pituitrin and Neuroblastoma

We report a mediastinal neuroblastoma in an octogenarian with paraneoplastic syndrome of inappropriate antidiuretic hormone secretion (SIADH). Neuroblastomas are very rare tumors in adults, with thoracic or mediastinal locations being especially uncommon. These neoplasms have been occasionally associated with the SIADH. Given the rarity of incidence and paucity of diagnostic and outcomes data, the significance of standard neuroblastoma prognostic characteristics is unclear, and no treatment paradigms exist for these patients. Further studies are needed to inform future clinical guidelines.

Topics: Adult; Aged, 80 and over; Humans; Inappropriate ADH Syndrome; Mediastinal Neoplasms; Neuroblastoma; Vasopressins

We describe a rare case of thymic neuroblastoma with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). A 60-year-old male patient was admitted to our hospital for further examination and treatment of anterior mediastinal tumor found at a regular health check-up. On examination there was hyponatremia, decrease in plasma osmolarity and elevation of plasma antidiuretic hormone (ADH) level. Thus, he underwent total thymectomy under the diagnosis of thymoma with SIADH. The tumor was located in the right lobe of the thymus and the final diagnosis was thymic neuroblastoma. To our knowledge, this is the first reported case of thymic neuroblastoma in which production of ADH by tumor cells is demonstrated immunohistochemically. This case highlights the need to consider functional activity of thymic neuroblastoma and complete resection of the tumor is warranted for treatment.

Topics: Biomarkers; Biopsy; Humans; Hyponatremia; Immunohistochemistry; Inappropriate ADH Syndrome; Magnetic Resonance Imaging; Male; Middle Aged; Neuroblastoma; Osmolar Concentration; Thymectomy; Thymus Neoplasms; Tomography, X-Ray Computed; Treatment Outcome; Up-Regulation; Vasopressins

Mutations in the human gene encoding the antidiuretic hormone vasopressin (VP) cause autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI), a rare inherited disorder that presents as polydipsia and polyuria as a consequence of a loss of secretion of VP from posterior pituitary nerve terminals. Work from our laboratories has shown that adFNDI, like other neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's, is associated with autophagy. We have recently shown that the activation of autophagy in mouse neuroblastoma Neuro2a cells after adenoviral vector-mediated delivery of an adFNDI mutant VP transgene (Cys67stop) is a cell survival mechanism; its inhibition induces apoptosis. We now show that expression of Cys67stop sensitizes Neuro2a cells to the lethal effects of dopamine. This mode of cell death exhibits features typically associated with classical apoptosis. Yet inhibition of autophagy reversed these effects and rescued cell viability. We propose that autophagy-mediated cell death is a "two-hit" process: Following the cellular stress of the accumulation of a misfolded mutant protein, autophagy is prosurvival. However, a second insult triggers an autophagy-dependent apoptosis.

Topics: Adenoviridae; Animals; Autophagy; Cadaverine; Caspases; Cell Death; Cell Line, Tumor; Cell Survival; Codon, Terminator; Cysteine; Diabetes Insipidus, Neurogenic; Disease Models, Animal; Dopamine; Fluorescent Dyes; Gene Expression; Genetic Vectors; Mice; Mutation; Neuroblastoma; Phagosomes; Transfection; Transgenes; Vacuoles; Vasopressins

Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a progressive, inherited neurodegenerative disorder that presents as polydipsia and polyuria as a consequence of a loss of secretion of the antidiuretic hormone vasopressin (VP) from posterior pituitary nerve terminals. VP gene mutations cause adFNDI. Rats expressing an adFNDI VP transgene (Cys67stop) show a neuronal pathology characterized by autophagic structures in the cell body. adFNDI has thus been added to the list of protein aggregation diseases, along with Alzheimer's, Parkinson's and Huntington's, which are associated with autophagy, a bulk process that delivers regions of cytosol to lysosomes for degradation. However, the role of autophagy in these diseases is unclear. To address the relationships between mutant protein accumulation, autophagy, cell survival, and cell death, we have developed a novel and tractable in vitro system. We have constructed adenoviral vectors (Ads) that express structural genes encoding either the Cys67stop mutant protein (Ad-VCAT-Cys67stop) or an epitope-tagged wild-type VP precursor (Ad-VCAT). After infection of mouse neuroblastoma Neuro2a cells, Ad-VCAT encoded material enters neurite processes and accumulates in terminals, while the Cys67stop protein is confined to enlarged vesicles in the cell body. Similar to the intracellular derangements seen in the Cys67stop rats, these structures are of ER origin, and colocalize with markers of autophagy. Neither Ad-VCAT-Cys67stop nor Ad-VCAT expression affected cell viability. However, inhibition of autophagy or lysosomal protein degradation, while having no effect on Ad-VCAT-expressing cells, significantly increased apoptotic cell death following Ad-VCAT-Cys67stop expression. These data suggest that activation of autophagy by the stress of the expression of an adFNDI mutant protein is a prosurvival mechanism.

Topics: Acridine Orange; Adenoviridae; Animals; Animals, Genetically Modified; Autophagy; Blotting, Western; Cadaverine; Cathepsin D; Cell Line, Tumor; Codon, Terminator; Cysteine; Diabetes Insipidus, Neurogenic; Fluorescent Antibody Technique; Fluorescent Dyes; Gene Expression; Genetic Vectors; Hydrogen-Ion Concentration; Lysosomes; Mutation; Neurites; Neuroblastoma; Neurons; Organelles; Rats; Recombinant Proteins; Transfection; Transgenes; Vacuoles; Vasopressins

The vasopressin gene is expressed in the suprachiasmatic nucleus where the basic helix-loop-helix (bHLH)-PAS factors CLOCK and MOP3 regulate circadian expression through interactions with E-box sequences. We have examined vasopressin gene regulation by HIF-1alpha, a bHLH-PAS factor involved in responses to hypoxia. By transfecting Neuro-2A cells with 5' flanking regions of vasopressin gene driving a luciferase reporter, we have shown that CLOCK and HIF-1alpha cooperate in the induction of expression from 1000 bp and 350 bp of the vasopressin promoter but do not activate a 120-bp promoter fragment. The region between -191 and -128 contains an E-box A that appears to be essential for HIF-1alpha/CLOCK-mediated transcriptional activity. However, gel-shift analysis shows that the cooperative effect of HIF-1alpha and CLOCK results in MOP3 binding, but does not involve heterodimerization of HIF-1alpha/CLOCK, at E-box A. These data indicate that cross-talk between mediators of hypoxic and circadian pathways can regulate target genes.

Topics: 5' Flanking Region; Animals; ARNTL Transcription Factors; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Circadian Rhythm; CLOCK Proteins; Dimerization; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Molecular Sequence Data; Neuroblastoma; Promoter Regions, Genetic; Protein Structure, Tertiary; Trans-Activators; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured; Vasopressins

It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct prohormone folding in the endoplasmic reticulum.

Topics: Animals; Cell Line, Tumor; Diabetes Insipidus; Endoplasmic Reticulum; Gene Expression; Mutagenesis; Neuroblastoma; Neurophysins; Protein Folding; Protein Structure, Tertiary; Protein Transport; Rats; Secretory Vesicles; Vasopressins

Mouse neuroblastoma Neuro-2a cells were examined for the expression of pro-enkephalin mRNA, protein, and Met-enkephalin ([Met]-Enk) peptide. Reverse transcriptase/polymerase chain reaction (RT/PCR) and in situ hybridization demonstrated the presence of pro-enkephalin mRNA in these cells. Immunocytochemistry using an antibody which recognizes pro-enkephalin and high pressure liquid chromatography (HPLC) followed by radioimmunoassay indicated that pro-enkephalin was synthesized in these cells and processed to yield the bioactive pentapeptide, [Met]-Enk. Furthermore, release studies showed that the [Met]-Enk was secreted from these cells with high K+ stimulation. Using double labeling, in situ hybridization combined with immunocytochemistry, we demonstrated that prohormone convertase 2 (PC2) mRNA is colocalized with pro-enkephalin in the same Neuro-2a cells, suggesting that this enzyme may be responsible for processing this precursor. we also showed the presence of vasopressin mRNA and arginine-vasopressin peptide in these cells using in situ hybridization and immunocytochemistry, respectively. Thus, the Neuro-2a cells are a multiple neuropeptide-producing cell line and an excellent model for studying the mechanisms involved in the synthesis, intracellular targeting and processing of endogenous pro-enkephalin and pro-vasopressin, as well as other transfected neuropeptide precursors.

Topics: Animals; Arginine Vasopressin; Chromatography, High Pressure Liquid; Enkephalin, Methionine; Enkephalins; Gene Expression; Immunohistochemistry; In Situ Hybridization; Mice; Neuroblastoma; Polymerase Chain Reaction; Proprotein Convertase 2; Protein Precursors; RNA-Directed DNA Polymerase; RNA, Messenger; Subtilisins; Tumor Cells, Cultured; Vasopressins

To obtain a model for the sorting and processing of preprovasopressin (preproVP), rat VP cDNA was transfected in murine Neuro2A neuroblastoma cells, which do not express VP. The precursor of VP was expressed and processed into the authentic VP gene products VP, neurophysin (NP) and glycopeptide (GP) as determined with reversed phase HPLC and radioimmunoassay. In addition, Neuro2A-specific forms of NP and GP were observed, which may be produced in the constitutive secretory pathway in these cells.

Topics: Animals; Base Sequence; Cell Line; Chromatography, High Pressure Liquid; DNA Primers; Gene Expression; Glycopeptides; Hypothalamus; Male; Molecular Sequence Data; Neuroblastoma; Neurophysins; Pituitary Gland; Polymerase Chain Reaction; Protein Precursors; Protein Processing, Post-Translational; Radioimmunoassay; Rats; Rats, Wistar; Tumor Cells, Cultured; Vasopressins

Synthetic peptides corresponding to the effector domain of the small molecular weight GTP-binding protein Rab3A are known to stimulate exocytosis in various secretory cells. In the present study, we report that Rab3A effector domain peptide (33-48) causes accumulation of inositol 1,4,5-trisphosphate (1,4,5-IP3) in permeabilized pancreatic acinar cells, hepatocytes, 3T3 fibroblasts, and SH-SY5Y neuroblastoma cells. A scrambled peptide of Rab3A had no effect showing specificity of the Rab3A peptide response. No effect was observed in intact cells indicating that the target of the peptide is located intracellularly. We conclude that Rab3 effector domain peptide-induced accumulation of 1,4,5-IP3 is a wide-spread phenomenon, suggesting regulation of phosphoinositide-specific phospholipase C by Rab3-like proteins.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Bombesin; Carbachol; Cell Membrane Permeability; Digitonin; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Liver; Male; Mice; Molecular Sequence Data; Neuroblastoma; Pancreas; Peptide Fragments; rab3 GTP-Binding Proteins; Rats; Rats, Wistar; Tumor Cells, Cultured; Vasopressins

Topics: Animals; Bradykinin; Cell Line; Enkephalins; Enzyme Activation; Glioma; Insulin-Like Growth Factor I; Interleukin-1; Kinetics; Nerve Growth Factors; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphotransferases; Transforming Growth Factor beta; Vasopressins