyruvate Hydratase/analysis , Wilms Tumor/analysis , Child , Child, Preschool , Female , Humans , Infant , MaleABSTRACT
SK-N-SH and SH-SY5Y human neuroblastoma cells treated by 12-)-tetradecanoyl-phorbol-13-acetate(TPA) express morphological and biochemical changes, which indicate that differentiation towards more mature cells has occurred. The most prominent morphological changes were the development in 40-60% of the cells of cell-surface projections longer than 50 micrometers and cytoplasmic neurosecretory granules demonstrated by electron microscopy. At the biochemical level, TPA induced a two-fold increase in the relative activity of neuron-specific enolase and 30- to 40-fold increase in noradrenaline and adrenaline concentrations. A decrease in proliferation rate of TPA-treated cells was observed. The biological effects of TPA were slightly potentiated by nerve growth factor.
Subject(s)
Neuroblastoma/pathology , Phorbols/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Cell Division/drug effects , Cell Line , Cells, Cultured , Cytoplasmic Granules/drug effects , Epinephrine/metabolism , Humans , In Vitro Techniques , Microscopy, Electron , Norepinephrine/metabolism , Phenotype , Phosphopyruvate Hydratase/metabolismABSTRACT
The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCl, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultured cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.
