Structural Features of the Nerve Growth Factor Inducible Large External Glycoprotein of PC12 Pheochromocytoma Cells and Brain

Abstract: We have examined the oligosaccharide structure of a major M_r= 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri- and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77–90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N -acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris , together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the α-linked mannose residues. NGF treatment of the PC12 cells had no significant effect on the oligosaccharide structure of the NILE glycoprotein. The greater molecular size of the PC12 cell NILE glycoprotein as compared to the immunochemically cross-reactive species present in brain (M_r= 205,000) is apparently due to the greater size of the PC12 cell tri- and tetraantennary complex oligosaccharides.     

Specific Binding of Glycosylated β-Galactosidase to Rat Clonal Pheochromocytoma PC12h Cells: Effects of Nerve Growth Factor and Gangliosides

Rat clonal pheochromocytoma PC12h cells were found to bind beta-galactosidase modified with specific glycosides. The enzyme modified with p-aminophenyl beta-D-glucoside was most effectively bound to the cells, followed by alpha-D-mannoside and alpha-D-glucoside. The binding was dependent on the number of PC12h cells, the incubation interval, and the pH; the maximal binding at 4 degrees C was obtained by incubation with 75 micrograms of cell protein for 15 min at pH 4.0. The binding proved to be a saturable and receptor-mediated process, and the apparent Km value and the maximal binding capacity of the cells with beta-D-glucosylated beta-galactosidase were 1.03 +/- 0.06 microM and 333 +/- 24 pmol/min/mg of protein, respectively. When the cells were cultured in the presence of nerve growth factor (NGF), GM1, GM2, and a ganglioside mixture, marked morphological differentiation was observed in the presence of NGF, and the specificity of the binding was also affected. By supplementation of NGF in the culture medium, the cells lost the selectivity of the glycoside binding, whereas cells cultured with GM1 supplement showed increased binding of the specific glycosides.     

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.     

The VHL gene is epigenetically inactivated in pheochromocytomas and abdominal paragangliomas

Pheochromocytoma (PCC) and abdominal paraganglioma (PGL) are neuroendocrine tumors that present with clinical symptoms related to increased catecholamine levels. About a third of the cases are associated with constitutional mutations in pre-disposing genes, of which some may also be somatically mutated in sporadic cases. However, little is known about inactivating epigenetic events through promoter methylation in these very genes. Using bisulphite pyrosequencing we assessed the methylation density of 11 PCC/PGL disease genes in 96 tumors (83 PCCs and 13 PGLs) and 34 normal adrenal references. Gene expression levels were determined by quantitative RT-PCR. Both tumors and normal adrenal samples exhibited low methylation index (MetI) in the EGLN1 (PDH2), MAX, MEN1, NF1, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127 promoters, not exceeding 10% in any of the samples investigated. Aberrant RET promoter methylation was observed in two cases only. For the VHL gene we found increased MetI in tumors as compared with normal adrenals (57% vs. 27%; P < 0.001), in malignant vs. benign tumors (63% vs. 55%; P < 0.05), and in PGL vs. PCC (66% vs. 55%; P < 0.0005). Decreased expression of the VHL gene was observed in all tumors compared with normal adrenals (P < 0.001). VHL MetI and gene expressions were inversely correlated (R = −0.359, P < 0.0001). Our results show that the VHL gene promoter has increased methylation compared with normal adrenals (MetI > 50%) in approximately 75% of PCCs and PGLs investigated, highlighting the role of VHL in the development of these tumors.     

Sequences Distal to the AP1/E Box Motif Are Involved in the Cell Type-Specific Expression of the Rat Tyrosine Hydroxylase Gene

Abstract: In order to define cell type-specific elements associated with the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), transient transfections of promoter deletion constructs were used to test relative reporter-gene activities in TH-expressing and-nonexpressing cell lines. Such assays demonstrated that a region between-503 and-578 contributed to rat TH promoter activity in the pheochromocytoma cell line PC12. Deletion of these sequences resulted in a 66% loss in cell type-specific activity. Mutations within the E box/dyad symmetry element (CAGGTGCCTGTGACAGTG) did not affect the basal and cell type-specific pattern of expression exhibited by the rat TH promoter. Promoter fusion constructs between the rat TH promoter (-741 and-197) and the human TH promoter (-197 and +1) exhibited reporter-gene activities equivalent to that of wild-type-741 rat TH constructs, further demonstrating that sequence elements upstream of the rat E box/dyad symmetry are important for cell type-specific expression. Gel-shift experiments indicated that a PC12 nuclear factor could bind to a 39-bp sequence within this region in a cell type-specific manner. The size of this factor was 52 kDa as determined by UV cross-linking experiments.     

Apport de la TEP/TDM au 18FDG dans le bilan préopératoire des phéochromocytomes. Étude rétrospective comparative par rapport à la scintigraphie à la MIBG

Pheochromocytomas (PHEO) are tumors arisen from the adrenal medulla, with a high secretory risk. Malignancy is rare and difficult to establish before metastastic spread. The risk of multifocality becomes greater whether genetic predisposition exists. MIBG scintigraphy is the reference functional imaging of PHEO. The place of 18FDG PET/CT is not well-established in literature. Our study retrospectively analyzes patients operated for a PHEO and who underwent both MIBG and 18FDG PET/CT before surgery, between 2007 and 2015. On the 49 patients included (52 lesions), among them 13 had genetic mutation, MIBG detected 39 lesions (75%) and 18FDG PET/CT forty-eight (92%), enabling a combined sensitivity of 98%. Sensitivity was not affected by the predominant secretion (metanephrine or normetanephrine), whereas MIBG-negative lesions showed a higher proliferation index (Ki67) than MIBG-positive lesions (6.6 vs. 2.8; P = 0.0044). FDG PET/CT semi-quantitative indices vary with the germline mutation status and were significantly higher for Cluster 1 lesions (SDHx, VHL or FH mutations) than for any other lesions (SUVmax = 27.73 vs. 5.92 for the others mutations (Cluster 2), 9.53 for lesions without mutation and 3.78 for undetermined lesions; P = 0.002). In conclusion, because of their capacity to take up FDG, 18FDG PET/CT could be useful in the preoperative imaging of PHEO, especially when MIBG is not contributive or when F-DOPA PET/CT is not available. An intense FDG uptake may orient to a Cluster 1 mutation.     

Dopamine-β-Hydroxylase: Structural Comparisons of Membrane-Bound Versus Soluble Forms from Adrenal Medulla and Pheochromocytoma

Dopamine-beta-hydroxylase (DBH) in membrane-bound (mDBH) and water-soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A-agarose column chromatography followed by DEAE-Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by two-dimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.     

Cell Cycle-Dependent Modulation of Biosynthesis and Stimulus-Evoked Release of Catecholamines in PC12 Pheochromocytoma Cells

Catecholamine biosynthesis and its stimulus-evoked release in PC12 pheochromocytoma cells were studied as a function of cell cycle by means of HPLC with electrochemical detection. We found that 3,4-dihydroxyphenylethylamine (dopamine) levels in PC12 cells remained constant throughout the period of cell cycle. In contrast, the noradrenaline content was dependent on the cell cycle: it increased during the S + G2 phase followed by a decrease in the M phase. These results were confirmed further by measuring the activities catalyzing the catecholamine biosynthesis. Thus, activities of tyrosine 3-monooxygenase and 3,4-dihydroxyphenylalanine decarboxylase were independent of the cell cycle, whereas both soluble and membrane-bound dopamine beta-monooxygenase activities were modulated during the cell cycle. On the other hand, release of the catecholamines stimulated with 50 mM KCl increased in the G1 phase, reached a maximum in the late G1, and then gradually decreased in later periods. We also found that carbamylcholine-induced release of the catecholamines occurred maximally in the early S + G2 phase followed by a decrease during the M phase. Cell cycle dependence of the catecholamine release was in good agreement with that of 45Ca2+ uptake. Thus, this study provides evidence that the catecholamine biosynthesis and its release in PC12 cells are modulated during the period of cell cycle.     

Clonal Variability of PC12 Pheochromocytoma Cells with Respect to Catecholamine Biosynthesis

Clonal variants of PC12 cells with respect to catecholamine biosynthesis were isolated, and the catecholamine content was measured by high performance liquid chromatography with electrochemical detection. The dopamine content of 13 subclones, which were selected and isolated in tyrosine-free medium, was substantially higher than the control level: 0.91 +/- 0.10 nmol/mg protein (mean +/- SEM; n = 3). In contrast, the noradrenaline content showed a marked heterogeneity: only two subclones contained noradrenaline levels similar to or higher than the control level: 0.40 +/- 0.05 (n = 5). The rest of them contained below the level of 0.20, and only negligible amounts of noradrenaline were found in four subclones. Thus, the noradrenaline-to-dopamine ratio varied widely between 0.003:1 and 0.53:1. This divergence of the noradrenaline content appears to be related to differing levels of dopamine beta-monooxygenase activity. The administration of ascorbate to the medium alone, however, did not restore the level of noradrenaline to the normal level in a subclone. Heterogeneity of the response to applied glucocorticoid was also demonstrated.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)