peptide-phi and Neuroblastoma

Topics: Amino Acid Sequence; Animals; Galanin; Humans; Immunohistochemistry; Molecular Sequence Data; Neuroblastoma; Peptide PHI; Peptides; Structure-Activity Relationship; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca2+ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10(-13) M and 10(-10) M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC1) receptor. However, binding experiments using 125I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cyclic AMP; Humans; Mice; Neuroblastoma; Neuropeptides; Nucleic Acid Synthesis Inhibitors; Peptide PHI; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Pituitary Hormone; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNAs (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt2cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt2cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt2cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.

Topics: Bucladesine; Cytoplasmic Granules; Enkephalin, Methionine; Hormones; Humans; Immunohistochemistry; In Situ Hybridization; Microscopy, Electron; Neuroblastoma; Neuropeptide Y; Peptide PHI; Protein Precursors; Radioimmunoassay; RNA, Messenger; Substance P; Tachykinins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

Using regional specific antisera the concentrations of vasoactive intestinal polypeptide (VIP) and the peptide with N-terminal histidine and C-terminal isoleucine (PHI) in various peripheral tissues and VIP producing tumours were compared with their immunohistochemical localization. In normal tissue the VIP levels were in general higher than the PHI levels, while the VIP/PHI ratio in tumour tissue varied considerably more than in normal tissue. By immunohistochemistry it was found that VIP and PHI immunoreactivity occurred in the same autonomic neurons. Gel chromatography revealed that VIP and PHI immunoreactivity in both normal and tumour tissue consisted of two larger molecular forms in addition to "authentic" peptides. These larger molecular forms which had overlapping elution positions probably represent VIP/PHI precursors. In tumour tissue the larger molecular forms constituted a larger proportion of the total immunoreactivity. Neurally induced relaxation of smooth muscle caused a simultaneous release of VIP and PHI which in combination with the observed relaxatory effect of the peptide suggest a role in the control of smooth muscle activity. Similarly VIP and PHI were co-secreted from tumour tissues as evidenced from elevated plasma levels in patients with VIP producing tumours. In conclusion VIP and PHI seem to co-exist and be co-secreted. Differences in posttranslational processing may create variable content and release of the two peptides.

Topics: Animals; Cats; Digestive System; Ganglioneuroma; Humans; Neuroblastoma; Pancreatic Neoplasms; Peptide PHI; Reference Values; Species Specificity; Swine; Tissue Distribution; Vasoactive Intestinal Peptide

The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0.1-0.3 pmol per mm2 with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB-OK-1 cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit anti-PHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bucladesine; Cell Line; Golgi Apparatus; Histocytochemistry; Humans; Immunochemistry; Immunoenzyme Techniques; Neuroblastoma; Peptide PHI; Peptides; Protein Precursors; Vasoactive Intestinal Peptide

The mechanism of N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) induction of pro-vasoactive intestinal polypeptide (VIP)/PHM-27 biosynthesis was investigated in human neuroblastoma cells in culture. When neuroblastoma cells were grown for 48 h in the presence of 1 mM Bt2cAMP, the synthesis of pro-VIP/PHM-27 was stimulated 11-fold. The amount of prepro-VIP/PHM-27 mRNA determined both by hybridization with cloned prepro-VIP/PHM-27 cDNA and a reticulocyte cell-free translation assay was also increased 11-fold in the Bt2cAMP-induced cells. Transcription of prepro-VIP/PHM-27 mRNA in isolated nuclei was observed in induced cells, but not in uninduced cells. Blot hybridization with prepro-VIP/PHM-27 cDNA of total nuclear RNA isolated from neuroblastoma cells revealed an RNA species corresponding to mature prepro-VIP/PHM-27 mRNA, and the amount of the RNA was markedly increased in the induced cells. The quantity of VIP/PHM-27 gene in the DNA of neuroblastoma cells was analyzed after hydrolysis with a restriction endonuclease, EcoRI. However, VIP/PHM-27 gene was not amplified in the induced cells. These results indicate that Bt2cAMP-induced pro-VIP/PHM-27 synthesis is achieved by enhancing the transcription rate of prepro-VIP/PHM-27 mRNA.

Topics: Animals; Bucladesine; Cell Line; Cell Nucleus; Cyclic AMP; DNA; DNA Restriction Enzymes; DNA Transposable Elements; Humans; Kinetics; Neuroblastoma; Nucleic Acid Hybridization; Peptide PHI; Plasmids; Protein Biosynthesis; Protein Precursors; Rabbits; Reticulocytes; RNA, Messenger; Transcription, Genetic; Vasoactive Intestinal Peptide