neuropeptide-y and Neuroblastoma

Currently available data on the involvement of neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) and their receptors (YRs) in cancer are updated. The structure and dynamics of YRs and their intracellular signaling pathways are also studied. The roles played by these peptides in 22 different cancer types are reviewed (e.g., breast cancer, colorectal cancer, Ewing sarcoma, liver cancer, melanoma, neuroblastoma, pancreatic cancer, pheochromocytoma, and prostate cancer). YRs could be used as cancer diagnostic markers and therapeutic targets. A high Y1R expression has been correlated with lymph node metastasis, advanced stages, and perineural invasion; an increased Y5R expression with survival and tumor growth; and a high serum NPY level with relapse, metastasis, and poor survival. YRs mediate tumor cell proliferation, migration, invasion, metastasis, and angiogenesis; YR antagonists block the previous actions and promote the death of cancer cells. NPY favors tumor cell growth, migration, and metastasis and promotes angiogenesis in some tumors (e.g., breast cancer, colorectal cancer, neuroblastoma, pancreatic cancer), whereas in others it exerts an antitumor effect (e.g., cholangiocarcinoma, Ewing sarcoma, liver cancer). PYY or its fragments block tumor cell growth, migration, and invasion in breast, colorectal, esophageal, liver, pancreatic, and prostate cancer. Current data show the peptidergic system's high potential for cancer diagnosis, treatment, and support using Y2R/Y5R antagonists and NPY or PYY agonists as promising antitumor therapeutic strategies. Some important research lines to be developed in the future will also be suggested.

Topics: Breast Neoplasms; Colorectal Neoplasms; Humans; Liver Neoplasms; Male; Neoplasm Recurrence, Local; Neuroblastoma; Neuropeptide Y; Pancreatic Neoplasms; Peptide YY; Prostatic Neoplasms; Receptors, Neuropeptide Y; Sarcoma, Ewing

Neuropeptide Y (NPY) is a sympathetic neurotransmitter with pleiotropic actions, many of which are highly relevant to tumor biology. Consequently, the peptide has been implicated as a factor regulating the growth of a variety of tumors. Among them, two pediatric malignancies with high endogenous NPY synthesis and release - neuroblastoma and Ewing sarcoma - became excellent models to investigate the role of NPY in tumor growth and progression. The stimulatory effect on tumor cell proliferation, survival, and migration, as well as angiogenesis in these tumors, is mediated by two NPY receptors, Y2R and Y5R, which are expressed in either a constitutive or inducible manner. Of particular importance are interactions of the NPY system with the tumor microenvironment, as hypoxic conditions commonly occurring in solid tumors strongly activate the NPY/Y2R/Y5R axis. This activation is triggered by hypoxia-induced up-regulation of Y2R/Y5R expression and stimulation of dipeptidyl peptidase IV (DPPIV), which converts NPY to a selective Y2R/Y5R agonist, NPY(3-36). While previous studies focused mainly on the effects of NPY on tumor growth and vascularization, they also provided insight into the potential role of the peptide in tumor progression into a metastatic and chemoresistant phenotype. This review summarizes our current knowledge of the role of NPY in neuroblastoma and Ewing sarcoma and its interactions with the tumor microenvironment in the context of findings in other malignancies, as well as discusses future directions and potential clinical implications of these discoveries.

Topics: Animals; Cell Proliferation; Child; Humans; Neovascularization, Pathologic; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y

Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be a potent growth and angiogenic factor. The peptide and its receptors are abundant in neural crest-derived tumors, such as sympathetic neuroblastomas and pheochromocytomas, as well as parasympathetic Ewing's sarcoma family of tumors. NPY regulates their growth directly, by an autocrine activation of tumor cell proliferation or apoptosis, and indirectly, by its angiogenic activity. The overall effect of the peptide on tumor growth depends on a balance between these processes and the type of receptors expressed in the tumor cells. Thus, NPY and its receptors may become targets for the treatment of neural tumors, directed against both tumor cell proliferation and angiogenesis.

Topics: Animals; Cell Proliferation; Humans; Models, Biological; Neoplasms, Nerve Tissue; Neovascularization, Pathologic; Neural Crest; Neuroblastoma; Neuropeptide Y; Sarcoma, Ewing

Neuroblastomas are pediatric tumors of sympathetic origin, expressing neuronal markers, such as NPY and its receptors. Due to this, neuroblastomas are often associated with elevated plasma levels of NPY, which correlates with poor clinical outcome of the disease. This clinical data corroborates the recent discovery of growth-promoting actions of NPY in neuroblastomas. The peptide has been shown to stimulate proliferation of neuroblastoma cells in an autocrine manner and induce tumor vascularization. Since both processes are mediated by the same Y2 and Y5 receptors, targeting this pathway may be a potential bidirectional therapy for these children's tumors.

Topics: Cell Division; Humans; Neovascularization, Pathologic; Neuroblastoma; Neuropeptide Y

Topics: Adrenal Gland Neoplasms; Alzheimer Disease; Anorexia Nervosa; Autonomic Nervous System Diseases; Biomarkers; Humans; Hypertension; Immunoradiometric Assay; Neuroblastoma; Neuropeptide Y; Pheochromocytoma; Radioimmunoassay; Reference Values

Topics: Amino Acid Sequence; Amino Acids; Animals; Cell Line; Cell Membrane; Kidney; Male; Molecular Sequence Data; Molecular Structure; Neuroblastoma; Neuropeptide Y; Peptides; Protein Conformation; Rabbits; Radioligand Assay; Receptors, Neuropeptide Y

Heterogeneity among NPY (and PYY) receptors was first proposed on the basis of studies on sympathetic neuroeffector junctions, where NPY (and PYY) can exert three types of action: 1) a direct (e.g., vasoconstrictor) response; 2) a postjunctional potentiating effect on NE-evoked vasoconstriction; and 3) a prejunctional suppression of stimulated NE release; the two latter phenomena are probably reciprocal, since NE affect NPY mechanisms similarly. It was found that amidated C-terminal NPY (or PYY) fragments, e.g., NPY 13-36, could stimulate selectively prejunctional NPY/PYY receptors, which were termed Y2-receptors. Consequently, the postjunctional receptors which were activated poorly by NPY/PYY fragments, were termed Y1-receptors. Later work has indicated that the Y2-receptor may occur postjunctionally in selected sympathetic effector systems. The central nervous system appears to contain a mixture of Y1- and Y2-receptors as indicated by functional as well as binding studies. For instance, NPY and NPY 13-36 produced diametrically opposite effects on behavioral activity, indicating the action of the parent peptide on two distinct receptors. Cell lines, most importantly neuroblastomas, with exclusive populations of Y1- or Y2-receptors, have been characterized by binding and second messenger studies. In this work, selective agonists for the two receptor subtypes were used. Work of many investigators has formed the basis for subclassifying NPY/PYY effects being mediated by either Y1- or Y2-receptors. A preliminary subclassification based on effects of NPY, PYY, fragments and/or analogs is provided in Table 6. It is, however, to be expected that further receptor heterogeneity will be revealed in the future. It is argued that mast cells possess atypical NPY/PYY receptors. The histamine release associated with stimulation of the latter receptors may, at least in part, underlie the capacity of NPY as well as of short C-terminal fragments to reduce blood pressure. Fragments, such as NPY 22-36, appear to be relatively selective vasodepressor agents because of their weak vasopressor properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Calcium; Cloning, Molecular; Colforsin; Cyclic AMP; Humans; Hypotension; In Vitro Techniques; Neuroblastoma; Neuropeptide Y; Norepinephrine; Peptide Fragments; Peptide YY; Peptides; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Phencyclidine; Receptors, sigma; Signal Transduction; Sympathetic Nervous System; Synaptic Transmission; Vasoconstriction

Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative diseases. Glutamate excitotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress are the common mechanisms associated with numerous neurodegenerative illnesses. The present study aimed to elucidate the protective effects of NPY against glutamate toxicity and tunicamycin-induced ER stress in the human neuroblastoma SH-SY5Y cell line. We exposed the SH-SY5Y cells to glutamate and tunicamycin for two different time points and analyzed the protective effects of NPY at different concentrations. The protective effects of NPY treatments were assessed by cell viability assay, and the signalling pathway changes were evaluated by biochemical techniques such as Western blotting and immunofluorescence assays. Our results showed that treatment of SH-SY5Y cells with NPY significantly increased the viability of the cells in both glutamate toxicity and ER stress conditions. NPY treatments significantly attenuated the glutamate-induced pro-apoptotic activation of ERK1/2 and JNK/BAD pathways. The protective effects of NPY were further evident against tunicamycin-induced ER stress. NPY treatments significantly suppressed the ER stress activation by downregulating BiP, phospho-eIF2α, and CHOP expression. In addition, NPY alleviated the Akt/FoxO3a pathway in acute oxidative conditions caused by glutamate and tunicamycin in SH-SY5Y cells. Our results demonstrated that NPY is neuroprotective against glutamate-induced cell toxicity and tunicamycin-induced ER stress through anti-apoptotic actions.

Topics: Cell Line, Tumor; Glutamic Acid; Humans; Neuroblastoma; Neuropeptide Y; Neuroprotective Agents; Tunicamycin

Epilepsy is a common neurological condition characterised by recurrent unprovoked seizures and often treatable with appropriate medication. However, almost 30% of cases are pharmacoresistant and while a proportion of these may be amenable to resective surgery, a gene therapy approach could be an attractive alternative option. Neuropeptide Y (NPY) has anticonvulsant and anti-epileptogenic properties in animal models of temporal lobe epilepsy when delivered by an adeno-associated viral (AAV) vector. Here we sought to demonstrate successful secretion of NPY from AAV-transduced human neuronal cells, which would be essential in planning any clinical trial.. A human neuroblastoma cell line (SH-SY5Y) was used to assess in vitro whether an AAV vector manufactured to clinical-grade protocols would be effective at transducing these cells to express NPY. Optimal transduction efficiency was first achieved with retinoic acid and tetradecanoylphorpol-13-acetate (TPA) treatment, prior to expose to AAV1-green fluorescent protein (GFP) reporter vector, AAV1-NPY therapeutic vector or sham treated with no vector. Levels of NPY in cell supernatants were determined using two antibody-based methods RESULTS: We found that the levels of NPY released into the cell culture media supernatant, and protein extracts of the cell pellet, were significantly higher following exposure to AAV1-NPY than when compared to either a control GFP reporter vector (AAV1-GFP) or sham treated controls.. This first demonstration that an AAV-NPY construct can successfully transduce human neuronal cells supports the pre-clinical development of a clinical trial using AAV-based NPY for pharmacoresistant epilepsy.

Topics: Cell Line, Tumor; Dependovirus; Drug Resistant Epilepsy; Genetic Therapy; Genetic Vectors; Humans; Neuroblastoma; Neurons; Neuropeptide Y

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)-mediated chemoresistance and Y2 receptor (Y2R)-mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.

Topics: Adolescent; Animals; Biomarkers; Cell Proliferation; Child; Child, Preschool; Disease Progression; Female; Humans; Infant; Infant, Newborn; Male; Mice; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y

Neuroblastoma (NB) is a pediatric tumor of neural crest origin with heterogeneous phenotypes. Although low-stage tumors carry a favorable prognosis, >50% of high-risk NB relapses after treatment with a fatal outcome. Thus developing therapies targeting refractory NB remains an unsolved clinical problem. Brain-derived neurotrophic factor (BDNF) and its TrkB receptor are known to protect NB cells from chemotherapy-induced cell death, while neuropeptide Y (NPY), acting via its Y2 receptor (Y2R), is an autocrine proliferative and angiogenic factor crucial for maintaining NB tumor growth. Here we show that in NB cells, BDNF stimulates the synthesis of NPY and induces expression of another one of its receptors, Y5R. In human NB tissues, the expression of NPY and Y5R positively correlated with the expression of BDNF and TrkB. Functionally, BDNF triggered Y5R internalization in NB cells, whereas Y5R antagonist inhibited BDNF-induced p44/42 mitogen-activated protein kinase activation and its pro-survival activity. These observations suggested TrkB-Y5R transactivation that resulted in cross-talk between their signaling pathways. Additionally, NPY and Y5R were upregulated in a BDNF-independent manner in NB cells under pro-apoptotic conditions, such as serum deprivation and chemotherapy, as well as in cell lines and tissues derived from posttreatment NB tumors. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis and inhibited their growth in vivo by augmenting cell death. In summary, the NPY/Y5R axis is an inducible survival pathway activated in NB by BDNF or cellular stress. Upon such activation, Y5R augments the pro-survival effect of BDNF via its interactions with TrkB receptor and exerts an additional BDNF-independent anti-apoptotic effect, both of which contribute to NB chemoresistance. Therefore, the NPY/Y5R pathway may become a novel therapeutic target for patients with refractory NB, thus far an incurable form of this disease.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Survival; Cells, Cultured; Child; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y

Okadaic acid (OA) and dinophysistoxins (DTXs) are a group of marine toxins that cause diarrheic shellfish poisoning (DSP) in humans and animals. These compounds are produced by dinoflagellates of the Prorocentrum and Dinophysis genera and can accumulate in filter-feeding bivalves, posing a serious health risk for shellfish consumers. The enteric nervous system (ENS) plays a crucial role in the regulation of the gastrointestinal tract. In addition, neuropeptides produced by ENS affects the epithelial barrier functions. In the present work we used a two-compartment human coculture model containing the SH-SY5Y neuroblastoma cell line and polarized colonic epithelial monolayers (Caco-2) to study the OA intestinal permeability. First, we have determined OA cytotoxicity and we have found that OA reduces the viability of SH-SY5Y in a dose-dependent way, even though DTX1 is 4 to 5 times more potent than OA. Besides DTX1 is 15 to 18 orders of magnitude more potent than OA in decreasing transepithelial electrical resistance (TEER) of caco-2 cells without inducing cytotoxicity. Permeability assays indicate that OA cross the monolayer and modulates the neuropeptide Y (NPY) secretion by neuroblastoma cells. This NPY also affects the permeability of OA. This offers a novel approach to establish the influence of OA neuronal action on their diarrheic effects through a cross talk between ENS and intestine via OA induced NPY secretion. Therefore, the OA mechanisms of toxicity that were long attributed only to the inhibition of protein phosphatases, would require a reevaluation.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Survival; Diarrhea; Electric Impedance; Humans; Neuroblastoma; Neurons; Neuropeptide Y; Okadaic Acid; Pyrans

Beta-amyloid (Aβ) peptides are secreted from neurons, resulting in extracellular accumulation of Aβ and neurodegeneration of Alzheimer's disease. Because neuronal secretion is fundamental for the release of neurotransmitters, this study assessed the hypothesis that Aβ undergoes co-release with neurotransmitters. Model neuronal-like chromaffin cells were investigated, and results illustrate regulated, co-secretion of Aβ(1-40) and Aβ(1-42) with peptide neurotransmitters (galanin, enkephalin, and NPY) and catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). Regulated secretion from chromaffin cells was stimulated by KCl depolarization and nicotine. Forskolin, stimulating cAMP, also induced co-secretion of Aβ peptides with peptide and catecholamine neurotransmitters. These data suggested the co-localization of Aβ with neurotransmitters in dense core secretory vesicles (DCSV) that store and secrete such chemical messengers. Indeed, Aβ was demonstrated to be present in DCSV with neuropeptide and catecholamine transmitters. Furthermore, the DCSV organelle contains APP and its processing proteases, β- and γ-secretases, that are necessary for production of Aβ. Thus, Aβ can be generated in neurotransmitter-containing DCSV. Human IMR32 neuroblastoma cells also displayed regulated secretion of Aβ(1-40) and Aβ(1-42) with the galanin neurotransmitter. These findings illustrate that Aβ peptides are present in neurotransmitter-containing DCSV, and undergo co-secretion with neuropeptide and catecholamine neurotransmitters that regulate brain functions.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Catecholamines; Cattle; Cells, Cultured; Chromaffin Cells; Colforsin; Cyclic AMP; Dopamine; Enkephalins; Epinephrine; Galanin; Ganglionic Stimulants; Humans; Neuroblastoma; Neurons; Neuropeptide Y; Neuropeptides; Neurotransmitter Agents; Nicotine; Norepinephrine; Peptide Fragments; Secretory Vesicles

Neuropeptide Y (NPY) is a peptide found in the brain and autonomic nervous system, which is associated with anxiety, depression, epilepsy, learning and memory, sleep, obesity and circadian rhythms. NPY has recently gained much attention as an endogenous antiepileptic and antidepressant agent, as drugs with antiepileptic and/or mood-stabilizing properties may exert their action by increasing NPY concentrations, which in turn can reduce anxiety and depression levels, dampen seizures or increase seizure threshold. We have used human neuroblastoma SH-SY5Y cells to investigate the effect of valproate (VPA) and amitriptyline (AMI) on NPY expression at therapeutic plasma concentrations of 0.6mM and 630nM, respectively. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA) known to differentiate SH-SY5Y cells into a neuronal phenotype and to increase NPY expression through activation of protein kinase C (PKC) was applied as a positive control (16nM). Cell viability after drug treatment was tested with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. NPY expression was measured using immunofluorescence and quantitative RT-PCR (qRT-PCR). Results from immunocytochemistry have shown NPY levels to be significantly increased following a 72h but not 24h VPA treatment. A further increase in expression was observed with simultaneous VPA and TPA treatment, suggesting that the two agents may increase NPY expression through different mechanisms. The increase in NPY mRNA by VPA and TPA was confirmed with qRT-PCR after 72h. In contrast, AMI had no effect on NPY expression in SH-SY5Y cells. Together, the data point to an elevation of human NPY mRNA and peptide levels by therapeutic concentrations of VPA following chronic treatment. Thus, upregulation of NPY may have an impact in anti-cancer treatment of neuroblastomas with VPA, and antagonizing hypothalamic NPY effects may help to ameliorate VPA-induced weight gain and obesity without interfering with the desired central effects of VPA.

Topics: Amitriptyline; Anticonvulsants; Cell Line, Tumor; Cell Survival; Gene Expression; Humans; Neuroblastoma; Neuropeptide Y; Tetradecanoylphorbol Acetate; Valproic Acid

This study was aimed to investigate the potential neuroprotective effect of neuropeptide Y (NPY) on the survival of dopaminergic cells in both in vitro and in animal models of Parkinson's disease (PD). NPY protected human SH-SY5Y dopaminergic neuroblastoma cells from 6-hydroxydopamine-induced toxicity. In rat and mice models of PD, striatal injection of NPY preserved the nigrostriatal dopamine pathway from degeneration as evidenced by quantification of (1) tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta, levels of (2) striatal tyrosine hydroxylase and dopamine transporter, (3) dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) as well as (4) rotational behavior. NPY had no neuroprotective effects in mice treated with Y(2) receptor antagonist or in transgenic mice deficient for Y(2) receptor suggesting that NPY effects are mediated through this receptor. Stimulation of Y(2) receptor by NPY triggered the activation of both the ERK1/2 and Akt pathways but did not modify levels of brain derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor. These results open new perspectives in neuroprotective therapies using NPY and suggest potential beneficial effects in PD.

Topics: Adrenergic Agents; Analysis of Variance; Animals; Animals, Newborn; Arginine; Autoradiography; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Enzyme Inhibitors; Female; Functional Laterality; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuroblastoma; Neurodegenerative Diseases; Neuropeptide Y; Neuroprotective Agents; Nortropanes; Oligopeptides; Oxidopamine; Parkinson Disease; Protein Binding; Rats; Rats, Wistar; Receptors, Neuropeptide Y; Substantia Nigra; Tyrosine 3-Monooxygenase

In the central nervous system, several neuropeptides are believed to be involved in the pathophysiology of Alzheimer's disease (AD). Among them, neuropeptide Y (NPY) is a small peptide widely distributed throughout the brain, where it serves as a neurotransmitter and/or a modulator of several neuroendocrine functions. More recently, NPY has generated interest because of its role in neuroprotection against excitotoxicity and modulation of neurogenesis. Interestingly, these effects are also influenced by neurotrophins, critical molecules for the function and survival of neurons that degenerate in AD.. Our purpose was to investigate whether NPY might be a neuroprotective agent in AD and whether neurotrophins are involved in NPY-induced neuroprotection.. To test this hypothesis, we exposed the SH-SY5Y neuroblastoma cell line to toxic concentrations of β-amyloid (Aβ) peptide fragment 25-35 (Aβ(25-35)) and measured cell survival and neurotrophin expression before and after a preincubation with NPY in the growth medium.. Our results demonstrated that preincubation with NPY prevented cell loss due to the toxic effect of Aβ(25-35). Moreover, while intracellular production of nerve growth factor and brain-derived neurotrophic factor were reduced by Aβ, NPY restored or even increased neurotrophin protein and mRNA in SH-SY5Y cells.. In conclusion, this study demonstrates that NPY increases the survival of SH-SY5Y neuroblastoma cells and counteracts the toxic effect of Aβ. In addition, NPY restores the neurotrophin levels in these cells. Although preliminary, these observations might be useful to understand the pathology of Alzheimer's and/or develop new therapeutic strategies.

Topics: Amyloid beta-Peptides; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Survival; Humans; Nerve Growth Factor; Nerve Growth Factors; Neuroblastoma; Neuropeptide Y; Neuroprotective Agents; Peptide Fragments

We describe the recombinant production of the human Y(1) receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y(1) receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Topics: Animals; Binding, Competitive; Brain Chemistry; Cattle; Cell Line, Tumor; Escherichia coli; Humans; Membrane Lipids; Neuroblastoma; Neuropeptide Y; Phosphorus Isotopes; Protein Folding; Receptors, Neuropeptide Y; Recombinant Proteins; Swine; Tritium

Chemokines expressed in neurons are important mediators in neuron-neuron and neuron-glia signaling. One of these chemokines is CCL21 that activates microglia via the chemokine receptor CXCR3. As neurons also express CXCL10, a main ligand for CXCR3, we have thus investigated in detail the expression pattern of CXCL10 in neurons. We show that CXCL10 is constitutively expressed by neurons, is stored in large dense-core vesicles and is not regulated by neuronal injury or stress. Neuronal CXCL10 release occurred constitutively at low level. In vivo CXCL10 expression was found in the developing brain at various embryonic stages and its peak expression correlates with the presence of CD11b- and GFAP-positive cells expressing CXCR3. These results suggest a possible role of neuronal CXCL10 in recruitment and homing of glial cells during embryogenesis.

Topics: Amyloid beta-Peptides; Animals; CD11b Antigen; Cells, Cultured; Cerebral Cortex; Chemokine CXCL10; Coculture Techniques; Embryo, Mammalian; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Glioma; Glutamic Acid; Green Fluorescent Proteins; Humans; Immunoprecipitation; Lipopolysaccharides; Mice; Microscopy, Immunoelectron; Neuroblastoma; Neuroglia; Neurons; Neuropeptide Y; Peptide Fragments; RNA, Messenger; Sodium Azide; Sodium Chloride; Synaptic Vesicles; Time Factors; Transfection; Vesicle-Associated Membrane Protein 2

Neuroblastomas are pediatric tumors that develop from sympathetic precursors and express neuronal proteins, such as neuropeptide Y (NPY). NPY is a sympathetic neurotransmitter acting via multiple receptors (Y1-Y5R). Both NPY and Y2Rs are commonly expressed in neuroblastoma cell lines and tissues. The peptide secreted from neuroblastomas stimulates tumor cell proliferation and angiogenesis. As both processes are Y2R-mediated, the aim of this study was to assess Y2R as a potential therapeutic target for neuroblastoma. In vitro, Y2R antagonist (BIIE0246) prevented activation of p44/42 mitogen-activated protein kinase (MAPK) induced by endogenous NPY, which resulted in decreased proliferation and induction of Bim-mediated apoptosis. Similar growth-inhibitory effects were achieved with NPY small interfering RNA (siRNA) and Y2R siRNA. In vivo, Y2R antagonist significantly inhibited growth of SK-N-BE(2) and SK-N-AS xenografts, which was associated with decreased activation of p44/42 MAPK, as well as reduced proliferation (Ki67) and increased apoptosis (TdT-mediated dUTP nick end labeling; TUNEL). The Y2R antagonist also exerted an antiangiogenic effect. In vitro, it reduced the proliferation of endothelial cells induced by neuroblastoma-conditioned media. Consequently, the Y2R antagonist-treated xenografts had decreased vascularization and a high degree of focal fibrosis. In human neuroblastoma tissues, the expression of Y2R was observed in both tumor and endothelial cells, while NPY was predominantly expressed in neuroblastoma cells. In summary, Y2R is a promising new target for neuroblastoma therapy affecting both cancer cells and tumor vasculature.

Topics: Animals; Apoptosis; Arginine; Benzazepines; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neovascularization, Pathologic; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Xenograft Model Antitumor Assays

Preproneuropeptide Y is a precursor peptide to mature neuropeptide Y (NPY), which is a universally expressed peptide in the central and peripheral nervous system. NPY is normally routed to endoplasmic reticulum and secretory vesicles in cells, which secrete NPY. In our previous studies, we found a functional Leucine7 to Proline7 (L7P) polymorphism in the signal peptide sequence of preproNPY. This polymorphism affects the secretion of NPY and causes multiple physiological effects in humans. The sequence of NPY mRNA contains two in frame kozak sequences that allow translation initiation to shift, and translation of two proteins. In addition to mature NPY(1-36) also a putative truncated NPY(17-36) with mitochondrial targeting signal is produced. The purpose of this study was to investigate the protein mobility of the putative mitochondrial fragment and the effect of the L7P polymorphism on the cellular level using GFP tagged constructs. The mobility was studied with fluorescence recovery after photobleaching technique in a neuronal cell line. We found that the mobility of the secretory vesicles with NPY(1-36) in cells with L7P genotype was increased in comparison to vesicle mobility in cells with the more abundant L7L genotype. The mobility in the cells with the putative mitochondrial construct was found to be very low. According to the results of the present study, the mitochondrial truncated peptide stays in the mitochondrion. It can be hypothesized that this could be one of the factors affecting energy balance of the membranes of the mitochondrion.

Topics: Cell Line, Tumor; Green Fluorescent Proteins; Humans; Leucine; Microscopy, Confocal; Mitochondria; Neuroblastoma; Neurons; Neuropeptide Y; Polymorphism, Genetic; Proline; Protein Transport; Transfection

To elucidate more precisely the biological characteristics of neuroblastomas, we examined four human neuroblastomas heterotransplanted into athymic nude mice NB-39 (undifferentiated type), NB-45 (poorly differentiated type with undifferentiated component), NB-52 (poorly differentiated type), and NB-726 (differentiating type) by electron microscopy, immunohistochemistry, and radioimmunoassay for the peptides in tumors. Ultrastructurally, NB-45, NB-52, and NB-726 contained more numerous and variously sized neurosecretory granules than did NB-39. Immunohistochemistry revealed neurofilament proteins, tyrosine hydroxylase, neuropeptide Y (NPY), and chromogranin A-positive cells in the four tumors in the following order of frequency: NB-726, NB-45, NB-52, and NB-39. NB-726, NB-45, and NB-52, but not NB-39, contained galanin-positive tumor cells. NB-45 and NB-726 harbored a few positive cells for calcitonin gene-related peptide. Furthermore, NB-726 exhibited positivity to leu-enkephalin, met-enkephalin, vasoactive intestinal peptide (VIP), and serotonin. Radioimmunoassay substantiated the results of immunohistochemistry, showing NPY in all tumors and either galanin or VIP in three tumors, excepting NB-39. Average doubling time of the tumor was as follows: 2 days in NB-39, 10 days in NB-45, 22 days in NB-52, and 45 days in NB-726. These results indicate that human neuroblastoma cells have different biological characteristics and reduced growth rate with differentiation in terms of ultrastructure and of peptide production abilities.

Topics: Animals; Calcitonin Gene-Related Peptide; Child; Child, Preschool; Enkephalin, Leucine; Enkephalin, Methionine; Female; Galanin; Humans; Infant; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Neuropeptide Y; Secretory Vesicles; Serotonin; Transplantation, Heterologous; Tyrosine 3-Monooxygenase; Vasoactive Intestinal Peptide

The biosynthesis and release of neuropeptide Y (NPY) is regulated by several factors. Here, the effect of the muscarinic agonist carbachol on NPY biosynthesis and release was analyzed utilizing the SH-SY5Y human neuroblastoma cell line. We observed that: (a) carbachol moderately increased the post-translational cleavage of proNPY to NPY; (b) carbachol treatment stimulated NPY accumulation into the medium in a time- and dose-related manner; (c) protein kinase C activation is involved in carbachol-mediated NPY synthesis/release (>6h). In conclusion, the present observations support the hypothesis that muscarinic receptor activation regulates the biosynthesis and secretion of NPY.

Topics: Carbachol; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Neuroblastoma; Neuropeptide Y; Protein Kinase C; Time Factors

Activation of the NPFF(2) receptor reduces the inhibitory effect of opioids on the N-type Ca(2+) channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and alpha(2)-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF(2) receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca(2+) transient by activation of G(i)-coupled receptors was either unaffected (micro-opioid), increased (NPY), reduced (NPFF(2)) or lost (alpha(2)-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the micro-opioid and NPFF(2) receptors to the G(i/o) proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.

Topics: Calcium; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Neuroblastoma; Neuropeptide Y; Oligopeptides; Receptors, Neuropeptide; Receptors, Opioid; Staurosporine

To correlate the immunophenotype of metastatic cells in the bone marrow of patients with neuroblastoma with early treatment failure.. The studies were performed on bone marrow material obtained from children treated in the department of paediatric oncology, haematology, and transplantology, Poznan University of Medical Sciences, Poland from 1996 to 2003. Immunocytochemical analysis of nervous tissue markers (using the immunomax technique) was performed on 108 bone marrow preparations obtained from 36 children diagnosed with neuroblastoma (stage IV with bone marrow metastases). The analysis included expression of PGP 9.5 protein, substance P, chromogranin A, bombesin, galanin, neuropeptide Y (NPY), and vasoactive intestinal peptide in neuroblastoma metastatic cells defined by the expression of neurone specific enolase.. Nineteen relapses occurred within 12 months of the end of treatment. Correlation between the various markers studied and early treatment failure, using Fisher's exact test, revealed that chromogranin A and NPY are strong indicators of an unfavourable prognosis in patients with stage IV neuroblastoma (p < 0.001 and p < 0.0002, respectively).. Determination of metastatic cell immunophenotypes in bone marrow (particularly chromogranin A and NPY) may help establish the short term prognosis in children with neuroblastoma.

Topics: Adolescent; Biomarkers, Tumor; Bone Marrow Examination; Bone Marrow Neoplasms; Child; Child, Preschool; Chromogranin A; Chromogranins; Humans; Immunophenotyping; Neoplasm Proteins; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Prognosis; Treatment Failure

Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be potently angiogenic and growth promoting for endothelial, vascular smooth muscle and neuronal cells. NPY and its cognate receptors, Y1, Y2 and Y5, are expressed in neural crest-derived tumors; however, their role in regulation of growth is unknown. The effect of NPY on the growth and vascularization of neuroendocrine tumors was tested using three types of cells: neuroblastoma, pheochromocytoma, and Ewing's sarcoma family of tumors (ESFT). The tumors varied in expression of NPY receptors, which was linked to differential functions of the peptide. NPY stimulated proliferation of neuroblastoma cells via Y2/Y5Rs and inhibited ESFT cell growth by Y1/Y5-mediated apoptosis. In both tumor types, NPY receptor antagonists altered basal growth levels, indicating a regulatory role of autocrine NPY. In addition, the peptide released from the tumor cells stimulated endothelial cell proliferation, which suggests its paracrine angiogenic effects. In nude mice xenografts, exogenous NPY stimulated growth of neuroblastoma tumors, whereas it increased apoptosis and reduced growth of ESFT. However, in both tumors, NPY treatment led to an increase in tumor vascularization. Taken together, this is the first report of NPY being a growth-regulatory factor for neuroendocrine tumors, acting both by autocrine activation of tumor cell proliferation or apoptosis and by angiogenesis. NPY and its receptors may become targets for novel approaches in the treatment of these diseases, directed against both tumor cell proliferation and angiogenesis.

Topics: Animals; Apoptosis; Cell Cycle; Cell Proliferation; Culture Media, Conditioned; Endothelial Cells; Humans; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Neuroblastoma; Neuropeptide Y; Pheochromocytoma; Rats; Receptors, Neuropeptide Y; Transplantation, Heterologous

Human SMS-KAN neuroblastoma cells endogenously express the neuropeptide Y (NPY) type 2 (Y(2)) receptor. Although ligand binding and GTPgammaS binding studies supported high functional Y(2) receptor expression, only weak coupling to the natural second messenger cyclic AMP was observed. The main reason was the low responsiveness of SMS-KAN cells to forskolin, a direct activator of adenylyl cyclases. In order to obtain a cell-based functional assay for the Y(2) receptor in SMS-KAN cells, the transient calcium (Ca(2+)) mobilization assay in the fluorimetric imaging plate reader (FLIPR) format was established by stably expressing a chimeric G protein Gq(i9). This manipulation resulted in robust mobilization of Ca(2+) after challenge with various NPY-related agonists in a 384-well format. The sensitivity of the FLIPR readout was in the low nanomolar range for NPY agonists and comparable to that of the recombinant Y(2) receptor. The selective Y(2) antagonist BIIE0246 competitively inhibited NPY-mediated Ca(2+) transients in SMS-KAN/Gq(i9) cells with a pA(2) value of 7.39+/-0.1. This is the first evidence that an endogenously expressed G protein-coupled receptor couples to an overexpressed chimeric G protein, thereby functionally responding in the FLIPR readout.

Topics: Calcium; Cell Line, Tumor; Colforsin; Cyclic AMP; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y; Recombinant Fusion Proteins

Expression of neuropeptide Y (NPY) receptors in human SK-N-MC neuroblastoma cells was investigated. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that Y1, Y4, and Y5 receptors were expressed in these cells. Expression was confirmed by Western blot and immunocytochemistry demonstrated abundant presence of all three receptors on cell membranes. NPY peptide was also expressed in these cells, but other members of the larger peptide family (peptide YY and pancreatic polypeptide) were not expressed. Incubation with U0126, a specific mitogen-activated protein kinase (MAPK) inhibitor, decreased cell number in serum-free medium culture. Since NPY activates MAPK via different subtypes of NPY receptors, results suggest that endogenously expressed NPY may control proliferation of these cells through a paracrine/autocrine mechanism.

Topics: Autocrine Communication; Butadienes; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neuroblastoma; Neuropeptide Y; Nitriles; Paracrine Communication; Receptors, Neuropeptide Y

Neuropeptide Y (NPY) is universally expressed in many different neuronal and non-neuronal cells. Human NPY gene has two in-frame kozak sequences and thus, has potentially two translation initiation sites producing two NPY peptides with different molecular weights. In the present study, the intracellular location of NPY was studied in endothelial cells endogenously expressing NPY, and in neuronal (SK-N-BE) and non-neuronal (CHO-K1) cells transfected with NPY-GFP-constructs. By mutating kozak sequences we discovered that kozak-1 directs the NPY peptide to secretory vesicles, and kozak-2 is a prerequisite for mitochondrial targeting. If both kozak sequences are present, non-neuronal cells seem to benefit leaky scanning to initiate translation at both initiation sites, in contrast to neuronal cells, which prefer the kozak-1. This finding suggests that both the kozak sequences of NPY mRNA can be used in the translation depending on the cell type. The size and the function of the novel NPY fragment routed to mitochondria remains to be determined.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Membrane; Endothelial Cells; Fibroblasts; Humans; Intracellular Space; Mitochondria; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Neurotransmitter Agents; Protein Transport; Receptors, Neuropeptide Y; Secretory Vesicles; Signal Transduction

Neuroblastoma represents one of the most frequently developing malignant solid tumours in children. At the time of diagnosis, in more than half of the cases, metastatic cells are also present in the bone marrow. The present study was aimed at immunocytochemical analysis of selected neuropeptide manifestation in metastatic cells of neuroblastoma in bone marrow and at comparing the obtained results with the immunophenotype of parental neuroblastoma cells. The studies were performed on bone marrow material obtained from children treated at the Department of Paediatric Haematology and Oncology, University of Medical Sciences, Poznań, Poland, in 1998-2000. Immunocytochemical analysis of nervous tissue markers (employing the immunomax technique) involved 36 bone marrow preparations obtained from 27 children. The analysis included expression of neuron-specific enolase (NSE), PGP 9.5 protein, substance P (SP), chromogranin A (ChA), bombesin (B), galanin (G), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). Close to 90% metastatic cells in bone marrow were found to exhibit NSE+SP+B+ phenotype and over a half of the cells manifested additionally expression of PGP 9.5+ChA+NPY+. Comparison of the obtained results with the immunophenotype of neuroblastoma cells obtained directly from the primary tumour demonstrated high correlation of NSE, SP and PGP 9.5 expression. Due to the relative ease of obtaining the bone marrow material and absence of neuromarkers in bone marrow metastatic cells in solid tumours other than neuroblastoma, determination of immunophenotype of the cells may represent a valuable supplementation in preliminary diagnosis of this tumour in children.

Topics: Adolescent; Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Child; Child, Preschool; Chromogranin A; Chromogranins; Female; Galanin; Humans; Immunohistochemistry; Immunophenotyping; Male; Neoplasm Metastasis; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Substance P; Vasoactive Intestinal Peptide

Von Hippel-Lindau (VHL) tumor suppressor protein is expressed in neurons of the central nervous system and plays an important role during the neuronal differentiation of central nervous system progenitor cells. To elucidate the neuronal differentiating potential of VHL protein in neuroblastoma cells, we overexpressed or inhibited VHL protein in human neuroblastoma cells (SY-SH5Y), and examined the morphological change, expressions of neuronal markers, and electrophysiological functions. Here we show that with VHL gene transduction SY-SH5Y cells stably expressing the VHL protein had neurite-like processes with varicosities, showed the distinct expression of the neuronal markers neuropeptide Y and neurofilament 200, acquired regulated neurosecretion competence in response to depolarizing and cholinergic stimuli, and had large voltage-gated fast sodium currents and delayed rectifier potassium (Kv) currents compatible with those of functional neurons. In addition, they displayed inactivated ether-á-go-go potassium channels related to the promotion of the cell cycle and to the termination of differentiation. Also, by treatment with retinoic acid, they rapidly underwent cell death related to apoptosis. These findings suggest that the induction of neuronal function by VHL protein is associated with down-regulation of the cell cycle. In contrast, the inhibition of endogenous expression of VHL protein with antisense-orientated VHL gene transduction reduced such neuronal properties inherent to these cells, including the capacity for activation of ether-á-go-go channels. In conclusion, VHL protein has a neuronal differentiating potential to transform neuroblastoma cells into functional neuron-like cells. Our finding of the neuronal differentiation of neuroblastoma cells under the control of the VHL gene may contribute to the development of clinical techniques for neuronal regeneration in the case of intractable neuronal diseases and for differentiation therapy against neuroblastomas.

Topics: Apoptosis; Cell Differentiation; Cell Transformation, Neoplastic; Down-Regulation; Ether-A-Go-Go Potassium Channels; Genetic Therapy; Humans; Ion Channel Gating; Ligases; Neuroblastoma; Neurofilament Proteins; Neurons; Neuropeptide Y; Potassium Channels; RNA, Messenger; Sodium Channels; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1 alpha and HIF-2 alpha are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2 alpha in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1 alpha and HIF-2 alpha proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Cell Hypoxia; DNA-Binding Proteins; Down-Regulation; Endothelial Growth Factors; Female; Gene Expression; HeLa Cells; Helix-Loop-Helix Motifs; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin-Like Growth Factor II; Lymphokines; Mice; Mice, Nude; Neoplasms, Experimental; Neural Crest; Neuroblastoma; Neuropeptide Y; Oxygen; Paraganglia, Chromaffin; Phenotype; Sympathetic Nervous System; Trans-Activators; Transcription Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A good in vitro model within which to investigate molecular interactions between feeding relevant neuropeptide systems has been lacking. Consequently, we began using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen various neuronal cell lines for the presence of feeding relevant neuropeptides and receptors. N1E-115 murine neuroblastoma cells have emerged as an attractive candidate for further analysis because they contain mRNA for a variety of key systems implicated in the regulation of energy homeostasis.

Topics: Agouti-Related Protein; Animals; Energy Metabolism; Intercellular Signaling Peptides and Proteins; Leptin; Mice; Nerve Tissue Proteins; Neuroblastoma; Neuropeptide Y; Neuropeptides; Pro-Opiomelanocortin; Proteins; Receptor, Insulin; Receptor, Melanocortin, Type 3; Receptor, Melanocortin, Type 4; Receptors, Corticotropin; Repressor Proteins; RNA, Messenger; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transcription Factors; Tumor Cells, Cultured

The human neuroblastoma cell line SH-SY5Y can differentiate into a functional sympathetic neuronal phenotype when treated with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. When TrkA is introduced into the cells, NGF also induces differentiation. In both cases, protein kinase C (PKC) is pivotal for induction and maintenance of the differentiated phenotype. We have recently shown that PKC activity is needed to enable the MAPK ERK to accumulate in the nucleus of SH-SY5Y cells and hence activate transcription. To find out whether this could be one reason for the PKC dependency in the differentiation process we have investigated the role of ERK during neuronal differentiation of these cells. The results show that ERK was needed for full upregulation of the neuronal marker genes NPY and GAP-43. However, ERK activity was not necessary for TPA-induced neurite formation. Neither was activation of ERK sufficient to promote neurite outgrowth. The results clearly show that there was no correlation between nuclear ERK activity, measured as SRE transactivation, and neurite formation in TPA-differentiated SH-SY5Y neuroblastoma cells.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Flavonoids; GAP-43 Protein; Gene Expression; Humans; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Mitogens; Neurites; Neuroblastoma; Neuropeptide Y; Nuclear Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-fos; ras Proteins; Receptor, trkA; Serum Response Factor; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCbeta isoforms in this process. The PKCbeta-specific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCbeta inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCbeta isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.

Topics: Cell Differentiation; Culture Media, Serum-Free; Enzyme Inhibitors; Flavonoids; GAP-43 Protein; Gene Expression Regulation; Genes, Reporter; Isoenzymes; Mesylates; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurites; Neuroblastoma; Neurons; Neuropeptide Y; Phosphorylation; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C beta; Pyrroles; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Retinoids are involved in the regulation of development and differentiation in many tissues, including the nervous system, where they have been associated with some neurotransmitter systems. In the present study, we evaluated the effects of all-trans retinoic acid (RA) on the biosynthesis and secretion of neuropeptide Y (NPY), a widely expressed neuroregulatory peptide. The SH-SY5Y human neuroblastoma cell line has been used as the in vitro model system. Treatment with 10 microM RA induced a marked decrease in NPY gene expression after as little as 3-6 h of incubation and resulted in its almost complete suppression at 12-24 h and after a 6-day differentiating treatment. The NPY content in cell extracts and the NPY secreted and accumulated in the culture medium were also reduced by exposure to 10 microM RA at 12 and 24 h and at 6 days. Moreover, RA treatment for 6 days, but not for 24 h, resulted in a marked stimulation of proNPY processing to mature NPY. The presence of negative retinoic acid-response elements in the human NPY promoter (up to -1078 bp) was excluded by a computer search. When SH-SY5Y cells were treated simultaneously with 20 nM TPA and 10 microM RA for 24 h, the marked stimulatory effect of TPA alone was completely suppressed. These observations suggest that the expression of NPY in SH-SY5Y human neuroblastoma cells is negatively regulated by RA at the level of gene expression, probably by mechanisms involving the interaction of activated RARs with transcription factors (such as AP-1).

Topics: Blotting, Northern; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Neuropeptide Y; Protein Precursors; Protein Processing, Post-Translational; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Neuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1-36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1-36) and the truncated form NPY (3-36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (n = 6), (41%, 12-100%, median, range), compared to the less aggressive stage 1, 2 and 4S tumours (n = 12), (93%; 69-100%), (P= 0.012). ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue.

Topics: Child; Child, Preschool; Chromatography, Gel; Chromatography, High Pressure Liquid; Female; Humans; Infant; Infant, Newborn; Male; Mass Spectrometry; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Protein Precursors; Protein Processing, Post-Translational; Sequence Analysis; Survival Rate

Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein-coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK-N-MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY were resistant to acid removal of cell surface-bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand-receptor complex is collected in pits and caveolae followed by endocytosis.

Topics: Amino Acid Sequence; Endocytosis; Humans; Microscopy, Confocal; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Protein Binding; Radioligand Assay; Receptors, Neuropeptide Y; Tumor Cells, Cultured

Previous studies in our laboratory using rat brain tissue have shown that neuropeptide Y (NPY) can enhance NMDA- and potassium-stimulated dopamine release from various brain regions and that this enhancement is reversed by sigma (sigma) receptor antagonists. In the current study, we sought to determine whether SH-SY5Y cells are suitable for investigating sigma receptor effects and whether any sigma receptors present are of the subtype responsive to NPY. We compare mechanisms by which the prototypical sigma receptor agonist (+)-pentazocine, and the proposed endogenous sigma receptor ligand NPY regulate potassium-stimulated [(3)H]dopamine release from SH-SY5Y cells. Both (+)-pentazocine and NPY inhibit potassium-stimulated [(3)H]dopamine release. Unlike our studies in rat brain tissue, the effect of NPY on [(3)H]dopamine release is not reversed by sigma receptor antagonists. SH-SY5Y cells appear to be an appropriate model to study the regulation of dopamine release by sigma receptors or by NPY receptors, but this population is not identical to that population identified in brain slices.

Topics: Analgesics, Opioid; Animals; Cyclohexylamines; Dopamine; Drug Interactions; Humans; Models, Neurological; Narcotic Antagonists; Neuroblastoma; Neuropeptide Y; Pentazocine; Potassium; Pyrrolidines; Receptors, sigma; Tumor Cells, Cultured

The objective of our study was to assess the gene expression of the antiproliferative systems neuronal nitric oxide synthase (nNOS) and adrenomedullin (AM) in human neuroblastoma. A novel real-time PCR method was evaluated using neuropeptide Y (NPY) for validation. Glyceraldehyd-3-phospate dehydrogenase (GAPDH) and NPY gene expression in neuroblastomas of 50 patients were measured in parallel by competitive quantitative and TaqMan real-time RT-PCR. AM and nNOS mRNA were determined by real-time PCR. Our results showed a linear relationship between competitive quantitative and real-time RT-PCR measurements of NPY and GAPDH (r = 0.87 and r = 0.92, respectively). AM and nNOS mRNA was found in all tumor samples. AM/GAPDH mRNA increased with higher differentiation according to Shimada (p = 0.013). There was no relation between MYCN amplification nor with the tumor stage (p = 0.78 and p = 0.30, respectively). AM/GAPDH did not relate to recurrence or death in a 5-year follow-up period. Neuronal NOS/GAPDH expression did not relate to any biological or clinical parameter of prognosis or differentiation. Similar results were obtained when the neuronal marker protein gene product 9.5 (PGP9.5) was used to normalize mRNA concentration. In conclusion, TaqMan real-time PCR appears to be a reliable method to quantify gene expression in neuroblastomas. Adrenomedullin mRNA in neuroblastoma is linked to tumor differentiation but not to prognostic markers.

Topics: Adolescent; Adrenomedullin; Child; Child, Preschool; Gene Expression Regulation, Neoplastic; Germany; Humans; Infant; Infant, Newborn; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Peptides; Polymerase Chain Reaction; Predictive Value of Tests; Recurrence; Reproducibility of Results; Survival Rate; Transcription, Genetic

Several studies have shown that neuropeptide Y (NPY) is involved in the stimulation of gonadotropin hormone releasing hormone (GnRH) and luteinizing hormone (LH) secretion and that these effects are modulated by gonadal steroid feedback. The NPY regulation of GnRH release is probably mediated by the activation of the Y(1) receptor subtype. In this study we examined the regulation of the Y(1) receptor gene transcription by estrogens in transiently transfected NG108-15 neuroblastoma glioma cells. A chimeric plasmid containing the murine Y(1) receptor promoter fused to the firefly luciferase reporter gene was induced by approximately 2-fold in response to 17 beta-estradiol treatment. The estrogen-mediated enhancement of luciferase activity was dose-dependent, blocked by the estrogen receptor (ER) antagonist ICI 182,780, and was strictly dependent on the presence of ER alpha, since it occurred only in NG108-15 cells cotransfected with an expression vector for the human ER. Mutational analysis was performed to investigate whether the hemipalindromic estrogen-responsive elements (EREs) flanking the Y(1) receptor gene are responsible for conferring estradiol inducibility to the Y(1) receptor gene promoter. Mutation of the ERE1 half site at position -932, or mutation of the ERE2 half site at position -809, relative to the ATG, failed to affect the 17 beta-estradiol-mediated enhancement of luciferase activity. Conversely, mutation of both ERE1 and ERE2 half sites completely abolished activation of luciferase activity induced by estrogen. We also examined whether 17 beta-estradiol stimulates the transcriptional activity of the Y(1) receptor gene by binding to ER beta. Results demonstrated that luciferase activity was not modulated by estrogens when cells were transfected with the expression plasmid bearing the human ER beta. Moreover coexpression of both ER alpha and ER beta completely abolished the estrogen-induced activation of luciferase activity observed in the presence of ER alpha. Our data suggest that estrogens activate Y(1) receptor gene transcription possibly via a direct interaction of ER alpha with the hemipalindromic EREs flanking the Y(1) receptor gene.

Topics: Animals; DNA Primers; Estradiol; Estrogen Receptor alpha; Gene Expression Regulation; Genes, Reporter; Glioma; Luciferases; Mice; Mutagenesis; Neuroblastoma; Neuropeptide Y; Promoter Regions, Genetic; Rats; Receptors, Estrogen; Receptors, Neuropeptide Y; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The mammalian achaete-scute homologue, MASH-1, is crucial for early development of the sympathetic nervous system and is transiently expressed in sympathetic neuroblasts during embryogenesis. Here we report that the human homologue (HASH-1) was expressed in all analyzed cell lines (6/6) derived from the sympathetic nervous system tumor neuroblastoma. The majority of small-cell lung carcinoma (4/5) cell lines tested expressed HASH-1, while other nonneuronal/non-neuroendocrine cell lines were negative. Induced differentiation of neuroblastoma cells resulted in HASH-1 downregulation. This occurred concomitant with induction of neurite outgrowth and expression of the neuronal marker genes GAP-43 and neuropeptide Y. Constitutive expression of exogenous HASH-1 did not alter the capacity of the neuroblastoma cells to differentiate in response to differentiation-inducing agents. It is concluded that moderate HASH-1 expression does not compromise the capacity of these cells to differentiate.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Northern; Carcinoma, Small Cell; Carrier Proteins; Cell Differentiation; Cell Size; DNA-Binding Proteins; Down-Regulation; GAP-43 Protein; Growth Substances; Humans; Lung Neoplasms; Membrane Proteins; Neurites; Neuroblastoma; Neurons; Neuropeptide Y; Receptor, trkA; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

To elucidate the possibility of utilizing protein kinase C (PKC) isoforms as target genes in neuroblastoma therapy, 5 neuroblastoma cell lines and neuroblastoma tumor specimens were examined for PKC isoform expression pattern and the cell lines were analyzed for sensitivity to PKC inhibition. All cell lines [IMR-32, LAN-2, LAN-5, SH-SY5Y and SK-N-BE(2)] expressed alpha, betaII, delta and epsilon isoforms of PKC, while no PKCeta or theta protein was detected in any cell line. PKCgamma was found only in LAN-2 cells. PKCalpha, betaII and delta were detected in 5 neuroblastoma tumors and PKCepsilon in 4 out of 5 tumors. Exposure to the PKC inhibitors GF109203X, Gö 6976 or Gö 6983 caused a decrease whereas activation of PKC with 12-O-tetradecanoyl phorbol 13-acetate caused an increase in the number of neuroblastoma cells. The effect of Gö 6976 was due to both inhibited proliferation and to increased apoptosis. While GF109203X suppressed neurite outgrowth induced by a growth factor combination, Gö 6976 potentiated neurite outgrowth. Our data suggest a role for classical PKC isoforms in neuroblastoma growth and survival and for novel isoforms in neurite outgrowth.

Topics: Cell Differentiation; Cell Division; Cell Survival; Fibroblast Growth Factor 2; GAP-43 Protein; Humans; Isoenzymes; Neuroblastoma; Neuropeptide Y; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.

Topics: Amino Acid Sequence; Animals; Blotting, Western; CHO Cells; Cricetinae; Cross-Linking Reagents; Humans; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Photoaffinity Labels; Photochemistry; Receptors, Neuropeptide Y; Transfection; Tumor Cells, Cultured

Neuropeptide Y (NPY) participates in the control of several functions in the nervous system. NPYergic neurons present in brain areas involved in cognitive processes are linked to ascending projections of the cholinergic system, a finding that suggests a role for acetylcholine in the control of these cells. In the present study, the effect of the activation of cholinergic muscarinic receptors on the expression of the human NPY gene was assessed. The SH-SY5Y neuroblastoma cell line was used as an in vitro model of human neurons; NPY mRNA levels were evaluated by Northern blot analysis. The results indicate that: (a) the expression of the human NPY gene in SH-SY5Y cells is stimulated by the cholinergic muscarinic agonist, carbachol; (b) this effect is mediated by the M3 muscarinic receptor subtype, as indicated by the inhibitory effect of the M3 antagonist 4-DAMP; (c) protein kinase C (PKC) activation plays an important role in the induction of NPY gene expression in this system, as suggested by experiments with the PKC activator, TPA, and the PKC inhibitor, GF 109203X; (d) the stimulation of NPY mRNA levels by TPA and by carbachol in SH-SY5Y cells requires de novo synthesis of RNA and protein. In conclusion, the present study shows that the activation of PKC-coupled muscarinic receptors as the M3 subtype positively modulates the expression of the human NPY gene in SH-SY5Y neuroblastoma cells, via PKC-related mechanisms.

Topics: Carbachol; Dactinomycin; Gene Expression Regulation; Humans; Muscarinic Agonists; Neuroblastoma; Neuropeptide Y; Nucleic Acid Synthesis Inhibitors; Protein Kinase C; Receptors, Muscarinic; RNA, Messenger; Tumor Cells, Cultured

The objective of this study was to relate plasma neuropeptide Y (NPY) concentrations of patients with neuroblastoma with the stage of the disease, the patients' age, the prognosis, the tumor mRNA expression and with the effect on tumor cell proliferation. Plasma NPY of 85 patients with neuroblastoma was measured by radioimmunoassay. The patients' median age was 18 months (range, three weeks-12 years). Ten children with childhood tumors that did not affect neurological or neuroendocrine structures and ten healthy children served as control groups. NPY mRNA expression in neuroblastoma tissue was assessed by Northern blot analysis. Proliferation of neuroblastoma cells (SK-N-MC and CHP 234) was evaluated by 5-bromo-2'-deoxyuridine incorporation during DNA synthesis in vitro. Plasma NPY levels were significantly higher in stage 3 (p < 0.05), 4 (p < 0.001) and 4S (p < 0.05) patients than in both control groups. Plasma levels above 8 pmol/l were only seen in stages 2 (17%), 3 (32%), 4 (45%) and 4S (44%). Seven of the 12 patients (58%) who died had NPY levels above 8 pmol/l (vs. 29% in survivors; p = 0.05). In patients with longer follow-up monitoring, relapse coincided with increasing NPY levels. There was no relationship between the patients' age and their plasma NPY concentrations (r = 0.08; p = 0.49). No relation was found between NPY mRNA expression in tumor tissue and NPY plasma concentrations of the ten patients (r = 0.08; p = 0.81). NPY in the supernatant of neuroblastoma cells did not alter the 5-bromo-2'-deoxyuridine incorporation during DNA synthesis. In summary, NPY plasma concentrations in patients with neuroblastoma relate to the stage of the disease. The relation to the prognosis is at the threshold of significance. No relation between tissue and plasma NPY, nor any effect of NPY on proliferation of tumor cells was found.

Topics: Age Factors; Case-Control Studies; Cell Division; Child; Child, Preschool; Gene Expression; Humans; Infant; Infant, Newborn; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Prognosis; Prospective Studies; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Four NPY receptor subtypes have been cloned, and shown to be coupled to both Ca2+ and cAMP. However, very little is known about the downstream elements mediating NPY actions. It has recently been demonstrated in our laboratory that intrahypothalamic (i.h.t.) administration of NPY induces hypothalamic CaM kinase activity, cyclic AMP response element binding protein (CREB) phosphorylation and cyclic AMP response element (CRE) binding activity in rat hypothalamic nuclear proteins. In the present study, we have investigated whether these changes in CRE binding transcriptional factors activated by NPY results in gene regulation using a human neuroblastoma cell line (SK-N-BE2). This cell line which expresses the Y2 subtype of NPY receptors was transfected with a fusion gene containing 1.305 kb of human CRF 5' flanking region with a perfect palindromic CRE site linked to firefly luciferase gene. NPY treatment increased CaM kinase II activity, CREB phosphorylation and CRE binding in these cells. In transfected cells, luciferase activity was also increased by NPY (1.8-4-fold) within 4 h of treatment. Moreover, forskolin (7-30-fold), which stimulates cAMP production, and thapsigargin (6-8-fold), which mobilizes intracellular calcium, also increased luciferase activity within 4 h of treatment. PMA (phorbol-12-myristate-13-acetate), an activator of protein kinase-C, induced luciferase activity by 1.8-fold. NPY augmented forskolin-stimulated luciferase activity from 11- to 15-fold, but had no significant effect on thapsigargin-induced luciferase activity. These findings suggest that activation of protein kinase A (PKA) or CaM kinase leads to the induction of fusion gene. NPY treatment upregulated fusion gene expression through Ca2+ pathway in SK-N-BE2 cell line. Pretreatment with CREB antisense, but not the sense oligodeoxynucleotides, inhibited forskolin-, thapsigargin- and NPY-stimulated luciferase activity. However, CREB sense or antisense oligodeoxynucleotide treatment had no effect on PMA-stimulated luciferase activity. Furthermore, NPY induced CRE binding activity and the expression of CRE containing Y1 receptor gene in SK-N-MC cell line. These findings suggest that NPY can upregulate CRE containing reporter gene including Y1 receptor gene and NPY-induced reporter gene regulation in SK-N-BE2 cells is mediated by intracellular Ca2+ and CREB protein.

Topics: Animals; Artificial Gene Fusion; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Humans; Neuroblastoma; Neuropeptide Y; Phosphorylation; Rats; Receptors, Neuropeptide Y; RNA, Messenger; Thapsigargin; Transfection; Tumor Cells, Cultured; Up-Regulation

Sucrose gradient centrifugation combined with electron microscopy revealed that undifferentiated SH-SY5Y cells contain predominantly one population of noradrenaline containing vesicles, i.e. large dense cored vesicles. These vesicles have been purified approximately twenty times using sucrose/D2O gradients. Electron microscopy of sucrose/D2O fractions confirms that large dense cored vesicles are enriched in the fractions containing predominantly dopamine- -hydroxylase, chromogranin A, noradrenaline and neuropeptide Y. The membranes of these vesicles contain the typical large dense cored vesicle markers dopamine- -hydroxylase, synaptotagmin, cytochrome b561 and rab 3. Stimulation of SH-SY5Y cells with carbachol and KCl shows that noradrenaline and neuropeptide Y are released in the same proportion as stored in the large dense cored vesicles. The immuno-blot pattern and intensity of chromogranin A and chromogranin B present in large dense cored vesicles and in the released material were definitely the same. This suggests that noradrenaline and the proteins/peptides are released in the same molar stoichiometry as they are stored in large dense cored vesicles. These data provide for the first time experimental evidence that the neuroblastoma cell line SH-SY5Y contains functionally active large dense cored vesicles similar to those of sympathetic neurons and indicate that this cell line is a suitable experimental cell model to study the exocytotic pathway of large dense cored vesicles.

Topics: Cell Differentiation; Chromogranins; Endocytosis; Exocytosis; Humans; Models, Biological; Neuroblastoma; Neurons; Neuropeptide Y; Norepinephrine; Stem Cells; Tumor Cells, Cultured

In the pursuit of potent analogues of neuropeptide Y (NPY) that are selective for the Y1 receptor subtype, two lactam bridge scans of a centrally truncated parent compound were synthesized. A single lactam bridge (gamma-carboxyl of Glu to epsilon-amino of Lys) extending from residues i to i + 3 or i to i + 4 of the proposed alpha-helical region (residues 25-31 of NPY) was introduced in des-AA7-24[Gly6]NPY. Cyclogues (contraction of cyclic analogues), which were approximately one-half the size of native NPY, were initially screened for binding affinity at two discrete NPY receptor types using human neuroblastoma cell membranes, SK-N-MC and SK-N-BE2. Exploitation of the subtle differences present on each receptor type allowed for the identification of cyclogues which bound specifically to Y1 receptors with increased affinity when compared to the corresponding linear parent analogue, while one short Y1 specific cyclogue, des-AA2,3,5,7-24cyclo-(26/29)[Gly6,Glu26,Lys2 9,Pro34]NPY, bound with Ki = 16 nM. Other cyclogues showed distinct preference for Y2 receptors and bound in the low-nanomolar range. Functionally, the compounds inhibited the norepinephrine-stimulated accumulation of cAMP indicating that all acted as agonists with varying potencies.

Topics: Amino Acid Sequence; Cell Membrane; Humans; Kinetics; Lactams; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y

The modulatory effects of neuropeptide Y (NPY) on ATP-induced increases in cytosolic free-calcium concentration ([Ca2+]i) were investigated in the CHP-234 human neuroblastoma cell line. Pretreatment of cells with 100 nM NPY potentiated the increase in [Ca2+]i evoked subsequently by 20 microM ATP, compared with initial application of ATP in a control experiment, whereas a similar pretreatment with 1 microM NPY attenuated the subsequent response to ATP. Both actions of NPY were completely blocked by H-89 [N-[2-((3-(4-bromo-phenyl)-2-propenyl)-amino)-ethyl]-5 isoquinoline sulphonamide dihydrochloride], a selective antagonist of protein kinase A. The effects of 100 nM NPY were mimicked by H-89, while forskolin and 8-Br-cAMP mimicked the effects of 1 microM NPY. Both basal and forskolin-stimulated cAMP levels were inhibited by 100 nM NPY and by 100 nM NPY(13-36), a selective agonist of the NPY Y2-receptor subtype. In contrast, at 1 microM such inhibition was not observed for either NPY or NPY(13-36). It is concluded that NPY has a biphasic modulatory effect on increases in [Ca2+]i produced by ATP, which probably involves the cAMP/protein kinase A cascade.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Adenylate Kinase; Calcium; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Humans; Intracellular Fluid; Neuroblastoma; Neuropeptide Y; Tumor Cells, Cultured

1. In this study we have investigated neuropeptide Y (NPY) and somatostatin (SRIF) receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line SH-SY5Y. 2. The Ca(2+)-sensitive dye fura 2 was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither NPY (30-100 nM) nor SRIF (100 nM) elevated [Ca2+]i when applied alone. However, when either NPY (300 pM-1 microM) or SRIF (300 pM-1 microM) was applied in the presence of the cholinoceptor agonist carbachol (1 microM or 100 microM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. The elevation of [Ca2+]i by NPY was independent of the concentration of carbachol. In the presence of 1 microM or 100 microM carbachol NPY elevated [Ca2+]i with a pEC50 of 7.80 and 7.86 respectively. 4. In the presence of 1 microM carbachol the NPY Y2 selective agonist peptide YY(3-36) (PYY(3-36)) elevated [Ca2+]i with a pEC50 of 7.94, the NPY Y1 selective agonist [Leu31, Pro34]-NPY also elevated [Ca2+]i when applied in the presence of carbachol, but only at concentrations > 300 nM. The rank order of potency, PYY(3-36) > or = NPY > > [Leu31, Pro34]-NPY indicates that an NPY Y2-like receptor is involved in the elevation of [Ca2+]i. 5. In the presence of 1 microM carbachol, SRIF elevated [Ca2+]i with a pEC50 of 8.24. The sst2 receptor-preferring analogue BIM-23027 (c[N-Me-Ala-Tyr-D-Trp-Lys-Abu-Phe]) elevated [Ca2+]i with a pEC50 of 8.63, and the sst5-receptor preferring analogue L-362855 (c[Aha-Phe-Trp-D-Trp-Lys-Thr-Phe]) elevated [Ca2+]i with a pEC50 of approximately 6.1. Application of the sst3 receptor-preferring analogue BIM-23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2, 1 microM) to SH-SY5Y cells in the presence of carbachol neither elevated [Ca2+]i nor affected the elevations of [Ca2+]i caused by a subsequent coapplication of SRIF. The rank order of potency, BIM-23026 > or = SRIF > > L-362855 > > > BIM-23026 suggests that an sst2-like receptor is involved in the elevation of [Ca2+]i. 6. Block of carbachol activation of muscarinic receptors with atropine (1 microM) abolished the elevation of [Ca2+]i by the SRIF and NPY. 7. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the NPY or SRIF response. The Ca2+ channel activator maitotoxin (2 ng ml-1) also elevated [Ca2+]i but subsequent application of either NPY or SRIF in the presence of maitotoxin caused no further changes in [Ca2+]i. 8. The elevations of [Ca2+

Topics: Brain Neoplasms; Calcium; Calcium Channels; Carbachol; Electrophysiology; Humans; Inosine Triphosphate; Muscarinic Agonists; Neuroblastoma; Neuropeptide Y; Pertussis Toxin; Receptors, Gastrointestinal Hormone; Receptors, Opioid, delta; Receptors, Somatostatin; Somatostatin; Tumor Cells, Cultured; Virulence Factors, Bordetella

The distribution of voltage-sensitive elevations of the level of Ca2+ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca2+ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 microM) and omega-conotoxin GVIA (100 nM) respectively. Up to ten times Ca2+ elevations were observed in varicosities of treated cells than in cell bodies of treated and cells. These elevations were insensitive to compounds known to release Ca2+ from intracellular stores. Elevations of Ca2+ were sustained, and they were insensitive to 5 microM nifedipine, 100 nM omega-agatoxin IVA and 100 nM omega-conotoxin GVIA, and partially sensitive to 2 microM omega-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca2+ channels different from those in the cell body.

Topics: Brain Neoplasms; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Culture Media; Electrophysiology; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Immunohistochemistry; Ion Channel Gating; Neuroblastoma; Neuropeptide Y; Protein Kinase Inhibitors; Staurosporine

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.

Topics: Amino Acid Sequence; Animals; Cell Membrane; Electron Spin Resonance Spectroscopy; GTP-Binding Proteins; Humans; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Spin Labels; Structure-Activity Relationship; Swine; Tryptophan; Tumor Cells, Cultured

The suppression of neuropeptide Y Y1 receptor gene expression by antisense oligonucleotides targeted to different gene regions was monitored on mRNA and protein level in the human neuroblastoma SK-N-MC cell line. The antisense oligonucleotide targeted to the junction of the first intron and second exon suppressed specifically Y1 receptor subtype number by more than 50%, but only if oligonucleotides were administered by electroporation. Also, the formation of Y1 receptor mRNA as shown by reverse transcription-polymerase chain reaction was markedly blocked in this case. Using the antisense oligonucleotide targeted to the start of translation, no effect, neither on the Y1 receptor number nor on Y1 receptor mRNA, could be observed. This finding suggests that besides sequence-specific effects of antisense oligonucleotides gene site-specific effects play a major role in the efficacy of suppression.

Topics: Blotting, Northern; Humans; In Vitro Techniques; Neuroblastoma; Neuropeptide Y; Oligonucleotides, Antisense; Polymerase Chain Reaction; Protein Binding; Radioligand Assay; Receptors, Neuropeptide Y; Tumor Cells, Cultured

We have shown previously that platelet-derived growth factor (PDGF) has trophic effects on dopaminergic neurons in vitro. We now examined a mouse neuroblastoma cell line, NB41, for its response to PDGF and studied their phenotypic characteristics following introduction of an antisense PDGF beta-receptor RNA. NB41 cells produce both PDGF-AA and -BB; however, they carry only PDGF beta-receptors, responding to BB but not to AA. Culturing the cells with PDGF-BB induced mRNA for c-fos and PDGF-beta receptor as well as that of neuron-specific enzyme, tyrosine hydroxylase. In contrast, mRNA of chromogranin A, which is produced by chromaffin cells, decreased. Introduction of an antisense PDGF beta-receptor RNA in NB41 cells completely suppressed neurite extension and cell growth. We compared the PDGF-beta receptor sense and antisense clones for their survival. Following serum withdrawal, NB41 cells showed a DNA ladder, which by an addition of the neurotoxin, 6-hydroxy dopamine (6-OHDA), resulted in a further enhancement of the DNA ladder. The addition of PDGF-BB prior to 6-OHDA rescued cells from undergoing apoptosis, seen as a reduction of the DNA ladder. The antisense clone, regardless of the presence of PDGF-BB in the culture, showed a pronounced DNA ladder after serum withdrawal, which was further enhanced by the addition of 6-OHDA.

Topics: Animals; Apoptosis; Becaplermin; Chromogranin A; Chromogranins; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, fos; Mice; Neuroblastoma; Neuropeptide Y; Oxidopamine; Platelet-Derived Growth Factor; Protein Kinases; Proto-Oncogene Proteins c-sis; Receptor, Platelet-Derived Growth Factor beta; Receptors, Platelet-Derived Growth Factor; RNA, Antisense; RNA, Messenger; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Hypothalamic neuropeptide Y (NPY) is thought to be important in the regulation of feeding and also in the release of Adrenocorticotrophic hormone (ACTH). Intracerebroventricular administration of NPY to male rats significantly increased plasma ACTH 10 min after injection and stimulated 2-h food intake. A series of analogues of NPY that have a greatly reduced affinity for the Y1 [human pancreatic polypeptide (human PP), NPY(3-36)], the Y2 ([Pro34]NPY, human PP), the Y3 (peptide YY), and the Y6 (human PP) receptor, all markedly stimulated ACTH release. Rat PP, which binds with high affinity to the Y4 receptor, was unable to stimulate ACTH release. A novel analogue fragment [Pro34]NPY(13-36) was synthesized as a ligand with low Y1 and Y2 receptor affinity. Interestingly, neither [Pro34]NPY(13-36) nor the selective Y5 receptor agonist [D-Trp32]NPY stimulated food intake, whereas both significantly increased plasma ACTH. Thus the hypothalamic NPY receptor mediating increases in plasma ACTH has a fragment activation profile unlike the Y1-Y4 or Y6 receptors and appears distinct from the NPY receptor controlling food intake.

Topics: Adrenocorticotropic Hormone; Animals; Cell Line; Cell Membrane; Cerebral Cortex; Cerebral Ventricles; Hippocampus; Humans; Injections, Intraventricular; Male; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Pituitary Gland, Anterior; Rats; Rats, Wistar; Receptors, Neuropeptide Y; Tumor Cells, Cultured

Neuropeptide Y (NPY) is widely distributed throughout the central and peripheral nervous system and exerts a wide range of physiological responses by activating specific receptors. In this study we have characterized the potency of the high affinity peptide dimer antagonist, GR231118, to displace radiolabeled NPY/PYY from different tissues and cell lines expressing Y1 or Y2 receptors and from CHO cells stably transfected with human cDNA encoding for Y1, Y2 and Y4 receptors. GR231118 displays high affinity for Y1 and Y4 receptors, equal or better than that of NPY itself, while its activity is several fold weaker for Y2 receptors. Displacement of radiolabeled PYY from rat hypothalamic membranes by GR231118, reveals the existence of high and low affinity binding sites which may be equated to Y1 and Y2 receptors respectively suggesting that the compound maybe used as a tool to dissect central NPY receptors.

Topics: Animals; Binding Sites; Binding, Competitive; CHO Cells; Cloning, Molecular; Cricetinae; DNA, Complementary; Humans; Hypothalamus; Neuroblastoma; Neuropeptide Y; Peptides, Cyclic; Rabbits; Radioligand Assay; Rats; Receptors, Neuropeptide Y; Transfection; Tumor Cells, Cultured

Xenopus oocytes were injected with RNAs for the two inward-rectifier potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (rcKATP or CIR) in addition to RNA from the neuroblastoma cell line KAN-TS. Potassium currents were evoked by neuropeptide Y in oocytes injected with polyadenylated RNA or with cRNA from pools of a neuroblastoma (KAN-TS) cDNA library, and progressive subdivision of responding pools yielded a single cDNA. The encoded protein contains 381 amino acids, has the seven hydrophobic domains characteristic of G protein-coupled receptors, and is 31% identical to the Y1 receptor: potassium currents were induced by neuropeptide Y (EC50=60pm) and Y2-selective analogues. Coexpression with potassium channel subunits will be a generally useful method for the cloning of G protein-coupled receptors.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Humans; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Oocytes; Potassium Channels; Receptors, Neuropeptide Y; RNA; RNA, Messenger; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Xenopus

The biosynthesis of neuropeptide Y (NPY) was investigated to determine the efficiency of synthesis and processing of the precursor. In brain tissues examined, the major product was amidated NPY(1-36). Although this was also the major product in adrenal and heart atrium, a minor portion of the immunoreactivity was identified as unprocessed precursor. NPY degradation was investigated using SK-N-MC and SMS-MSN cells in conjunction with iodinated NPY tracers, labeled in either the tyrosine-1 or the tyrosine-36 position. Similar patterns of degradation were observed with both cell lines, and it would appear that the initial proteolytic attack on NPY(1-36) generates predominately N-terminal fragments.

Topics: Adrenal Glands; Animals; Binding Sites; Brain; Heart Atria; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Protein Precursors; Protein Processing, Post-Translational; Radioimmunoassay; Swine; Tissue Distribution; Tumor Cells, Cultured

In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100-fold increase in affinity compared to the C-terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS-KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH-SY5Y cells and cyclic [Lys28-Glu32] NPY Ac-25-36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D-NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.

Topics: Amino Acid Sequence; Calcium; Calcium Channels; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Neuroblastoma; Neuropeptide Y; Patch-Clamp Techniques; Peptide Fragments; Peptides, Cyclic; Protein Structure, Secondary; Receptors, Neuropeptide Y; Tumor Cells, Cultured

Topics: Cytoplasmic Granules; Humans; Neuroblastoma; Neuropeptide Y; Norepinephrine; Subcellular Fractions; Tumor Cells, Cultured

Neuropeptide Y (NPY), a 36-residue polypeptide produced abundantly in both nervous and peripheral tissues, appears to play a significant role in the regulation of diverse biological processes, including feeding behavior and cardiovascular and psychotropic functions. The actions of NPY are mediated through effective binding to specific receptors of which two, designated Y1 and Y2, have been well characterized. A shortened cyclic analogue of NPY, des-AA10-17-cyclo-7/21[Cys7,21]NPY, was shown to retain high affinity for both human neuroblastoma SK-N-MC and SK-N-BE2 cell types (expressing Y1 and Y2 receptors, respectively). Increasing the size of the ring (des-AA10-17-cyclo-2/27[Cys2,27]NPY) in the present study produced a high-affinity analogue (Ki = 3.0 vs 0.3 nM for NPY) that bound exclusively to Y2 receptors. Using the feedback from structure-activity relationships, we also describe the optimization of specific substitutions and bridging arrangements leading to the production of other truncated, high-affinity Y1 selective analogues which bind, as does NPY itself, in the low-nanomolar range. Of greatest significance, des-AA10-17-cyclo-7/21[Cys7,21,Pro34]NPY (11) was found to possess agonistic properties with an affinity comparable to that of the native NPY molecule when tested for its ability to inhibit norepinephrine-stimulated cAMP release in SK-N-MC human neuroblastoma cells. Compound 11 also caused an increase in blood pressure in anesthetized rats. However, in two central nervous system models of Y1 receptor function, stimulation of feeding and anxiolytic activity, this analogue was inactive, which suggests the presence of a new subclass of receptors. In summary, the present results demonstrate that residues 10-17 of NPY are not directly involved in either Y1 or Y2 receptor recognition or activation. This suggests that the selectivity of NPY receptors is highly dependent on subtle conformational changes such as the substitution of residue 34 to a proline or the introduction of intramolecular constraints. Additionally, we have produced an analogue of NPY that selectively activates peripheral NPY Y1 receptors.

Topics: Amino Acid Sequence; Animals; Blood Pressure; Cyclic AMP; Diazepam; Dose-Response Relationship, Drug; Eating; Humans; Male; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Norepinephrine; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Tumor Cells, Cultured

In the present study, the subtype specificity and species selectivity of the nonpeptide BIBP 3226, as well as its in vitro antagonism of neuropeptide Y (NPY)-mediated second messengers have been investigated. Radiolabeled NPY is potently displaced by BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenylmethyl]-D- arginine amide] on human Y1 receptor expressing Chinese hamster ovary-K1 cells (Ki = 0.47 +/- 0.07 nM). SK-N-MC human neuroblastoma cells (Ki = 5.1 +/- 0.5 nM) and the rat parietal cortex membranes (Ki = 6.8 +/- 0.7 nM). The interaction of BIBP 3226 with the Y1 receptor is stereoselective, because the (S)-enantiomer of the (R)-configured BIBP 3226 displays almost no affinity (Ki > 10,000 nM). In contrast, concentrations up to 10 microM BIBP 3226 do not displace [125I]NPY from the human Y2 receptor (neuroblastoma cell line SMS-KAN), the rabbit Y2 receptor (kidney) and the rat Y2 receptor (hippocampus). Functional antagonism could be shown for the human Y1 receptor: 0.1 microM BIBP 3226 antagonizes the NPY induced Ca++ mobilization (pKb = 7.5 +/- 0.17) as well as the NPY-mediated inhibition of cyclic AMP synthesis (pKb = 8.2 +/- 0.24) in SK-N-MC cells. In contrast, none of the formerly described putative antagonists PYX-2, [D-Trp32]NPY and benextramine could be characterized as high affinity Y1 receptor antagonists. The 18 amino acid NPY analog EXBP 68 Ile-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic (2,4'), (2',4')-diamide] displayed Y1-selective affinity with in vitro antagonistic properties (Ki = 0.33 +/- 0.04 nM and pKb = 8.4 +/- 0.07) in SK-N-MC cells. Therefore, BIBP 3226 is the first potent and subtype-selective nonpeptide Y1 receptor antagonist.

Topics: Adipocytes; Amino Acid Sequence; Animals; Arginine; Brain; Calcium; CHO Cells; Cricetinae; Dogs; Humans; Kidney; Male; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Rabbits; Rats; Receptors, Neuropeptide Y; Stereoisomerism; Substrate Specificity; Tumor Cells, Cultured

Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.

Topics: Amino Acid Sequence; Animals; Brain; Calcium; Cell Line; Humans; Kidney; Kinetics; Leukemia, Erythroblastic, Acute; Male; Mammals; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Organ Specificity; Peptides; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Structure-Activity Relationship; Swine; Tumor Cells, Cultured

Whole-cell Ca2+ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using the perforated-patch technique with 10 mM Ba2+ as charge carrier. Neuropeptide Y (NPY; 10 nM to 1 microM) caused concentration-dependent inhibition of Ca2+ channel currents which were associated with a slowing in current activation kinetics. [Ala31]NPY, a residue 31 L-alanine substituted analogue of NPY, also inhibited Ca2+ channel currents and caused slowing of activation kinetics, but with approximately 6-fold lower potency. In the presence of 100 nM [Ala31]NPY (which itself had little or no effect on currents), the actions of NPY were similar in magnitude to its effects in the absence of the analogue. Our results suggest that substitution of isoleucine for alanine at residue 31 results in a NPY analogue which is a full agonist but with lower affinity for Y2 receptors.

Topics: Barium; Calcium Channels; Dose-Response Relationship, Drug; Evoked Potentials; Humans; Neuroblastoma; Neuropeptide Y; Patch-Clamp Techniques; Receptors, Neuropeptide; Time Factors; Tumor Cells, Cultured

We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite out-growth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E1 plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.

Topics: Glioma; Hybrid Cells; Immunohistochemistry; Laminin; Neuroblastoma; Neuropeptide Y; Synapsins; Transfection; Tumor Cells, Cultured

Human neuropeptide Y (NPY) gene expression occurs exclusively in the central and peripheral nervous systems requiring complex cell-specific regulation. In this study we have examined the effect of modulating the second messenger systems involving protein kinase A and protein kinase C on the expression of the NPY gene in different neuronal cell types. We report that the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin on a neuroblastoma cell line (LA-N-5) and a pheochromocytoma cell line (PC12) are mediated through both increased transcription of the NPY gene and through stabilization of NPY messenger RNA (mRNA). After 8 h of treatment TPA and forskolin increase the steady-state level of NPY mRNA 10- and 12-fold in LA-N-5 and PC12 cells, respectively. This response in neuroblastoma cells is due to an increase in the half-life of NPY mRNA. The response in PC12 cells is mediated by both increased mRNA stability and increased transcription. Transient transfection analyses using PC12 cells indicate that only 51 base pairs 5' to the transcription start site are necessary for the TPA and forskolin induced transcriptional response. Thus, these experiments demonstrate that TPA and forskolin effect the regulation of the NPY gene via transcriptional and posttranscriptional mechanisms in a cell-specific manner.

Topics: Animals; Colforsin; Dichlororibofuranosylbenzimidazole; Drug Stability; Gene Expression Regulation; Neuroblastoma; Neuropeptide Y; PC12 Cells; Promoter Regions, Genetic; Protein Processing, Post-Translational; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Whole-cell Ca2+ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using conventional and perforated-patch techniques. Neuropeptide Y (NPY, 10-1000 nM) caused concentration-dependent inhibition of Ca2+ channel current amplitudes which was pertussis toxin-sensitive, voltage-dependent and associated with slowing of channel activation kinetics, regardless of which recording configuration was used. Inhibition was observed in all cells tested. Similar current inhibitions were observed with NPY (18-36) and peptide YY, but not with [Leu31, Pro34]NPY, indicating that the effects were mediated by Y2 receptors. Pancreatic polypeptide (100 nM) was without effect on Ca2+ channel currents. The effects of NPY were additive with nifedipine (at a supramaximal concentration of 5 microM), suggesting that NPY predominantly inhibits N-type Ca2+ channels present in these cells, and not L-type. The effects of NPY were mimicked by a novel, cyclic analogue of NPY which is 40-fold more selective for Y2 receptors than other commonly used Y2-selective peptides. The cyclic analogue was also more potent than NPY itself, causing maximal current inhibition at approx 300 nM, whereas the response to NPY was not fully saturated at 1 microM. Our results indicate that SH-SY5Y cells represent an excellent model system for studying the coupling of Y2 receptors to N-type channel inhibition. Furthermore, in the absence of selective antagonists for NPY receptor subtypes, the highly selective Y2 agonist cyclic NPY derivative may prove a useful tool for probing the various roles of Y2 receptors in the nervous system.

Topics: Calcium Channels; Electrophysiology; GTP-Binding Proteins; Humans; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Receptors, Neuropeptide Y; Tumor Cells, Cultured

Exposure of human SK-N-MC neurotumor cells to 4 beta-phorbol 12-myristate 13-acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down-regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein Gi was unlikely. Cholate extracts of membranes from control and PMA-treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc- membranes, which lack the stimulatory G protein subunit Gs alpha; thus, Gs did not appear to be the target of PMA action. Membranes from PMA-treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn2+ and Mn2+ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK-N-MC cells. PMA treatment also resulted in a shift to less sensitivity in the K(act) for isoproterenol but not for dopamine or CGP-12177 (a beta 3-adrenergic agonist) stimulation. Thus, the beta 1 but not the D1 or beta 3 receptors were being desensitized by PKC activation. Analysis of SK-N-MC cells by western blotting with antibodies against different PKC isozymes revealed that both the alpha and zeta isozymes were present in these cells. Whereas PKC-alpha was activated and translocated from cytosol to membrane by phorbol esters, the zeta isozyme was not. Thus, PKC-alpha, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.

Topics: Adenylyl Cyclases; Adrenergic beta-Antagonists; Alkaloids; Aluminum Compounds; Animals; Brain; Cell Line; Cell Membrane; Cholera Toxin; Colforsin; Cyclic AMP; Dopamine; Drug Synergism; Fluorides; GTP-Binding Proteins; Humans; Isoenzymes; Isoproterenol; Kinetics; Manganese; Neuroblastoma; Neuropeptide Y; Propanolamines; Protein Kinase C; Rats; Sphingosine; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The ability to express exogenous mammalian genes stably in post-mitotic cells such as neurons remains an important goal for those attempting to modulate neurotransmission through gene delivery. We therefore investigated how differentiation to a post-mitotic state affected the expression of an exogenous gene encoding for neuropeptide Y (NPY) following transfection with an adeno-associated virus (AAV) derived vector. This vector (pJDT95npy) was constructed with rat NPY cDNA (551 bp) inserted downstream from the indigenous AAV p5, p19 and p40 promoters to characterize their relative abilities to drive NPY mRNA expression. Transfection of dividing neuroblastoma CHP126 cells with pJDT95npy resulted in the differential expression of chimeric NPY mRNAs derived from each promoter. P40-driven species became dominant after 1 month post-transfection. Vector integration into chromosomal DNA was demonstrated by Southern blot analyses, indicating at least some region-selective integration. In dividing cell extracts, only a low level of pro-NPY immunoreactivity and no mature NPY immunoreactivity was recovered. However, after differentiation of the pJDT95npy-transfected CHP 126 cells to a post-mitotic state, significant levels of pro-NPY and mature NPY were recovered in the cells and media. Differentiation also had a time-dependent effect on mRNA expression: a spike of p5 driven expression on day 3 was followed predominantly by p40-driven expression on day 5. This study indicates that AAV-derived vectors using the p40 promoter may be used to express genes in post-mitotic cells such as neurons.

Topics: Animals; Blotting, Northern; Blotting, Southern; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Dependovirus; Gene Expression; Genetic Vectors; Humans; Mitosis; Neuroblastoma; Neuropeptide Y; Plasmids; Rats; Recombinant Proteins; Restriction Mapping; RNA, Messenger; Transfection; Tumor Cells, Cultured

Four children with advanced or relapsed neuroblastoma were treated with oral 13-cis-retinoic acid 0.75 mg/kg/day. Clinical response to retinoic acid was noted only in the two children with tumors coexpressing trk protooncogene mRNA, encoding an essential part of the nerve growth factor (NGF) high affinity receptor, and low affinity NGF receptor gene (LNGFR) mRNA. Clinical stage or age, plasma neuropeptide Y, tumor DNA ploidy and N-myc amplification did not as accurately predict response to retinoic acid as NGF receptor mRNAs. In vitro data have shown that retinoic acid up regulates LNGFR expression and NGF sensitivity via interaction with specific regulatory elements in the LNGFR gene promoter. We hypothesize that part of the therapeutic effect of retinoic acid in neuroblastoma in vivo may be exerted via increased NGF receptor expression and NGF sensitivity. Analysis of trk and LNGFR mRNA may be useful to predict clinical response to retinoic acid in these children.

Topics: Administration, Oral; Child; Child, Preschool; DNA, Neoplasm; Female; Gene Expression; Genes, myc; Humans; Male; Neuroblastoma; Neuropeptide Y; Ploidies; Proto-Oncogene Proteins; Receptors, Nerve Growth Factor; RNA, Messenger; Treatment Outcome; Tretinoin

In this study we have investigated DNA-protein interactions at an AP1-like motif of the neuropeptide tyrosine (NPY) promoter during in vitro differentiation of human neuroblastoma cells SH-SY5Y to mature nonproliferative sympathetic neuron-like cells. These neuroblast-like cells originate from the parental cell line SK-N-SH from which two phenotypically distinct major cell types have been subcloned: the neuroblast-like SH-SY5Y cells and the epithelial-like SH-EP cells. SH-SY5Y cells can be induced to differentiate towards mature noradrenergic ganglion-like cells by the protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate). Interestingly, the effects of TPA are mimicked by the protein kinase inhibitor, staurosporine, which induces the expression of TPA target genes such as the neuronal differentiation-associated gene NPY in SH-SY5Y cells. Following activation of PKC, the effects of TPA are known to act through the transcription factor AP-1. To study transcriptional regulation during sympathetic differentiation of human neuroblastoma cells by TPA as well as by staurosporine, we focussed on protein complexes at an evolutionarily conserved AP-1 like motif located at nucleotide positions -70 to -65 within the 5'-flanking region of the NPY gene. We show that both c-Jun and c-Fos are part of the protein complexes that bind to this sequence in SH-SY5Y cells. Both staurosporine and TPA enhanced and modulated the binding of these DNA-protein complexes concomitant with the NPY mRNA expression. On the other hand, the absence of these complexes in the SH-EP subclone was associated with the absence of NPY mRNA expression and a lack of differentiation-associated morphological changes. The data suggest that Fos and Jun heterodimers are part of the protein complexes that bind to the AP-1 regulatory element of the NPY promoter in the neuroblast-like SH-SY5Y cells. These protein complexes appear to contribute to the cell specific expression of the NPY gene and seem to be required during differentiation of SH-SY5Y human neuroblastoma cells further along the sympathetic neuronal lineage induced by either TPA or staurosporine.

Topics: Alkaloids; Base Sequence; Cell Differentiation; Humans; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine

Stimulation of neuropeptide Y (NPY) Y2 receptors induced an intracellular free Ca2+ ([Ca2+]i) increase in a human neuroblastoma cell line, CHP-234. When NPY in a Ca(2+)-free solution was applied, this increase was abolished. Depolarization with high KCl evoked no response, suggesting that the responses were not mediated by voltage-gated Ca2+ channels. There was no evidence that the NPY response consisted of a capacitative Ca2+ entry sensitive to internal Ca2+ store levels. The [Ca2+]i elevation was diminished by Ni2+, a blocker of Ca2+ entry. Mn2+ induced a quench of the fura-2 fluorescence, which ceased promptly upon the removal of NPY, indicating that Ca2+ entry was linked tightly to receptor activation. Although thapsigargin- and ryanodine-sensitive Ca2+ stores were present, NPY-induced responses were not impaired by pretreatment with either drug. Furthermore, NPY had no effect on the thapsigargin-sensitive store. Pertussis toxin did not affect the NPY-stimulated [Ca2+]i increase, although it abolished the NPY-dependent inhibition of cAMP production. It is concluded that the Y2 receptors couple directly to receptor-operated Ca2+ channels without the involvement of intracellular Ca2+ stores. The results also indicate that Y2 receptors can activate both pertussis toxin-sensitive and -insensitive mechanisms in the same cell.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Cell Line; Cyclic AMP; Fluorescent Dyes; Fura-2; Humans; Kinetics; Microscopy, Fluorescence; Neuroblastoma; Neuropeptide Y; Nickel; Pertussis Toxin; Potassium Chloride; Receptors, Neuropeptide Y; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

Neuropeptide Y (NPY) attenuated angiotensin II (AII)-or bradykinin (BK)-induced Ca2+ release from intracellular stores and inhibited forskolin-stimulated cAMP accumulation and omega-conotoxin-sensitive high K(+)-induced Ca2+ influx in the human neuroblastoma cell line SMS-KAN. All three NPY actions were mediated via Y2 receptors. Pretreatment with pertussis toxin completely abolished all of the NPY actions. Activation or down-regulation of protein kinase C had no effect on any NPY-mediated effect; herbimycin A, a tyrosine kinase inhibitor, only abolished the inhibitory effect of NPY on AII- or BK-induced Ca2+ mobilization. Herbimycin A also blocked platelet-derived growth factor-induced Ca2+ mobilization, which involves tyrosine kinase activation, and there was a good correlation in the concentration dependency between the two effects of herbimycin A, strongly suggesting that its ability to cancel the NPY effect is due to inhibition of tyrosine kinase activity. NPY attenuated AII- or BK-induced inositol 1,4,5-trisphosphate production, and herbimycin A reversed this NPY effect. These results provide the first evidence that Y2 receptors negatively couple to AII- or BK-induced phosphoinositide turnover leading to Ca2+ mobilization through pertussis toxin-sensitive GTP-binding protein(s). Inhibition of phospholipase C-beta activity by NPY seems to be mediated by activation of protein-tyrosine kinase or phosphotyrosine-containing protein(s).

Topics: Angiotensin II; Benzoquinones; Bradykinin; Calcium; Cyclic AMP; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Lactams, Macrocyclic; Neuroblastoma; Neuropeptide Y; Pertussis Toxin; Potassium; Quinones; Receptors, Neuropeptide Y; Rifabutin; Second Messenger Systems; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Virulence Factors, Bordetella

In an effort to gain insight into the bioactive conformation of neuropeptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y1 and Y2 receptor binding affinities were evaluated using the human neuroblastoma cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesis (Boc strategy) followed by preparative HPLC purification produced analogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring glycine at position 9 was replaced by Ala and D-Ala), Y1 receptor binding was most perturbed by chiral inversion of residues at the C-terminus (residues 20, 27, 29-35, Ki > or = 300 nM). Substitutions at residues 2-5, 28, and 36 had Ki values ranging from 40 to 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y2 class of receptors all measured in the low or sub-nanomolar concentrations, with the exception of C-terminally modified isomers (residues 30-35). Only [D-Arg33]- and [D-Gln34]NPY displayed no measurable binding affinity to Y2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their binding affinities and position in the sequence for structural analysis using circular dichroism (CD) spectroscopy. Of the nine peptide evaluated ([D-Pro5]-, [Ala9]-, [D-Glu10]-, [D-Asp11]-, [D-Ala18]-, [D-Tyr20]-, [D-Tyr27]-, and [D-Arg33]NPY), only [D-Tyr27]NPY expressed a definitive correlation between loss of binding affinity and disruption of secondary structure by having the propensity to form beta-sheets at the expense of alpha-helical content. It was concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, the receptor interaction was only affected when certain critical residues were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.

Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Circular Dichroism; Humans; Molecular Sequence Data; Neuroblastoma; Neuropeptide Y; Peptides; Protein Conformation; Protein Structure, Secondary; Receptors, Neuropeptide Y; Structure-Activity Relationship; Tumor Cells, Cultured

Previous attempts to inhibit the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) -induced differentiation of SH-SY5Y neuroblastoma cells by non-specific inhibitors of protein kinases have failed. In the present study we have used the bisindolylmaleimide GF 109203X, which is a potent and selective inhibitor of protein kinase C (PKC). GF 109203X effectively antagonized TPA-stimulated phosphorylation of an endogenous 80 kDa PKC substrate. The compound blocked neurite outgrowth and rounding up of cells induced by the phorbol ester. In addition, GF 109203X completely inhibited TPA-induced increase in cellular content of noradrenaline as well as stimulation of expression of neuropeptide Y, growth-associated protein-43 and c-fos proto-oncogene mRNA by TPA. The inhibition of the TPA-induced effects by GF 109203X was dose-dependent.

Topics: Blotting, Northern; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; GAP-43 Protein; Gene Expression; Genes, fos; Humans; Indoles; Kinetics; Maleimides; Membrane Glycoproteins; Molecular Weight; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neuropeptide Y; Phosphoproteins; Phosphorylation; Protein Kinase C; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

1. The increase in perfusion pressure induced by neuropeptide Y (NPY), peptide YY (PYY) and related peptides were compared in the perfused rat tail artery precontracted by a submaximal concentration (1 microM) of the vasoconstrictor, phenylephrine. 2. NPY, PYY, [Leu31,Pro34]NPY, [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) and the centrally truncated and stabilized analogues [D-Cys5,8-aminooctanoic acid7-20, Cys24]-NPY (D-Cys5-NPY) and [D-Cys7, 8-aminooctanoic acid8-17,Cys20]-NPY (D-Cys7-NPY) produced a concentration-dependent enhancement of the vasoconstrictor response induced by 1 microM phenylephrine. PYY was two times more potent than NPY and [Leu31,Pro34]NPY while ESALL-NPY, D-Cys7-NPY and D-Cys5-NPY were approximately 3, 5 and 16 times less potent than NPY respectively. NPY, D-Cys5-NPY and D-Cys7-NPY gave similar maximal responses whereas those observed for PYY, [Leu31,Pro34]NPY and ESALL-NPY were much greater than that of NPY. 3. NPY 13-36 and [des-Ser3,Lys4,Cys2,8-aminooctanoic acid3-24, D-Cys27]-NPY ([es-Ser3,Lys4]Cys2-NPY) were practically inactive at concentrations up to 3 microM, whereas [des-Ser3,Lys4,D-Cys2,8-aminooctanoic acid3-24,Cys27]-NPY ([des-Ser3,Lys4]D-Cys2-NPY), which differs from [des-Ser3,Lys4]Cys2-NPY in the disulphide bridge (a D-Cys in position 2 for [des-Ser3,Lys4]D-Cys2-NPY instead of an L-CYs for [des-Ser3,Lys4]Cys2-NPY) was a weak agonist the maximal effect of which could not be ascertained. 4. The contractile effects of [des-Ser3,Lys4]D-Cys2-NPY were additive with those of NPY and [Leu31,Pro34]NPY demonstrating that it is not a partial agonist but may simply not interact competitively with the receptor binding site for NPY. NPY and PYY interacted in a manner expected of agonists competing for the same binding site.5. PYY, NPY and [Leu31,Pro34]NPY were equipotent in displacing the I125-labelled PYY from binding sites on membranes from Y1-receptor expressing SK-N-MC cells, while the centrally truncated analogues were much less potent. The rank order of potencies for displacement of the I125-PYY binding by these peptides in SK-N-MC cells correlated with their activity in enhancing the vasoconstrictor response of phenylephrine in the tail artery. For the [des-Ser3,Lys4]D-Cys2-NPY analogue, the displacement pattern was more complex in that the displacement analysis revealed the presence of two binding sites.6. In conclusion, these data provide no evidence for other than postjunctional Y1-receptors mediating the enhancement of th

Topics: Amino Acid Sequence; Animals; Arteries; Binding Sites; Cell Membrane; Drug Interactions; Humans; Iodine Radioisotopes; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth, Vascular; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Phenylephrine; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Receptors, Neuropeptide Y; Tail; Tumor Cells, Cultured; Vasoconstrictor Agents

Some childhood malignant neoplasms are thought to arise in embryonic tissues. These tumors present unique opportunities for studying the maturation of specific cellular lineages and examining the possible role of alterations in the regulation of differentiation in tumor development. Several features of neuroblastoma, a tumor thought to arise in cells originating in the embryonic neural crest, suggest that it may be particularly useful in this regard. The identification of a series of markers that characterize the various cell types of the peripheral nervous system that are detectable in neuroblastoma tumor tissues has made it possible to recognize that the cells of neuroblastoma tumor cell lines and tissues correspond to specific stages of adrenal gland development. Experiments directed at understanding the cellular signals by which neural crest cell maturation is mediated may provide insights of therapeutic import because neuroblastoma tumors corresponding to some stages of differentiation respond very differently to nonspecific cytotoxic therapies than tumors corresponding to other stages.

Topics: Adrenal Medulla; Biomarkers; Biomarkers, Tumor; Cell Differentiation; Child; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor II; Neural Crest; Neuroblastoma; Neuropeptide Y

Neuropeptide Y (NPY) was studied as a marker for neuroblastoma in 12 children. All but one patient with neuroblastoma had elevated plasma NPY concentrations at diagnosis. During treatment NPY values returned to normal in 9 of 12 children. All three children without normalization of plasma NPY values died; two of them had a relapse and the third died of toxic effects. Plasma NPY appears to be a sensitive marker of neuroblastoma.

Topics: Adolescent; Biomarkers, Tumor; Child; Child, Preschool; Female; Homovanillic Acid; Humans; Infant; Male; Neuroblastoma; Neuropeptide Y; Phosphopyruvate Hydratase; Vanilmandelic Acid

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

Neuropeptide Y (NPY) and peptide YY (PYY) are homologous 36 amino acid amidated peptides that often, but not always, exert similar actions and binding profiles. The present study of cultured cells confirms that both peptides as well as radioiodinated analogs, i.e. 125I-Bolton-Hunter-NPY (125I-BH-NPY) and 125I-peptide YY (125I-PYY), show high affinity to binding sites/receptors of the previously proposed Y1- and Y2-subtypes, selectively expressed by the human neuroblastoma cell lines, SK-N-MC and SK-N-BE(2), respectively. In contrast, bovine adrenal chromaffin cells did not bind 125I-PYY, while displaying high affinity 125I-BH-NPY sites, and may therefore represent a cell type expressing a recently proposed Y3-type of (NPY-preferring) receptors. Several non-labeled fragments/analogs have been used in displacement experiments to further characterize the structural requirements for Y1-, Y2-, and Y3-type binding. In every instance, specific binding was reduced by addition of 5'-guanylylimidodiphosphate [Gpp(NH)p], indicating that the three receptor subtypes belong to the G-protein-coupled superfamily of receptors. Moreover, in both neuroblastoma cell lines, the peptides elicited, with appropriate orders of potency, reduction of forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Finally, NPY-evoked 45Ca2+ influx was observed in SK-N-MC and in chromaffin cells. A common dual coupling mechanism of NPY/PYY receptors, i.e. to reduction of cAMP and to Ca2+ elevation, is therefore suggested to exist, although both phenomena could not be demonstrated in every cell type.

Topics: Animals; Calcium; Cattle; Cell Membrane; Chromaffin System; Cyclic AMP; Humans; Neuroblastoma; Neuropeptide Y; Peptide YY; Peptides; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Signal Transduction; Tumor Cells, Cultured

Gene expression of nerve growth factor receptor (NGFR), epidermal growth factor receptor(EGFR), chromogranin A (CGA) and neuropeptide Y (NPY) in 4 neuroblastoma cell Lines without N-myc amplification was studied by using Northern blot technique. N type cells expressed more NGFR mRNA than S type cell's and have only little or no EGFR expression. S type cells had stronger expression of EGFR mRNA than that of N type cells accompanying with only less or even no NGFR expression. The results indicated that difference of gene expression of these growth factor receptors might be due to the various directions of tumor cell differentiation. Cells differentiating toward neurons gave more NGFR expression and cells prepared to be differentiating toward other direction might give more EGFR gene expression. Various gene expression of CGA and NPY in neuroblastoma cell lines might be due to the presence of different stages of tumor cell differentiation and NGF only induced differentiation of those neuroblastoma cells ready to be differentiating to neurons afterwards.

Topics: Blotting, Northern; Chromogranin A; Chromogranins; DNA Probes; ErbB Receptors; Gene Expression; Humans; Nerve Growth Factors; Neuroblastoma; Neuropeptide Y; Receptors, Cell Surface; Receptors, Nerve Growth Factor; RNA, Messenger; Tumor Cells, Cultured

The physiological significance of multiple Y receptors has not been determined since until recently only one form of endogenous agonists was known, namely PYY1-36 and NPY1-36. Recently, a new molecular form of PYY was characterized as des(Tyr-Pro)PYY (PYY3-36 or PYY-II). Its ability to interact at various Y receptors was not characterized. Analytical chromatography of fresh canine colon extracts shows two peaks of immunoreactivity eluting in the positions of PYY-II and PYY1-36 (PYY). PYY-II was about 40% of the total PYY immunoreactivity indicating that it is one of the major forms of PYY expressing its biological activity. It is shown that PYY-II will not displace label from the Y1 receptors found on a human neuroblastoma cell line. It is further shown that PYY-II is as potent as PYY for the inhibition of pancreatic secretion, which must occur through Y2 receptors. The enzymatic removal of Tyr-Pro from PYY to form PYY-II must therefore regulate the relative expression of a non-selective agonist (PYY) to a highly selective Y2 agonist (PYY-II). Amino terminal processing of PYY represents a novel type of regulation of peptide hormone specificity. It has important biological implications for PYY and potential relevance for other peptide hormone receptor systems.

Topics: Animals; Binding, Competitive; Cell Line; Ceruletide; Dogs; Female; Gastrointestinal Hormones; Humans; Male; Neuroblastoma; Neuropeptide Y; Pancreas; Peptide YY; Peptides; Protein Processing, Post-Translational; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Secretin; Substrate Specificity

1. We investigated the usefulness of neuropeptide Y as a plasma marker for phaeochromocytoma, ganglioneuroblastoma and neuroblastoma using a simple and highly sensitive r.i.a. for human neuropeptide Y. 2. Plasma immunoreactive neuropeptide Y concentrations were measured without extraction in plasma samples (100 microliters) from patients with various diseases. 3. The plasma immunoreactive neuropeptide Y concentration in patients with phaeochromocytoma (172.3 +/- 132.4 pmol/l, mean +/- SD, n = 23) was significantly higher than that in healthy adult subjects (40.1 +/- 10.1 pmol/l, n = 40, P < 0.0001). The plasma immunoreactive neuropeptide Y concentrations in patients with ganglioneuroblastoma (590.7 +/- 563.6 pmol/l, n = 6) and patients with neuroblastoma (566.9 +/- 524.4 pmol/l, n = 15) were significantly higher than those in control children (1-9 years old, 82.2 +/- 39.9 pmol/l, n = 72, P < 0.0001). 4. The plasma immunoreactive neuropeptide Y concentration in patients with essential hypertension (34.0 +/- 3.7 pmol/l, n = 18) was within the normal range, but in patients with chronic renal failure undergoing maintenance haemodialysis (192.1 +/- 68.0 pmol/l, n = 25) and in non-dialysed patients with chronic renal failure (85.1 +/- 23.1 pmol/l, n = 7) it was significantly higher than that in healthy adult subjects (P < 0.0001). 5. Eighty-seven per cent of the patients with phaeochromocytoma, 67% of the patients with ganglioneuroblastoma and 80% of the patients with neuroblastoma showed plasma immunoreactive neuropeptide Y concentrations higher than the upper limits in the control subjects [62 pmol/l (adult) and 160 pmol/l (children)].(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adrenal Gland Neoplasms; Adult; Aged; Biomarkers, Tumor; Child; Child, Preschool; Female; Ganglioneuroma; Humans; Hypertension; Infant; Kidney Failure, Chronic; Male; Middle Aged; Neuroblastoma; Neuropeptide Y; Pheochromocytoma; Radioimmunoassay

We have demonstrated that the mouse neuroblastoma N18Tg2 cell line and several clones of hybrid ND cells (ND7, ND9 and ND21), derived from the fusion of neonatal rat sensory neurons with that neuroblastoma, show immunostaining to protein gene product 9.5, neuropeptide Y, C-flanking peptide of neuropeptide Y, tyrosine hydroxylase and chromogranins. Synaptophysin could only be detected in ND cells. Immunoreactivities to substance P, calcitonin gene-related peptide, galanin and somatostatin could not be detected in any of these cell lines. After three days of incubation in a differentiation medium, cell processes of various lengths were observed both in neuroblastoma and ND cell cultures. In ND7 cells there was also a redistribution of neuropeptide Y and its C-flanking peptide to the tips of cell processes. The differentiation of cell processes was also accompanied by the appearance of immunostaining to rat chromogranins in their tips. In contrast, synaptophysin expression was found mainly in cell bodies. Neuropeptide Y, its C-flanking peptide and chromogranins have been associated with secretory granules, whereas synaptophysin is a marker for small synaptic-like vesicles. Therefore, our morphological findings further support and expand the view that these markers are primarily associated with different subcellular structures. Moreover, they indicate that the regulated secretory pathway associated with chromogranins is segregated into nerve processes at an early stage of differentiation, when the synaptophysin-associated pathway is not yet mature. ND7 cells thus provide a useful model system for studying changes in the distribution of neuropeptides, cytoskeletal elements and proteins associated with cell secretion during neuronal differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Chromogranins; Clone Cells; Hybrid Cells; Immunohistochemistry; Mice; Neuroblastoma; Neurofilament Proteins; Neurons, Afferent; Neuropeptide Y; Neuropeptides; Rats; Synaptophysin; Tyrosine 3-Monooxygenase

Truncated, branched, and/or cyclic neuropeptide Y (NPY) analogues were tested for their ability to bind to the neuroblastoma cells, SK-N-MC (Y1 receptor) and SK-N-BE(2) (Y2 receptor). The design of such analogues was inspired by models of NPY based on the crystal structure of avian pancreatic polypeptide. The minimum length of the backbone was investigated using the following truncated analogues [binding affinity (nM) for Y1 and Y2 receptor subtypes respectively are given in parentheses]: des-AA10-17[D-Ala9]NPY (100, 0.9), des-AA7-23[D-Ala6]NPY (> 1000, 1.2), des-AA4-26[D-Ala3]NPY (> 1000, 120), cyclo(7,20)-des-AA10-17[Glu7,D- Ala9,D-Dpr20]NPY (100, nd), cyclo(2,27)-des-AA7-23[Glu2,D-Ala6,D-Dpr27]NPY (> 1000, 3.6), cyclo(2,30)- des-AA7-23[Glu2,D-Ala6,-D-Dpr30]NPY (> 1000, nd), cyclo(1,30)-des-AA4-26[Glu1,D-Ala3,D-Dpr30]NPY (> 1000, > 1000). A new family of branched NPY analogues corresponding to the partial deletion of the polyproline helix with conservation of the N-terminus was also examined: des-AA7-23[(Ac-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 2.1), des-AA7-23[(Ac-NPY7-22)-epsilon-D-Lys6]NPY (> 1000, 5.1), des-AA7-23-[(Ac-LEALEG-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 4.8). Finally, the role played by the flexible tail (residues 32-36) was studied with the following cyclic analogues: cyclo(30,34)-[Lys30,Glu34]NPY18-36 (> 1000, 360), cyclo(30,34)-[Orn30,Gly34]NPY18-36 (> 1000, 950), cyclo(30,34)-[Dpr30,Glu34]NPY18-36 (> 1000, 590), cyclo(33,36)-[Lys33,Glu36]NPY (> 1000, > 1000), cyclo(33,36)-[Lys33,Glu36]NPY18-36 (> 1000, > 1000). These results suggest that the Y1 receptor is highly discriminatory since deletion of residues 10-17, shown to have little effect on Y2 binding affinity, reduces Y1 affinity 50-fold. Bridging sites and constructs have been identified that may serve as useful leads in the design of more potent and selective analogues. We have identified two positions (9 and 6) where the introduction of a D amino acid is not detrimental to binding affinity. Whether this modification leads to the stabilization of a yet unidentified turn compatible with high Y2 receptor affinity will have to be determined by spectroscopic methods. Finally, stabilizing a putative alpha-helical conformation of the C-terminal heptapeptide of NPY18-36 has a deleterious effect on the Y1 and Y2 receptors.

Topics: Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Humans; Models, Molecular; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y; Structure-Activity Relationship

We have studied [125I]neuropeptide Y-binding sites and neuropeptide Y-mediated second messenger responses in human SK-N-MC neuroblastoma cells with special reference to the role of G-proteins. Neuropeptide Y stimulated two second messenger responses in SK-N-MC cells, inhibition of cAMP accumulation and mobilization of Ca2+ from intracellular stores. Both effects were completely abolished by pretreatment with pertussis toxin. Binding of [125I]neuropeptide Y to intact cells or SK-N-MC cell membranes was rapid, reversible, characterized by high affinity and low capacity, and had pharmacological characteristics of a homogeneous population of Y1-like neuropeptide Y receptors. In permeabilized cells, [125I] neuropeptide Y binding was inhibited by GTP gamma S in a concentration-dependent manner. Saturation experiments in the absence and presence of GTP gamma S demonstrated a reduction in the number of high-affinity [125I]neuropeptide Y-binding sites without a decrease in affinity of the remaining sites. Pretreatment of intact cells with pertussis toxin completely abolished the inhibition of [125I]neuropeptide Y binding by GTP gamma S. Moreover, pertussis toxin treatment reduced the number of high-affinity [125I]neuropeptide Y binding sites. We conclude that the agonist ligand [125I]neuropeptide Y identifies functional neuropeptide Y receptors in SK-N-MC cells; however, the number of specific [125I]neuropeptide Y-binding sites may not necessarily reflect the number of neuropeptide Y receptors, because the former is affected by the functional state of cellular G-proteins.

Topics: Calcium; Cyclic AMP; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intracellular Fluid; Iodine Radioisotopes; Kinetics; Neuroblastoma; Neuropeptide Y; Pertussis Toxin; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Second Messenger Systems; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

D-myo-Inositol-1,2,6-trisphosphate (PP56) is a novel experimental drug which is structurally related to the intracellular second messenger IP3. Among other pharmacological effects, PP56 has been shown to antagonize neuropeptide Y (NPY) induced vasoconstriction with a high degree of specificity. We examined the effects of i.c.v. PP56 on locomotor activity and food intake in rats, and on the hypoactivity and hyperphagia induced by NPY. In the open field, PP56 given alone increased locomotor activity by up to 85%. It did not prevent NPY induced hypoactivity to any extent. PP56 given alone did not affect food intake except for a small increase after the highest dose tested (200 nmol). When NPY was given after pretreatment with PP56, NPY induced hyperphagia was significantly reduced. A similar effect, however, was seen with regard to the hyperphagia produced by another orexigenic peptide, galanin. PP56 did not affect the binding of 125I-NPY to brain membranes in vitro, or to cells of two different neuroblastoma cell lines which selectively express NPY Y1 or Y2 receptors. In summary, PP56 acted as a locomotor stimulant per se. Only one of the two tested central effects of NPY could be antagonized by PP56, and then only partially and in a non-specific manner. The central effects of PP56 do not seem to be produced at the level of NPY receptors.

Topics: Animals; Behavior, Animal; Binding, Competitive; Brain; Eating; Humans; Hyperphagia; Immobilization; Injections, Intraventricular; Inositol Phosphates; Locomotion; Male; Membranes; Neuroblastoma; Neuropeptide Y; Rats; Rats, Inbred Strains; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Tumor Cells, Cultured

SH-SY5Y neuroblastoma cells undergo neuronal differentiation and their proliferation is inhibited when they are treated with phorbol 12-myristate 13-acetate (PMA). Insulin and insulin-like growth factor I (IGF-I) are mitogens for the nontreated SH-SY5Y cells, whereas the proliferative response to such factor stimulation is lost upon differentiation, in spite of the fact that the receptors for insulin and IGF-I remain expressed and functional in the differentiated cells. Here we show that the PMA-induced differentiation of SH-SY5Y cells grown in a serum-free medium is strongly potentiated by nanomolar concentrations of IGF-I, as judged by morphology and markers for neuronal differentiation--e.g., neuropeptide tyrosine and growth-associated protein 43. Also, insulin and IGF-II potentiated the phorbol ester-induced differentiation, although less efficiently than IGF-I. Using blocking anti-receptor antibodies, it could be shown that the differentiation induced by these factors, in combination with PMA, was primarily mediated through the IGF-I receptor.

Topics: Blotting, Northern; Blotting, Western; Cell Differentiation; Cell Division; Cell Line; Drug Synergism; Gene Expression; Genes, myc; Humans; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Kinetics; Neuroblastoma; Neurons; Neuropeptide Y; RNA, Neoplasm; Tetradecanoylphorbol Acetate

Topics: Biomarkers, Tumor; Blotting, Southern; Child; Child, Preschool; Chromatography, Gel; DNA, Neoplasm; Female; Ganglioneuroma; Humans; Infant; Male; Neoplasm Staging; Neuroblastoma; Neuropeptide Y

Topics: Biomarkers, Tumor; Child; Follow-Up Studies; Ganglioneuroma; Humans; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Prognosis; Recurrence; Reference Values

Topics: Adrenal Gland Neoplasms; Aging; Biomarkers, Tumor; Child, Preschool; Fetal Blood; Follow-Up Studies; Humans; Infant; Infant, Newborn; Neuroblastoma; Neuropeptide Y; Prognosis; Radioimmunoassay; Reference Values; Sarcoma; Soft Tissue Neoplasms

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.

Topics: Adenylyl Cyclase Inhibitors; Animals; Binding Sites; Calcium; Colforsin; Cytosol; Fluorescent Dyes; Intracellular Membranes; Neuroblastoma; Neuropeptide Y; Plant Extracts; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Swine; Thapsigargin; Tumor Cells, Cultured

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Binding, Competitive; Cholera Toxin; Colforsin; Cyclic AMP; Dopamine; Humans; Isoproterenol; Neuroblastoma; Neuropeptide Y; Peptide YY; Peptides; Pertussis Toxin; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Calcitonin gene-related peptide (CGRP) stimulated cyclic adenosine monophosphate (cAMP) levels in SK-N-MC human neuroblastoma cells in a time- and concentration-dependent manner. The efficacy order for CGRPs was human alpha-CGRP = human beta-CGRP = chick CGRP greater than rat CGRP greater than human [Tyr0]CGRP. Calcitonin (CT) failed to influence cAMP production in SK-N-MC cells. [Tyr0]CGRP27-37 which by itself did not affect cAMP levels antagonized CGRP action. Saturation analysis using [125I]CGRP showed a homogeneous population of binding sites. CGRP but not CT, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) inhibited radioligand binding. Our results provide evidence that human neuroblastoma SK-N-MC cells contain highly specific CGRP receptors which are positively coupled to cAMP generation.

Topics: Binding Sites; Binding, Competitive; Calcitonin Gene-Related Peptide; Cyclic AMP; Humans; Neuroblastoma; Neuropeptide Y; Receptors, Cell Surface; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The similarity of neuropeptide Y (NPY) to pancreatic polypeptide (PP), whose X-ray crystallographic structure is known, has allowed computer-assisted molecular modelling of NPY and predictions of its three-dimensional structure. Utilizing these techniques, Krstenansky et al. (Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 4377-4381) reported that a centrally truncated analog of porcine NPY, [D-Cys7-Aoc8-17-Cys20]pNPY, which was designed to maintain the tertiary structure of the native molecule, bound to sites on membranes from mouse brain with even higher affinity than native NPY. As brain membranes may represent a heterogeneous mixture of receptor subtypes, we decided to characterize the activity of this analog on a defined cell line. SK-N-MC cells are a human epithelioma cell line with high-affinity receptors of the Y1 subtype which are coupled to inhibition of adenylate cyclase. (D-Cys7-Aoc8-17-Cys20]pNPY bound to receptors on SK-N-MC cells, but in contrast to membranes from mouse brain, with a lower affinity than pNPY. Furthermore, [D-Cys7-Aoc8-17-Cys20]pNPY was able to inhibit isoproterenol-stimulated cAMP production in these cells. Therefore, it appears that the central amino acids deleted from this analog are not involved in NPY binding, and biological activity can be maintained by conservation of the tertiary structure of NPY around the binding surface.

Topics: Animals; Binding, Competitive; Cats; Cyclic AMP; Humans; Isoproterenol; Neuroblastoma; Neuropeptide Y; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Tumor Cells, Cultured

The present investigation provides three lines of evidence for the presence of a pro-form of neuropeptide Y (NPY) in plasma and follicular fluid. First, by the demonstration of NPY-immunoreactive material of a size corresponding to the estimated mol wt of pro-NPY. Second, an antiserum specific for the C-terminal tyrosine amide of NPY and peptide YY does not react with this material. Third, it was possible to convert the pro-NPY extracted from plasma and follicular fluid using the protease, Endoproteinase-Lys C, to a NPY-immunoreactive form eluting slightly before NPY on a G-50 column. The size of the digested product was consistent with a cleavage of pro-NPY resulting in an immunoreactive species, NPY-Gly-Lys. Pro-NPY was also found in tissue culture media from the human neuroendocrine cell line SH-SY5Y. As in the case of plasma and follicular fluid, another NPY immunoreactive species eluted from a G-50 gel filtration column slightly before synthetic human NPY. Analysis of this material with an antibody directed against the tyrosine amide of NPY in combination with isoelectric focusing revealed that this peak consisted of at least two immunoreactive forms of NPY. In conclusion, at least three different forms of NPY immunoreactivity are likely to be present in plasma, follicular fluid, and cell tissue culture media; pro-NPY, a degradation form of pro-NPY, or a biosynthetic intermediate and NPY.

Topics: Chromatography, Gel; Endopeptidases; Female; Follicular Fluid; Humans; Male; Metalloendopeptidases; Neuroblastoma; Neuropeptide Y; Ovary; Protein Precursors; Tumor Cells, Cultured

The activation of regulatory peptides by post-translational modification of their biosynthetic precursors is generally thought to occur only in neuroendocrine cells. We have selected clones of Chinese hamster ovary cells, a non-neuroendocrine cell line, which were transfected with a eukaryotic expression vector coding for the precursor for neuropeptide Y. Although the majority of the immunoreactive NPY was found in the form of pro-NPY, some degree of intracellular proteolytic processing of the precursor occurred in all clones. Part of the intracellular NPY immunoreactivity was even correctly amidated. Extracellular degradation of pro-NPY in the tissue culture medium generated immunoreactivity which corresponded in size to NPY. It is concluded that precursor processing can occur in non-neuroendocrine cells both as a biological process within the cells and as apparent processing, degradation in the tissue culture medium.

Topics: Animals; Cell Line; Chromatography, Gel; Cloning, Molecular; Cricetinae; Genetic Vectors; Humans; Neuroblastoma; Neuropeptide Y; Protein Precursors; Protein Processing, Post-Translational; Transfection; Tumor Cells, Cultured

Neuropeptide Y (NPY) was investigated as a possible tumor marker in pediatric patients with tumors of the sympathetic nervous system. Seven patients with neuroblastoma, 3 patients with ganglioneuroblastoma, and 2 with ganglioneuroma, were compared with 12 matched healthy controls and 34 tumor controls. NPY-like immunoreactivity (NPYLI) was analyzed in extracted plasma using a competitive radioimmunoassay. At diagnosis, plasma NPYLI was significantly increased (p less than .001) in the neuroblastoma patients (352 +/- 99 pM; mean +/- SEM) when compared with healthy controls (36 +/- 4 pM) and tumor controls (30 +/- 2 pM). Ganglioneuroblastoma and ganglioneuroma patients had lower levels (57 +/- 8 pM) than neuroblastoma patients but still significantly higher than the controls. In all patients with sympathetic tumors, the NPYLI level was decreased after treatment. Five neuroblastoma patients relapsed; all had increasing NPYLI levels. In 3 of these patients, incresing NPYLI was the first sign of relapse. Plasma NPYLI correlated well to urinary levels of homovanillic acid. NPY in plasma (NPYLI) may be a clinically useful marker of pediatric neuroblastoma for diagnosis and differential diagnosis. NPYLI correlates well with the clinical course and can be the first sign of relapse. Plasma determinations of NPYLI make it possible to monitor rapid alterations of disease.

Topics: Biomarkers, Tumor; Child, Preschool; Ganglioneuroma; Humans; Infant; Infant, Newborn; Neoplasm Recurrence, Local; Nervous System Neoplasms; Neuroblastoma; Neuropeptide Y; Radioimmunoassay; Reference Values; Sympathetic Nervous System

Neuropeptide Y (NPY) expression is limited to tissues of the central and peripheral nervous system. In the adrenal gland, NPY is found in a subset of cells of the adrenal medulla. Using in situ hybridization analysis, NPY mRNA expression was characterized during human fetal adrenal medullary development. We found a biphasic pattern of NPY mRNA expression during the development of the human adrenal medulla. NPY mRNA is detectable at the earliest evaluable time point (7.5 weeks of gestational age) through 18 weeks of gestational age, and is then not detectable until 8 months after birth. We also analyzed NPY mRNA expression in neuroblastoma tumors, which often arise in the adrenal medulla. Thirty-eight neuroblastoma tumors were analyzed for NPY mRNA expression using in situ hybridization. We found NPY mRNA expression in 30 of 38 tumors; 15 of 15 Stage IVS tumors from children under 1 year of age at diagnosis expressed NPY mRNA, whereas 0 of 4 Stage IV tumors from children less than 1 year of age at diagnosis expressed NPY mRNA. These data suggest that in children under 1 year of age at diagnosis, Stage IVS and Stage IV neuroblastoma may be marked by the presence or absence, respectively, of NPY mRNA expression. Moreover, since NPY is expressed for only a short period of time during embryogenesis, these tumors may arise from different neuroblast populations occurring during the course of adrenal medullary development.

Topics: Adrenal Glands; Female; Gene Expression; Humans; Neoplasm Staging; Neuroblastoma; Neuropeptide Y; Pregnancy; RNA, Messenger

In order to develop an immunoradiometric assay for human neuropeptide (hNPY), a recently discovered and potent vasoconstrictor 36-amino-acid peptide, we used hNPY and some of its subpeptides to prepare monoclonal anti-NPY antibodies. Two monoclonal antibodies with high affinity for hNPY, that is affinity constants in the range of 10(10) mol/L-1, which respectively, reacted with the 9-18 portion and the 32-36 portion of hNPY were used in the immunoradiometric system. The assay was highly specific, NPY-related peptides such as pancreatic polypeptide and peptide YY not being detected. The lower limit of sensitivity was 0.5 pmol/L. In 303 normal subjects, plasma NPY concentrations were less than 0.5 pmol/L in 67%, 0.5 to 5.0 pmol/L in 25% and 5.1 to 30 pmol/L in the remaining 8%. A value of 7.5 pmol/L (95th percentile value in the normal group) was considered as the upper limit of normal. Among 111 patients with various neuroendocrine tumors, elevated plasma NPY concentrations were found in patients with pheochromocytomas and neuroblastomas, the highest plasma levels being found in patients with malignant pheochromocytomas. We conclude that patients with neuroendocrine tumors, especially secreting and or malignant tumors of the sympathochromaffin system, often have elevated plasma NPY concentrations.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoid Tumor; Carcinoma; Child; Child, Preschool; Epitopes; Female; Humans; Infant; Infant, Newborn; Middle Aged; Neuroblastoma; Neuropeptide Y; Pheochromocytoma; Radioimmunoassay

By using monoiodinated radioligands of both intact neuropeptide Y (NPY) and of a long C-terminal fragment, NPY13-36, two subtypes of binding sites, which differ in affinity and specificity, have been characterized. The Y1 type of binding site, characterized on a human neuroblastoma cell line, MC-IXC, and a rat pheochromocytoma cell line, PC-12, binds NPY with a dissociation constant (Kd) of a few nanomolar but does not bind NPY13-36. The Y2 type of binding site, characterized on porcine hippocampal membranes and on another human neuroblastoma cell line, SMS-MSN, is of higher affinity and binds both NPY and NPY13-36. None of the binding sites distinguish between NPY and the homologous peptide YY (PYY). It is concluded that NPY/PYY-binding sites occur in two subtypes which may represent two types of physiological receptors.

Topics: Adrenal Gland Neoplasms; Animals; Binding Sites; Cell Membrane; Hippocampus; Humans; Neuroblastoma; Neuropeptide Y; Peptide Fragments; Pheochromocytoma; Rats; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Swine; Tumor Cells, Cultured

The expression of the potent vasoactive peptide neuropeptide Y (NPY) was studied in 16 clinically and/or histologically diagnosed human pheochromocytomas and 3 human neuroblastoma tumors. All tumors contained NPY in concentrations ranging from 21 pmol/g of tissue, similar to that found in normal adrenal tissue, to 91,000 pmol/g (median, 1,700 pmol/g). Three control tumors of Cushing's type did not contain NPY. An almost total proteolytic processing of pro-NPY to normal NPY was observed in the tumors (median, 93%; range, 72-100%). A positive correlation between the processing efficiency and the NPY content was also observed. The small amount of pro-NPY found in the tumors was characterized by "in vitro conversion" with endoproteinase Lys-C. In the tumor extracts, the majority of the NPY immunoreactivity, corresponding in size to the NPY standard, also behaved like synthetic NPY by high performance liquid chromatography and isoelectric focusing. As assessed by both its elution position in isoelectric focusing and its reaction with an antiserum specific for the COOH-terminal amidated sequence, the peptide produced by the tumors was found to be efficiently amidated, a modification which is essential for the biological activity of NPY. It is concluded that although only a subset of chromaffin cells express NPY, a very high number of pheochromocytomas and neuroblastomas produce correctly amidated and thus biologically active NPY in large amounts, and that this is of potential importance for tumor-related cardiovascular symptoms and for autocrine stimulation of tumor cells.

Topics: Adolescent; Adrenal Cortex; Adrenal Gland Neoplasms; Adult; Aged; Child; Child, Preschool; Chromatography, Gel; Chromatography, High Pressure Liquid; Humans; Isoelectric Focusing; Male; Middle Aged; Neuroblastoma; Neuropeptide Y; Pheochromocytoma; Protein Precursors; Radioimmunoassay

The synthesis and processing of the precursor for neuropeptide Y (NPY) were studied in 16 human and murine neuroendocrine cell lines. Eight of the cell lines, NS-20Y, PC12, LA-N-5, CHP-234, SMS-KCNR, SH-SY5Y, SMS-KCN, and BE(2)-M17, produced sufficient quantities to permit chromatographic characterization of the NPY immunoreactivity. Although the cell lines varied in the amount of NPY they produced, both within and between cell lines, they displayed a relatively constant pattern of posttranslational modifications. In contrast to observations in tumor extracts (M. M. T. O'Hare and T. W. Schwartz, Cancer Res., 49: 7010-7014, 1989), all cell lines studied contained a substantial amount of the intracellular NPY in the form of the unprocessed propeptide, 57% (range, 33-72%) as characterized by both gel filtrations (32 experiments in 8 cell lines) and "in vitro conversion" with endoproteinase Lys-C. In the majority, 4 of 6 cell lines studied, almost all of the NPY, which by size corresponded to the mature 36-amino acid form, was amidated as assessed by isoelectric focusing and by a radioimmunoassay specific for the COOH-terminal amide group of the peptide. Both the propeptide and smaller molecular forms of NPY were secreted from the cell cultures; however, proteolytic degradation in the tissue culture medium prevented a detailed, meaningful characterization of these peptides. It is concluded that many neuroendocrine cell lines, especially those derived from human neuroblastomas, express the NPY gene; the cells display a partly impaired dibasic processing capacity but they generally amidate the products efficiently.

Topics: Adrenal Gland Neoplasms; Animals; Chromatography, Gel; Chromatography, High Pressure Liquid; Humans; Isoelectric Focusing; Mice; Neuroblastoma; Neuropeptide Y; Pheochromocytoma; Protein Precursors; Protein Processing, Post-Translational; Radioimmunoassay; Tumor Cells, Cultured

Neuropeptide Y (NPY)- and somatostatin (SS)-like immunoreactivities (LI) were investigated in tumor tissues of one ganglioneuroma (GN), 3 ganglioneuroblastomas (GNB) and one neuroblastoma (NB) by radioimmunoassay. NPY-LI was detected from all 5 tumor tissues (16.4-1247 pmol/g wet tissue). Sephadex G-50 column chromatography and reverse phase high performance liquid chromatography (HPLC) revealed that most of the NPY-LI in tumor extracts was eluted in an identical position to synthetic human NPY except one GNB (case 2). In this case, most of the NPY-LI was eluted in a higher molecular weight region than synthetic human NPY in Sephadex G-50 column chromatography and in a more hydrophobic position in HPLC. SS-LI was detected from 4 tumor extracts except one GNB (case 2) (21.3-787 pmol/g wet tissue). Sephadex G-25 column chromatography and reverse phase HPLC revealed that SS-LI in tumor extracts was eluted just after the void volume and then in the same positions as SS-28 and SS-14. These results suggest that NPY, SS-14 and SS-28 exist in tumor tissues of GN, GNB and NB, and most of the NPY-LI in one GNB was a higher molecular and more hydrophobic form of NPY-LI.

Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Ganglioneuroma; Humans; Neuroblastoma; Neuropeptide Y; Peptides; Radioimmunoassay

Monoiodinated radioligands of the homologous 36-amino acid peptides, neuropeptide Y (NPY) and peptide YY, were prepared by reverse phase high performance liquid chromatography with isocratic elution. [125I-Tyr1]- and [125I-Tyr36]monoiodoNPY bound equally well to a single class of high affinity binding sites on synaptosomal membranes prepared from porcine hippocampus (Kd = 1.0 X 10(-10) M) whereas iodine substitution in Tyr27, for example, partly interfered with the receptor binding. The receptors on the hippocampal membranes did not distinguish between neuropeptide Y and peptide YY either in their monoiodinated or in their unlabeled forms. Six out of twelve human neuroblastoma cell lines had high affinity binding sites for monoiodinated NPY ranging from 2 to 145 X 10(3) sites per cell. The NPY binding to three of the cell lines, SMS-MSN, SMS-KAN, and CHP-234 was of relatively high affinity (Kd = 1.3 to 6.1 X 10(-10) M), and, as in the hippocampal membranes, the long C-terminal fragment, NPY(13-36)peptide was also a relatively potent ligand for these receptors. Two other neuroblastoma cell lines, MC-IXC and CHP-212, expressed NPY receptors characterized by a lower affinity (Kd = 4.8 and 24.6 X 10(-9) M) and negligible cross-reactivity with the C-terminal fragment. It is concluded that monoiodinated radioligands of the tyrosine-rich neuropeptide Y can be prepared and that receptors for these ligands in two apparently different subtypes are found on a series of human neuroblastoma cell lines.

Topics: Animals; Binding Sites; Hippocampus; Humans; Iodoproteins; Kinetics; Neuroblastoma; Neuropeptide Y; Peptide YY; Peptides; Swine; Tumor Cells, Cultured; Tyrosine

High concentrations of a newly-identified biologically potent peptide, neuropeptide Y, have been demonstrated in 3 related mouse neuroblastoma-derived clonal cell lines, N18TG2 0.35 pmol/mg protein, NG108-15 0.44 pmol/mg protein and NCB-20 0.39 pmol/mg protein. The NG108-15 cell line was chosen for further evaluation. Dexamethasone (10 microM) and nerve growth factor (10 ng/ml) resulted in a 2-fold increase in cellular neuropeptide Y concentrations. The response to dexamethasone was demonstrated to be dose-dependent. Exposure to both agents in combination resulted in a more than additive effect, indicating synergism.

Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Dexamethasone; Glioma; Hybrid Cells; Kinetics; Mice; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neuropeptide Y; Rats