epidermal-growth-factor and Neuroblastoma

Topics: Animals; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Cytokines; Epidermal Growth Factor; Humans; Immunoconjugates; Immunotherapy, Adoptive; Interleukin-2; Killer Cells, Lymphokine-Activated; Lymphotoxin-alpha; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms; Neuroblastoma; Recombinant Fusion Proteins; Superantigens; T-Lymphocytes, Cytotoxic

Neuroblastoma is a malignant extracranial solid tumor arising from the sympathoadrenal lineage of the neural crest and is often associated with

Topics: Cathepsin D; Cell Cycle; Child; Epidermal Growth Factor; ErbB Receptors; Humans; Lysosomes; Neuroblastoma; Peptide Hydrolases

Neuroblastoma, a type of cancer that is common in children, is composed of two genetically clonal but epigenetically distinct cell types: mesenchymal (MES) and adrenergic (ADRN) types, controlled by super-enhancer-associated lineage-specific transcription factor networks. Mesenchymal-type cells are more migratory, resistant to chemotherapy, and prevalent in relapse tumors. Importantly, both cell types spontaneously transdifferentiate into one another, and this interconversion can be induced by genetic manipulations. However, the mechanisms of their spontaneous transdifferentiation and extracellular factors inducing this phenomenon have not yet been elucidated. Using a unique approach involving gene set enrichment analysis, we selected six ADRN and 10 MES candidate factors, possibly inducing ADRN and MES phenotypes, respectively. Treatment with a combination of 10 MES factors clearly induced the MES gene expression profile in ADRN-type SH-SY5Y neuroblastoma cells. Considering the effects on gene expression profile, migration ability, and chemoresistance, a combination of tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF) was sufficient to synergistically induce the ADRN-to-MES transdifferentiation in SH-SY5Y cells. In addition, human neuroblastoma cohort analysis revealed that the expression of TNF and EGF receptors was strongly associated with MES gene expression signatures, supporting their important roles in transdifferentiation in vivo. Collectively, we propose a mechanism of neuroblastoma transdifferentiation induced by extracellular growth factors, which can be controlled in clinical situations, providing a new therapeutic possibility.

Topics: Adrenergic Neurons; Cell Line, Tumor; Cell Transdifferentiation; Epidermal Growth Factor; Humans; Neuroblastoma; Tumor Necrosis Factor-alpha

Cultured cancer cells serve as important models for preclinical testing of anti-cancer compounds. However, the optimal conditions for retaining original tumor features during in vitro culturing of cancer cells have not been investigated in detail. Here we show that serum-free conditions are critical for maintaining an immature phenotype of neuroblastoma cells isolated from orthotopic patient-derived xenografts (PDXs). PDX cells could be grown either as spheres or adherent on laminin in serum-free conditions with retained patient-specific genomic aberrations as well as tumorigenic and metastatic capabilities. However, addition of serum led to morphological changes, neuronal differentiation and reduced cell proliferation. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were central for PDX cell proliferation and MYCN expression, and also hindered the serum-induced differentiation. Although serum induced a robust expression of neurotrophin receptors, stimulation with their cognate ligands did not induce further sympathetic differentiation, which likely reflects a block in PDX cell differentiation capacity coupled to their tumor genotype. Finally, PDX cells cultured as spheres or adherent on laminin responded similarly to various cytotoxic drugs, suggesting that both conditions are suitable in vitro screening models for neuroblastoma-targeting compounds.

Topics: Animals; Biomarkers, Tumor; Biopsy; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Culture Media, Conditioned; Disease Models, Animal; Epidermal Growth Factor; Fibroblast Growth Factor 2; Heterografts; Humans; Immunohistochemistry; Mice; N-Myc Proto-Oncogene Protein; Neoplasm Metastasis; Neural Stem Cells; Neuroblastoma

Neuroblastoma is the most common extracranial solid tumor in children. The ErbB family of proteins is a group of receptor tyrosine kinases that promote the progression of various malignant cancers including neuroblastoma. Thus, targeting them with small molecule inhibitors is a promising strategy for neuroblastoma therapy. In this study, we investigated the anti-tumor effect of afatinib, an irreversible inhibitor of members of the ErbB family, on neuroblastoma. We found that afatinib suppressed the proliferation and colony formation ability of neuroblastoma cell lines in a dose-dependent manner. Afatinib also induced apoptosis and blocked EGF-induced activation of PI3K/AKT/mTOR signaling in all neuroblastoma cell lines tested. In addition, afatinib enhanced doxorubicin-induced cytotoxicity in neuroblastoma cells, including the chemoresistant LA-N-6 cell line. Finally, afatinib exhibited antitumor efficacy in vivo by inducing apoptosis in an orthotopic xenograft neuroblastoma mouse model. Taken together, these results show that afatinib inhibits neuroblastoma growth both in vitro and in vivo by suppressing EGFR-mediated PI3K/AKT/mTOR signaling. Our study supports the idea that EGFR is a potential therapeutic target in neuroblastoma. And targeting ErbB family protein kinases with small molecule inhibitors like afatinib alone or in combination with doxorubicin is a viable option for treating neuroblastoma.

Topics: Afatinib; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Doxorubicin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mice; Mice, Nude; Neuroblastoma; Quinazolines; Radiation-Sensitizing Agents; Signal Transduction; Xenograft Model Antitumor Assays

Low free T levels in men are associated with age-related cognitive decline and increased risk for neurotoxicity, resulting in disease. The mechanisms underlying these observations remain poorly defined. Although rapid, androgen receptor-dependent activation of ERK has been postulated as a neurotrophic and neuroprotective mechanism, actions of T metabolites such as 5α-androstane-3α,17β-diol (3α-diol) may also be involved. We investigated the influence of 3α-diol on the induction of ERK phosphorylation in SH-SY5Y human female neuroblastoma cells and primary cortical neurons from male and female mice. In SH-SY5Y cells, ERK phosphorylation was induced by 10 nM DHT, epidermal growth factor, hydrogen peroxide (H

Topics: Acetylcholine; Amyloid beta-Peptides; Androstane-3,17-diol; Animals; Caspase 3; Cell Line, Tumor; Cerebral Cortex; Dihydrotestosterone; Epidermal Growth Factor; Female; Humans; Hydrogen Peroxide; Male; MAP Kinase Signaling System; Mice; Neuroblastoma; Neurons; Phosphorylation

Grb2-associated regulator of Erk/MAPK1 (GAREM) is an adaptor molecule in the EGF-mediated signaling pathway. GAREM is expressed ubiquitously in human organs and cultured cells. Two GAREM homologues are encoded by the human genome. Therefore, previously identified GAREM is named GAREM1. Here we characterized a new subtype of GAREM, GAREM2, that is specifically expressed in the mouse, rat, and human brain. Three GAREM2 tyrosines (Tyr-102, Tyr-429, and Tyr-551) are phosphorylated upon EGF stimulation and are necessary for binding to Grb2. Furthermore, GAREM2 and Shp2 regulate Erk activity in EGF-stimulated cells. These characteristics are similar to those of GAREM1. GAREM2 is expressed in some neuroblastoma cell lines and is also tyrosine-phosphorylated and bound to Grb2 after treatment with EGF. Eventually, GAREM2 regulates Erk activation in the presence of EGF or insulin like growth factor 1. GAREM2 also regulates insulin-like growth factor 1-induced neuronal differentiation of the SH-SY5Y neuroblastoma cell line. Although the structure and function of both GAREM subtypes are similar, GAREM1 is recruited into the nucleus and GAREM2 is not. Nuclear localization of GAREM1 might be controlled by a GAREM1-specific nuclear localization sequence and 14-3-3ε binding. The N-terminal 20 amino acids of GAREM1 make up its nuclear localization sequence that is also a 14-3-3ε binding site. The GAREM family is a new class of adaptor molecules with subtype-specific biological functions.

Topics: Amino Acid Sequence; Animals; Brain; Cell Line, Tumor; Cell Nucleus; Chlorocebus aethiops; COS Cells; Epidermal Growth Factor; GRB2 Adaptor Protein; HEK293 Cells; HeLa Cells; Humans; Immunoblotting; MAP Kinase Signaling System; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Neurites; Neuroblastoma; Neurons; Phosphorylation; Protein Isoforms; Rats; Rats, Wistar; RNA Interference; Sequence Homology, Amino Acid

Previous studies have shown that tumor-driving glioma stem cells (GSC) may promote radio-resistance by constitutive activation of the DNA damage response started by the ataxia telangiectasia mutated (ATM) protein. We have investigated whether GSC may be specifically sensitized to ionizing radiation by inhibiting the DNA damage response. Two grade IV glioma cell lines (BORRU and DR177) were characterized for a number of immunocytochemical, karyotypic, proliferative and differentiative parameters. In particular, the expression of a panel of nine stem cell markers was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Overall, BORRU and DR177 displayed pronounced and poor stem phenotypes, respectively. In order to improve the therapeutic efficacy of radiation on GSC, the cells were preincubated with a nontoxic concentration of the ATM inhibitors KU-55933 and KU-60019 and then irradiated. BORRU cells were sensitized to radiation and radio-mimetic chemicals by ATM inhibitors whereas DR177 were protected under the same conditions. No sensitization was observed after cell differentiation or to drugs unable to induce double-strand breaks (DSB), indicating that ATM inhibitors specifically sensitize glioma cells possessing stem phenotype to DSB-inducing agents. In conclusion, pharmacological inhibition of ATM may specifically sensitize GSC to DSB-inducing agents while sparing nonstem cells.

Topics: Ataxia Telangiectasia; Cell Line, Tumor; DNA Breaks, Double-Stranded; Enzyme Inhibitors; Epidermal Growth Factor; Fibroblast Growth Factors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Humans; Intermediate Filament Proteins; Karyotyping; Mutation; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Neuroblastoma; Radiation, Ionizing

Neuronal leucine-rich repeat protein-1 (NLRR1), a type-1 transmembrane protein highly expressed in unfavorable neuroblastoma, is a target gene of MYCN that is predominately expressed in primary neuroblastomas with MYCN amplification. However, the precise biological role of NLRR1 in cell proliferation and tumor progression remains unknown. To investigate its functional importance, we examined the role of NLRR1 in EGF and insulin growth factor-1 (IGF-1)-mediated cell viability. We found that NLRR1 positively regulated cell proliferation through activation of extracellular signal-regulated kinase mediated by EGF and IGF-1. Interestingly, EGF stimulation induced endogenous MYCN expression through Sp1 recruitment to the MYCN promoter region, which was accelerated in NLRR1-expressing cells. The Sp1-binding site was identified on the promoter region for MYCN induction, and phosphorylation of Sp1 was important for EGF-mediated MYCN regulation. In vivo studies confirmed the proliferation-promoting activity of NLRR1 and established an association between NLRR1 expression and poor prognosis in neuroblastoma. Together, our findings indicate that NLRR1 plays an important role in the development of neuroblastoma and therefore may represent an attractive therapeutic target for cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; N-Myc Proto-Oncogene Protein; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Promoter Regions, Genetic; Sp1 Transcription Factor; Transcription, Genetic

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC(50) = 6-25 μM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150 mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAPKs (SAPK/JNK), caspase-3, and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G(2)/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10 mM) pre-treatment rescued cell viability of RKS262 (23 µM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100 μM) and a MAPK inhibitor SB203580 (20 μM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50 nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50 nM), EGF (100 nM) and IGF-1 (100 nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G(2)/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals, and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAPK and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Brain-Derived Neurotrophic Factor; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Coumarins; Cyclic S-Oxides; Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor I; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Nude; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Burden; Xenograft Model Antitumor Assays

Bromoacetoxy-calcidiol (B3CD), a pro-apoptotic and cytotoxic agent in neuroblastoma (NB) cell lines, displayed therapeutic potential in vivo as an anticancer drug in a NB xenograft mouse model. Tumors of all animals treated intraperitoneally with B3CD went into regression within 10-30 days of treatment, while tumors in control animals grew aggressively. The response mechanisms of NB cells to B3CD in vitro were studied and included differential targeting of cell cycle key regulators p21 and cyclin D1 on the transcriptional and expression level leading to arrest in G0/G1 phase. In contrast to the effect in ovarian cancer cells, B3CD-induced cell death in SMS-KCNR NB cells was only marginally mediated by the p38 MAPK signaling pathway. Signaling induced by exogenous recombinant EGF leads to a partial restoration of the negative effects of B3CD on SMS-KCNR cell proliferation and survival. Upon combinational treatment of SMS-KCNR cells with B3CD and recombinant EGF, the EGF receptor (EGF-R) was highly activated. We suggest future studies to include analysis of the effects of B3CD in combination therapy with pharmacological inhibitors of cell cycle regulators or with EGF-R-targeting inhibitors, -toxins or -antibodies in vitro and their translation into in vivo models of tumor development.

Topics: Animals; Antineoplastic Agents; Calcifediol; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Epidermal Growth Factor; G1 Phase; Humans; Mice; Neuroblastoma; p38 Mitogen-Activated Protein Kinases; Resting Phase, Cell Cycle; Signal Transduction; Xenograft Model Antitumor Assays

Nitric oxide (NO) donors inhibit the epidermal growth factor (EGF)-dependent auto(trans)phosphorylation of the EGF receptor (EGFR) in several cell types in which NO exerts antiproliferative effects. We demonstrate in this report that NO inhibits, whereas NO synthase inhibition potentiates, the EGFR tyrosine kinase activity in NO-producing cells, indicating that physiological concentrations of NO were able to regulate the receptor activity. Depletion of intracellular glutathione enhanced the inhibitory effect of the NO donor 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) on EGFR tyrosine kinase activity, supporting the notion that such inhibition was a consequence of an S-nitrosylation reaction. Addition of DEA/NO to cell lysates resulted in the S-nitrosylation of a large number of proteins including the EGFR, as confirmed by the chemical detection of nitrosothiol groups in the immunoprecipitated receptor. We prepared a set of seven EGFR(C --> S) substitution mutants and demonstrated in transfected cells that the tyrosine kinase activity of the EGFR(C166S) mutant was completely resistant to NO, whereas the EGFR(C305S) mutant was partially resistant. In the presence of EGF, DEA/NO significantly inhibited Akt phosphorylation in cells transfected with wild-type EGFR, but not in those transfected with C166S or C305S mutants. We conclude that the EGFR can be posttranslationally regulated by reversible S-nitrosylation of C166 and C305 in living cells.

Topics: Catalytic Domain; Cell Line, Tumor; Cell Proliferation; Cysteine; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Humans; Mutagenesis, Site-Directed; Mutation; Neuroblastoma; Nitric Oxide; Nitric Oxide Synthase; Oxidative Phosphorylation; Quaternary Ammonium Compounds; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transgenes

Neurospheres can be generated from the mouse fetal forebrain by exposing multipotent neural stem cells (NSCs) to epidermal growth factor (EGF). In the presence of EGF, NSCs can proliferate continuously while retaining the potential to differentiate into neurons, astrocytes and oligodendrocytes. We examined the expression pattern of the neurotrophin (NT) receptors tropomysin-related kinase (TRK)-A, TRK-B, TRK-C and p75 neurotrophin receptor (p75(NTR)) in NSCs and the corresponding lineage cells. Furthermore, we analyzed the action of the NT Brain-Derived Neurotrophic Factor (BDNF) on NSCs' behavior. The effects of BDNF on NSC proliferation and differentiation were examined together with the signaling pathways by which BDNF receptors transduce signaling effects. We found that all the known NT receptors, including the truncated isoforms of TRK-B (t-TRK-B) and TRK-C (t-TRK-C), were expressed by Nestin-positive cells within the neurosphere. Proliferation was enhanced in Nestin-positive and BrdU-positive cells in the presence of BDNF. In particular, we show that t-TRK-B was abundantly expressed in NSCs and the corresponding differentiated glia cells while full length TRK-B (fl-TRK-B) was expressed in fully differentiated post-mitotic neurons such as the neuronal cells of the newborn mouse cortex, suggesting that BDNF may exert its proliferative effects on NSCs through the t-TRK-B receptor. Finally, we analyzed the cell fates of NSCs differentiated with BDNF in the absence of EGF and we demonstrate that BDNF stimulated the formation of differentiated cell types in different proportions through the MAP kinase, AKT and STAT-3 signaling pathways. Thus, the in-vivo regulation of neurogenesis may be mediated by the summation of signals from the BDNF receptors, in particular the t-TRK-B receptor, regulating physiological fates as diverse as normal neural replacement, excessive neural loss, or tumor development.

Topics: Analysis of Variance; Animals; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Proliferation; Cerebral Cortex; Embryo, Mammalian; Embryonic Stem Cells; Enzyme Inhibitors; Epidermal Growth Factor; Female; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Kinases; Nerve Tissue Proteins; Neuroblastoma; Neurons; Oncogene Protein v-akt; Pregnancy; Rats; Receptor, trkB; Signal Transduction; STAT3 Transcription Factor; Transfection

In this report we show that exogenous NO added to human neuroblastoma NB69 cells inhibits cell proliferation and downregulates the epidermal growth factor receptor (EGFR) and its downstream signaling pathways. These comprise the 3-phosphoinositide-dependent kinase 1/Akt/glycogen synthase kinase-3beta pathway, the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinases 1 and 2 pathway, and the phospholipase Cgamma pathway. In contrast, NO enhances the EGFR-controlled p38MAPK pathway. We also show that NO enhances the activation of the cAMP-responsive element binding protein, a transcription factor controlled by p38MAPK, as demonstrated using 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190), a p38MAPK inhibitor. These processes are accompanied by the NO-mediated hypophosphorylation of the retinoblastoma protein (pRb), preferentially at Ser795 compared to Ser780 and Ser807/811, and the downregulation of p27(KIP1), p21(CIP1/WAF1), and p16(INK4a), although NO downregulated p16(INK4a) only when the p38MAPK activity was suppressed. The p38MAPK pathway controls the phosphorylation status of pRb as SB202190 enhances the hypophosphorylation of pRb. We reverted the inhibitory action of NO on EGFR and pRb phosphorylation in living cells using cell-permeable reducing agents, which suggested that reversible S-nitrosation controls these proteins. Our results support the notion that NO negatively modulates the p38MAPK-controlled phosphorylation of pRb, inducing the subsequent arrest of the cell cycle at the G1/S transition.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Imidazoles; Neuroblastoma; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Retinoblastoma Protein; Signal Transduction

In previous studies, incubation of doxorubicin-resistant neuroblastoma SK-N-SH (Dox-R) cells with epidermal growth factor (EGF) decreased extracellular signal-regulated kinase activation. Because extracellular signal-regulated kinase activation is associated with cell proliferation, we hypothesized that EGF could induce apoptosis and decrease the rate of cell growth in these cells.. The growth of wild-type (WT) SK-N-SH and Dox-R cells after incubation with EGF concentrations ranging from 1 to 100 ng/mL was determined by a colorimetric assay. Apoptosis was assessed by Hoechst staining and DNA laddering in WT, Dox-R, and cisplatin-resistant cells treated with EGF (100 ng/mL). Cleaved caspase-3 and EGF receptor (human epidermal growth factor receptor [HER1-HER4]) expression were verified by Western blot and reverse transcriptase-polymerase chain reaction.. Epidermal growth factor decreased WT cell growth at concentrations between 50 and 100 ng/mL; Dox-R cell growth was attenuated at all EGF concentrations. Apoptosis was observed in WT and Dox-R cells incubated with EGF. Maximal cleaved caspase-3 expression occurred in WT cells treated with EGF 100 ng/mL and in Dox-R treated with EGF 5 to 10 ng/mL. Epidermal growth factor did not induce apoptosis in cisplatin-resistant cells. HER2 and HER3 transcription was maximal in WT and Dox-R cells, respectively.. Wild-type and Dox-R cells exhibited decreased cell growth after EGF treatment because of apoptosis. This involved caspase-3 activation and could work through HER2 and HER3 receptors.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Epidermal Growth Factor; Humans; Neuroblastoma

We previously reported that epidermal growth factor (EGF) induced cleaved caspase 3 expression and apoptosis in human neuroblastoma cells. We hypothesized that EGF upregulates total caspase 3 expression, thereby enhancing the cytotoxic potency of chemotherapeutic agents.. Wild-type (WT) and doxorubicin-resistant (Dox-R) SK-N-SH neuroblastoma cells were incubated with EGF 0 to 250 ng/mL for 24 hours or with 5 ng/mL for 0 to 5 days, and total caspase 3 expression and transcription were determined by immunoblots and reverse transcription and polymerase chain reaction, respectively. Cell proliferation was determined after EGF (5 ng/mL) incubation for 1 to 5 days. Cells incubated with EGF (5 ng/mL) for 24 hours before doxorubicin treatment were used to determine the effect of EGF on cell replication and cleaved caspase 3 expression.. Wild-type and Dox-R cells had maximal total caspase 3 expression after incubation with EGF (5 ng/mL) for 3 and 5 days, respectively, and a corresponding increase in DNA transcription. Treating the cells with or without EGF (5 ng/mL) resulted in similar replication rate. However, cell death was increased by EGF pretreatment and doxorubicin when compared with that by doxorubicin alone (WT, 53% +/- 8%; Dox-R, 44% +/- 9%; P < .05). Cleaved caspase 3 expression was upregulated when cell death was increased.. Epidermal growth factor upregulates the expression and transcription of total caspase 3 in WT and Dox-R cells in a concentration- and time-dependent manner. Epidermal growth factor pretreatment augments the cytotoxic effect of doxorubicin.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Synergism; Epidermal Growth Factor; Humans; Neuroblastoma; Up-Regulation

Neuroblastoma is an extremely malignant solid tumor in children, characterized by spontaneous differentiation and regression. An epidermal growth factor-like homeotic protein, delta-like (dlk), has been involved in differentiation of neuroblastoma cell lines, but is unknown in in vivo expression of neuroblastoma. By using in situ hybridization and immunohistochemistry, dlk mRNA and protein expression were studied in formalin-fixed archival tissues from 10 patients with neuroblastoma, five with ganglioneuroblastoma, and five with ganglioneuroma. Three adrenal tissues from children died of diseases other than adrenal tumors and one from an adult with pheochromocytoma were severed as normal and disease controls. The results showed strong immunoreactive dlk staining in endothelial cells in neuroblastoma, ganglioneuroblastoma and ganglioneuroma. Dlk was detectable in mature neuromatous stroma and gangliocytes of ganglioneuroma, but not in neuroblasts of neuroblastoma and ganglioneuroblastoma, neither in gangliocytes of ganglioneuroblastoma. In contrast, dlk mRNA expression was mainly observed in the gangliocytes, but was less intense in the neuroblasts and neuromatous stroma cells. Endothelial cells were essentially devoid of dlk mRNA expression. The findings indicated that there is differential expression of dlk gene and protein among neuroblastoma, ganglioneuroblastoma and ganglioneuroma. The stronger expression of dlk in gangliocytes in ganglioneuroma, in contrast to weaker or no expression in gangliocytes in ganglioneuroblastoma and neuroblasts in neuroblastoma, suggests upregulation of dlk during differentiation of neuroblastoma into more benign form. Furthermore, higher dlk protein expression in the tumor endothelium than in the endothelium of normal adrenal gland implies that dlk may regulate the endothelial function in neuroblastic tumors.

Topics: Adolescent; Adult; Child; Child, Preschool; Epidermal Growth Factor; Female; Ganglioneuroblastoma; Ganglioneuroma; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Infant; Infant, Newborn; Male; Neoplasms, Neuroepithelial; Neuroblastoma; RNA, Messenger

The novel cyclic dinucleotide, 3',5'-cyclic diguanylic acid, cGpGp (c-di-GMP), is a naturally occurring small molecule that regulates important signaling mechanisms in prokaryotes. Recently, we showed that c-di-GMP has "drug-like" properties and that c-di-GMP treatment might be a useful antimicrobial approach to attenuate the virulence and pathogenesis of Staphylococcus aureus and prevent or treat infection. In the present communication, we report that c-di-GMP (50 microM) has striking properties regarding inhibition of cancer cell proliferation in vitro. c-di-GMP inhibits both basal and growth factor (acetylcholine and epidermal growth factor)-induced cell proliferation of human colon cancer (H508) cells. Toxicity studies revealed that exposure of normal rat kidney cells and human neuroblastoma cells to c-di-GMP at biologically relevant doses showed no lethal cytotoxicity. Cyclic dinucleotides, such as c-di-GMP, represent an attractive and novel "drug-platform technology" that can be used not only to develop new antimicrobial agents, but also to develop novel therapeutic agents to prevent or treat cancer.

Topics: Acetylcholine; Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Dose-Response Relationship, Drug; Epidermal Growth Factor; Growth Substances; Humans; Kidney; Models, Molecular; Neuroblastoma; Rats; Staphylococcus aureus

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.

Topics: Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Dopamine Agonists; Dopamine Antagonists; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Genetic Vectors; Humans; Indole Alkaloids; Microscopy, Confocal; Neuroblastoma; Piperazines; Platelet-Derived Growth Factor; Quinazolines; Quinpirole; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptors, Dopamine D2; Simplexvirus; Spiperone; Transcriptional Activation; Transfection; Tritium; Tyrphostins

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Cell Adhesion; Cell Size; Crk-Associated Substrate Protein; Epidermal Growth Factor; ErbB Receptors; Fibronectins; HeLa Cells; Humans; Integrins; Mice; Molecular Sequence Data; Neuroblastoma; Phosphotyrosine; Proteins; Rats; Retinoblastoma-Like Protein p130; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tumor Cells, Cultured

Secreted protein acidic and rich in cysteine (SPARC) is a multifunctional matricellular glycoprotein. In vitro, SPARC inhibits the proliferation and migration of endothelial cells stimulated by growth factors and induces endothelial cell apoptosis. We previously showed that SPARC also inhibits angiogenesis in vivo and impairs the growth of the pediatric tumor neuroblastoma (NB). SPARC comprises three domains that are independently folded by a complex pattern of disulfide bonds and have a high degree of structural conservation. In this study, separate modules of the SPARC domains were synthesized as cysteine-linked peptides and tested for their ability to inhibit angiogenesis. Peptide FS-E, representing the epidermal growth factor (EGF)-like module of the follistatin (FS) domain, did not cause endothelial cell apoptosis but strongly inhibited basic fibroblast growth factor (bFGF)-induced endothelial cell migration with an ED(50) = 10 pmol/L. In vivo, peptide FS-E blocked bFGF-stimulated angiogenesis and neovascularization induced by NB cells. The EGF-like conformation was essential for peptide FS-E function because reduction of its two disulfide bonds completely abrogated peptide activity. Peptides FS-K and EC-N, corresponding to part of the Kazal module of the FS domain and the conserved alpha-helix in the extracellular calcium-binding domain, respectively, had minimal to no inhibitory activity. Our data show that the EGF-like module of the SPARC FS domain is angiosuppressive, and its structural conformation is critical for antiangiogenic activity.

Topics: Amino Acid Sequence; Animals; Cell Movement; Conserved Sequence; Endothelium, Vascular; Epidermal Growth Factor; Humans; Molecular Sequence Data; Neovascularization, Pathologic; Neuroblastoma; Osteonectin; Peptide Fragments; Protein Folding; Protein Structure, Tertiary

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homologue-1 (MASH-1 in mouse and HASH-1 in humans) is expressed in specific subsets of embryonic neuronal precursors of both the peripheral and central nervous systems. This gene is essential for development of olfactory and most peripheral autonomic neurons. Neuro-blastoma is a pediatric malignancy derived from sympathetic nervous system precursors and HASH-1 is expressed in a majority of neuroblastoma tumors and cell lines, indicating the immature phenotype of these cells. Using a human neuroblastoma cDNA library and the yeast two-hybrid system to identify novel HASH-1-interacting proteins, we isolated ubiquilin-1 (DA41, hPLIC-1), a gene that contains multiple ubiquitin-related domains. Further analyses showed that ubiquilin-1 interacts not only with HASH-1, but also with other tissue-specific bHLH proteins, including HES-1. Overexpression of ubiquilin-1 led to accumulation of HASH-1 and HES-1. Moreover, ubiquilin-1 was translocated from the cytoplasm to the nucleus upon co-expression with HASH-1. These results indicate that ubiquilin-1 plays an active role in the precise regulation of HASH-1 and of other tissue-specific bHLH proteins.

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy-Related Proteins; Basic Helix-Loop-Helix Transcription Factors; Carrier Proteins; Cell Cycle Proteins; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Gene Library; Humans; Neuroblastoma; Neurons; Nuclear Proteins; PC12 Cells; Rats; Transcription Factors; Tumor Cells, Cultured; Yeasts

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF-induced response, leading to differentiation of almost all cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.

Topics: Animals; Antibodies, Blocking; Apoptosis; Blotting, Western; Brain-Derived Neurotrophic Factor; Carrier Proteins; Caspase 1; Caspase 3; Caspases; Cell Differentiation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Nerve Growth Factor; Neurites; Neuroblastoma; PC12 Cells; Phosphoric Monoester Hydrolases; Phosphorylation; Precipitin Tests; Protein Folding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; PTEN Phosphohydrolase; Rats; Receptor, Nerve Growth Factor; Receptor, trkA; Receptors, Nerve Growth Factor; Recombinant Fusion Proteins; Time Factors; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.

Topics: Apoptosis; Blotting, Western; Cell Division; Cell Survival; Culture Media; Cyclic GMP; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrazines; Immunohistochemistry; Isoenzymes; Neuroblastoma; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrogen Oxides; Phosphorylation; Signal Transduction; Tumor Cells, Cultured

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.

Topics: Animals; Carbohydrate Sequence; Cell Division; Dogs; Epidermal Growth Factor; ErbB Receptors; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Molecular Sequence Data; Neuroblastoma; Phosphorylation; Structure-Activity Relationship; Tumor Cells, Cultured; Tyrosine

The spin-trap, alpha-phenyl-N-tert-butylnitrone (PBN) has been shown to have neuroprotective properties and may prevent oxidative injury in vivo and in cultured cells. Although PBN quenches reactive oxygen species, the direct mechanism of neuroprotective action is unknown. In the present study, we examined the effects of PBN on the regulation of the mitogen activated kinase Erk and as well as Src family tyrosine kinases, enzymes known to be activated by oxygen species such as H2O2. In SH-SY5Y human neuroblastoma cells, H2O2 induced activation of Erk and Src kinases was markedly potentiated by treatment with PBN. The potentiation by PBN of the Erk and Src kinase activation by H2O2 required extracellular Ca2+ and appeared dependent on voltage sensitive Ca2+ channels. In contrast, PBN did not affect depolarization-dependent or growth factor-dependent Erk and Src kinase phosphorylation. Our results suggest that PBN might have a protective effect on cells by potentiating the anti-apoptotic Erk and Src kinase pathways responding to H2O2, an effect apparently distinct from its ability to trap oxygen free radicals.

Topics: Calcium; Calcium Channels, L-Type; Central Nervous System; Cyclic N-Oxides; Drug Interactions; Epidermal Growth Factor; Extracellular Space; Humans; Hydrogen Peroxide; Membrane Potentials; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Nitrogen Oxides; Oxidative Stress; Potassium Chloride; src-Family Kinases; Tumor Cells, Cultured

Polypeptide growth factors secreted from the target tissue determine the choice of transmitter synthesis in the innervating nerves. We have investigated whether they also influence the expression of chromogranins and neuropeptide Y, components co-stored with the neurotransmitters within large dense-core vesicles. IMR-32 and SH-SY5Y human neuroblastoma cells were treated for up to six days with various neurotrophic growth and differentiation factors. For chromogranins A and B, no significant changes at the mRNA level were observed and for chromogranin A this was confirmed at the protein level. The expression of secretogranin II/pro-secretoneurin mRNA, however, was considerably enhanced in both cell lines after basic fibroblast growth factor treatment. In IMR-32 cells we determined a fast and continuous induction, whereas the up-regulation in SH-SY5Y cells was more delayed. A transient elevation of secretogranin II/pro-secretoneurin mRNA levels was seen in SH-SY5Y cells in response to epidermal growth factor. In these cells we also measured the amounts of secretogranin II/pro-secretoneurin protein which were increased by both growth factors. In addition to the above described changes in secretogranin II/pro-secretoneurin biosynthesis we extended and confirmed data available on neuropeptide Y. We found a qualitatively similar pattern of biosynthesis regulation as for secretogranin II/pro-secretoneurin, indicating that the ultimately increased expression of the two proteins may be characteristic of the phenotypic differentiation after growth factor treatment. Moreover, this finding of a concomitant regulation further emphasizes the concept of secretogranin II/pro-secretoneurin being a neuropeptide precursor from which the functional peptide secretoneurin is proteolytically liberated.

Topics: Cell Differentiation; Chromogranin A; Chromogranins; Ciliary Neurotrophic Factor; Cytoplasmic Granules; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neuropeptides; Protein Biosynthesis; Protein Precursors; Proteins; RNA, Messenger; RNA, Neoplasm; Secretogranin II; Tumor Cells, Cultured

The mitogen-activated protein kinase (MAPk) signaling pathway, which plays a critical role in the proliferation of mammalian cells, is frequently up-regulated in human tumors and may contribute to the transformed phenotype. Since a major limitation of current cancer chemotherapy is prevalent resistance to cytotoxic drugs, this study determined whether alterations in growth factor signaling through MAPk may contribute to this phenomenon in human neuroblastoma cell lines. Drug-resistant SKNSH cell lines were established by long-term incubation with increasing concentrations to 10(-6) M doxorubicin (SKNSH rDOX6) or MDL 28842 (SKNSH rMDL6). The expression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-induced EGFR tyrosine phosphorylation were lower in drug-resistant SKNSH cells than their wild-type counterparts. In SKNSH rDOX6 cells, decreased activation and reduced nuclear translocation of MAPk in response to EGF, or lysophosphatidic acid (LPA), or phorbol 12-myristate 13-acetate (PMA), were observed. In SKNSH rMDL6 cells, although MAPk could be activated to wild-type levels by ligand stimulation, the translocation of active MAPk to the nucleus was also reduced. These results suggest that resistance to cytotoxic drugs in human neuroblastoma cell lines is associated with a decrease in growth factor signaling through the MAPk pathway.

Topics: Active Transport, Cell Nucleus; Adenosine; Antineoplastic Agents; Cell Nucleus; Down-Regulation; Doxorubicin; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Lysophospholipids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Neuroblastoma; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In the present study, we first examined the expression of T-cadherin in human CNS by northern blot analysis, immunohistochemical staining, and in situ hybridization. Northern blot analysis demonstrated expression of T-cadherin in human adult cerebral cortex, medulla, thalamus, and midbrain. Immunohistochemical staining with a newly generated monoclonal antibody, designated MA-511, revealed strong expression of T-cadherin in neural cell surface membrane and neurites in adult cerebral cortex, medulla oblongata, and nucleus olivaris. Little or no expression of T-cadherin was found in spinal cord. We further examined T-cadherin expression in various developing nervous systems, and found that T-cadherin expression was lower in developing brain than in adult brain. In situ hybridization revealed that neural cells in medulla oblongata and nucleus olivaris, but not in spinal cord, possessed T-cadherin molecules. We transfected T-cadherin-negative TGW and NH-12 neuroblastoma cells with a T-cadherin cDNA-containing expression vector. T-cadherin-expressing neuroblastoma cells lost mitogenic proliferative response to epidermal growth factor. Epidermal growth factor is known to be required for proliferation of neural stem cells. This finding, together with those of the present study, suggests that T-cadherin functions as a negative regulator of neural cell growth.

Topics: Adult; Antibodies, Monoclonal; Blotting, Northern; Brain Chemistry; Cadherins; Cell Division; Cloning, Molecular; DNA, Complementary; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Molecular Sequence Data; Neuroblastoma; RNA, Messenger; Sequence Homology, Amino Acid; Transfection; Tumor Cells, Cultured

Regulatory interactions among individual receptor-coupled signal transduction systems are critically important for establishing cellular responses in the face of multiple stimuli. In this study, potential regulatory interactions between signal transduction systems activated by growth factor receptors and by G-protein-coupled receptors were examined using human neuroblastoma SH-SY5Y cells which express endogenous epidermal growth factor (EGF) and muscarinic M3 receptors. Activation of muscarinic receptors with carbachol was found to inhibit EGF-induced signaling, including tyrosine phosphorylation of the adaptor protein Cbl and of the EGF receptor, and complex formation between Shc proteins and the EGF receptor and Grb2. Protein kinase C, which is activated by muscarinic M3 receptors, mediated this inhibitory cross-talk. Activation of EGF receptors was found to inhibit muscarinic receptor-induced tyrosine phosphorylation of focal adhesion kinase and paxillin. Reactive oxygen species, which are formed as components of the EGF signaling cascade, mediated this inhibitory cross-talk. These mutual inhibitory interactions demonstrate novel mechanisms for neuronal integration of multiple signals generated by activation of receptors by neurotransmitters and growth factors.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Carbachol; Cell Adhesion Molecules; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; GRB2 Adaptor Protein; Humans; Neuroblastoma; Oncogene Protein v-cbl; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proteins; Receptor, Muscarinic M3; Receptors, Muscarinic; Retroviridae Proteins, Oncogenic; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

This study's goals were to more fully define the activation of protein tyrosine phosphorylation stimulated by muscarinic receptors, to test if this signaling process is affected by oxidative stress induced by H2O2, and to compare the effects of H2O2 on protein tyrosine phosphorylation activated by epidermal growth factor (EGF) receptors. Experiments used human neuroblastoma SH-SY5Y cells which express endogenous M3 muscarinic and EGF receptors. Carbachol induced time-dependent increases in phosphotyrosine immunoreactivity of several protein bands, which were quantitated, and immunoprecipitation was used to identify the adhesion-related proteins focal adhesion kinase, p130Cas/HEF1, and paxillin, and three shc adapter proteins. Carbachol-induced tyrosine phosphorylation of the adhesion-related proteins was mediated by muscarinic receptors, and was inhibited by a src family kinase inhibitor, PP1. That carbachol can activate src family kinases was indicated further by the finding that carbachol induced an increase in tyrosine phosphorylation of p120-src substrate, which was inhibited by PP1. Oxidative stress induced by H2O2 concentration dependently inhibited carbachol-induced tyrosine phosphorylation of each of the adhesion-related proteins. EGF increased the phosphotyrosine immunoreactivity of 180- and 116-kDa proteins, identified as the EGF receptor and Cbl, respectively. In contrast to the results with carbachol, H2O2 potentiated EGF-induced tyrosine phosphorylation. These results demonstrate that muscarinic receptor activation induces previously unrecognized increases in tyrosine phosphorylation, and that this signaling process is impaired by H2O2, whereas protein tyrosine phosphorylation stimulated by EGF is increased by H2O2. Thus, oxidative stress can oppositely modulate protein tyrosine phosphorylation induced by activation of G protein-coupled and growth factor receptors in the same cells.

Topics: Carbachol; Cell Adhesion Molecules; Cytoskeletal Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Hydrogen Peroxide; Muscarinic Agonists; Neuroblastoma; Oxidative Stress; Paxillin; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Receptor, Muscarinic M3; Receptors, Muscarinic; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

The major substrates for the type I insulin-like growth factor (IGF-I) receptor are Shc and insulin receptor substrate (IRS) proteins. In the current study, we report that IGF-I induces a sustained tyrosine phosphorylation of Shc and its association with Grb2 in SH-SY5Y human neuroblastoma cells. The time course of Shc tyrosine phosphorylation parallels the time course of IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK). Transfection of SH-SY5Y cells with a p52 Shc mutant decreases Shc tyrosine phosphorylation and Shc-Grb2 association. This results in the inhibition of IGF-I-mediated ERK tyrosine phosphorylation and neurite outgrowth. In contrast, IGF-I induces a transient tyrosine phosphorylation of IRS-2 and an association of IRS-2 with Grb2. The time course of IRS-2 tyrosine phosphorylation and IRS-2-Grb2 and IRS-2-p85 association closely resembles the time course of IGF-I-mediated membrane ruffling. Treating cells with the phosphatidylinositol 3'-kinase inhibitors wortmannin and LY294002 blocks IGF-I-induced membrane ruffling. The ERK kinase inhibitor PD98059, as well as transfection with the p52 Shc mutant, has no effect on IGF-I-mediated membrane ruffling. Immunolocalization studies show IRS-2 and Grb2, but not Shc, concentrated at the tip of the extending growth cone where membrane ruffling is most active. Collectively, these results suggest that the association of Shc with Grb2 is essential for IGF-I-mediated neurite outgrowth, whereas the IRS-2-Grb2-phosphatidylinositol 3'-kinase complex may regulate growth cone extension and membrane ruffling.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Androstadienes; Cell Membrane; Chromones; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; GRB2 Adaptor Protein; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Models, Biological; Morpholines; Neurites; Neuroblastoma; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Phosphotyrosine; Proteins; Receptor, Insulin; Recombinant Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; src Homology Domains; Transfection; Tumor Cells, Cultured; Wortmannin

The studies on the factors that regulate the biology of the neuroblastoma cell lines may offer important information on the development of tissues and organs that derive from the neural crest. In the present paper we study the action of epidermal growth factor (EGF) on two human neuroblastoma cell lines: SK-N-SH which is composed at least of two cellular phenotypes (neuroblastic and melanocytic/glial cells), and its pure neuroblastic subclone SH-SY5Y. The results show that EGF (10 ng/ml) significantly stimulates the incorporation of [3H]-thymidine in the SK-N-SH cells only in the presence of fetal bovine serum (FBS) (control = 58,285 +/- 9327 cpm; EGF = 75,523 +/- 4457, p < 0.05). Such effect is not observed in the presence of a chemical defined medium, that is, in the absence of FBS (control = 100,997 +/- 4375; EGF = 95,268 +/- 4683; NS) In the SH-SY5Y cells the EGF does not modify the incorporation of [3H]-thymidine either in the presence of 10% of BFS (control = 113,838 +/- 6978; EGF = 119,434 +/- 9441; NS) or in its absence (control = 46,197 +/- 3335; EGF = 44,472 +/- 3493; NS). The results here reported suggest that: a) EGF may affect the proliferation of cells derived from a primary human neuroblastoma; b) this is evident by the EGF-induced increase of [3H]-thymidine incorporation in SK-N-SH cells; c) it is required the presence of other growth factors, present in the FBS, for the mitogenic action to be accomplished; d) since the pure neuroblastic SH-SY5Y cell line are refractory to the EGF, the effects observed in SK-N-SH cells probably occur on the melanocytic/glial cell subpopulation.

Topics: Epidermal Growth Factor; Humans; Neuroblastoma; Thymidine; Tumor Cells, Cultured

The human neuroblastoma line, SK-N-SH, has been subcloned into SH-SY5Y, a neuroblast N cell line, and SH-EP, an epithelial Schwann S cell line. We have previously shown that SH-SY5Y neuroblastoma cells produce insulin-like growth factor II (IGF-II), which acts by an autocrine mechanism to stimulate cell growth. In the current study, we examined the effect of IGF-II on SH-EP neuroblastoma cells. Northern blot and reverse transcriptase-polymerase chain reaction analyses indicate that SH-EP cells do not produce IGF-I or IGF-II but express the type I and type II IGF receptors (IGF-IR and IGF-IIR). Cell surface expression of IGF-IR, assessed by fluorescence-activated sorting, was lower in SH-EP cells than in SH-SY5Y cells. Immunoprecipitation of IGF-IR, followed by anti-phosphotyrosine or anti-IGF-IR immunoblotting, demonstrated functional expression of these receptors in both cell types and confirmed the lower level of IGF-IR expression in SH-EP cells. IGF-II promoted SH-EP cell growth in the presence of low concentrations of calf serum (0.1-0.3%) or 10 ng/ml epidermal growth factor (EGF). IGF-II stimulation of SH-EP growth was eliminated by the IGF-IR blocking antibody (alpha IR-3) but not by an IGF-IIR blocking antibody. Stimulation of cell growth via this receptor was also indicated by the ligand specificity for IGF analogs and insulin (IGF-II approximately IGF-I approximately des(1-3)IGF-I >> insulin). These results indicate that in the presence of a permissive factor such as calf serum or EGF, IGF-II stimulates SH-EP cell growth via the IGF-IR. Collectively, these data suggest that within primary neuroblastomas, IGF-II may act as a paracrine factor to contribute to the promotion of S cell growth.

Topics: Base Sequence; Blotting, Northern; Cell Division; Epidermal Growth Factor; Gene Expression; Humans; Insulin-Like Growth Factor II; Molecular Sequence Data; Neuroblastoma; Phosphorylation; Receptor, IGF Type 1; Receptor, IGF Type 2; Tumor Cells, Cultured; Tyrosine

Growth factors can induce both proliferation or differentiation of neuroblastoma (NB) cells through interaction with specific receptors. Using two automated colorimetric assays for determinations of cell numbers, the present study demonstrates that a) different NB and neuroepithelioma cell lines show distinct responses, both qualitatively and quantitatively, to basic FGF (bFGF), NGF, and EGF; b) even closely related NB cell lines (e.g., SK-N-SH, SH-SY5Y, and SHEP) do not respond uniformly to these factors; c) responses of the two neuroepithelioma cell lines employed (SK-N-MC and CHP-100) differ, but match those of certain NB cell lines; and d) two growth factors, bFGF and EGF, may both stimulate or inhibit proliferation, depending on the cell line studied. Specifically, IMR-32, SK-N-SH, and SH-SY5Y showed a mitogenic response to each growth factor. Maximal proliferative responses ranged from 204-355% as compared to controls (100%). GICAN was stimulated by NGF (199%), and SK-N-MC and NMB by EGF (282 and 140%, respectively), but other factors were ineffective. CHP-100 and GIMEN were inhibited by bFGF. NGF and EGF were not effective on CHP-100 cells, while EGF caused an arrest of mitogenic activity in GIMEN cells, and NGF stimulated their proliferation. Cell lines SHEP and LAN1 did not respond to any factor. To begin to analyze putative relationships of growth factor responsiveness and growth factor/growth factor receptor expressions, IMR-32, GIMEN, and LAN1 cell lines were studied for the presence of bFGF, NGF, FGF receptors (R)-1 (flg) and FGFR-4, trk, and low-affinity NGF receptor (p75) mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Division; Epidermal Growth Factor; Fibroblast Growth Factor 2; Filaggrin Proteins; Growth Substances; Humans; Nerve Growth Factors; Neuroblastoma; Receptors, Growth Factor; Tumor Cells, Cultured

The influence of five anti-hormone and/or anti-growth factor neutralizing antibodies on the in vitro proliferation of four human astrocytic tumor cell lines (U87, U138, U373, H4) is quantitatively described by means of a new tool which makes it possible to evaluate cell growth and cell clone architecture concomitantly. This tool relies upon the combined use of the digital cell image analyses of Feulgen-stained nuclei and the Delaunay and Voronoi mathematical triangulation and paving techniques. Of the five anti-hormone and/or anti-growth factors tested here, the anti-luteinizing hormone-releasing hormone (LHRH) antibody induced the most marked perturbation in the U138 and U373 cell lines, whereas this role was played by the anti-epidermal growth factor (EGF) antibody in the U87 and H4 cell lines. The anti-gastrin (G) antibody significantly modified the growth and/or cell clone architecture of the U138, U87 and H4 cell lines, as did the anti-transforming growth factor alpha (TGFalpha) antibody. The anti-transforming growth factor beta (TGFbeta) antibody modified the growth and/or cell clone architecture of the four cell lines under study. If the five antibodies are taken into consideration, the results strongly suggest that four (the anti-G, the anti-EGF, the anti-LHRH and the anti-TGFalpha) act as inhibitory agents on some glioma cell line proliferation, while the fifth one, i.e. the anti-TGFbeta, act as a stimulator of cell proliferation, perhaps by abrogating the inhibitory effects of TGFbeta on proliferation. A comparison of cell growth data with cell clone architecture characteristics provided further evidence of some specific influence exercised by a given hormone and/or growth factor on glioma cell proliferation. Indeed, the anti-LHRH antibody caused the most pronounced perturbations in the U138 and U373 cell clone architecture; this feature was observed in the H4 cell line and, to a lesser extent in the U87 one after the anti-EGF antibody had been used.

Topics: Antibodies, Monoclonal; Cell Division; Epidermal Growth Factor; Gastrins; Glioblastoma; Gonadotropin-Releasing Hormone; Humans; Neuroblastoma; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Activation of the G-protein-coupled muscarinic (M3) receptor in human neuroblastoma SH-SY5Y cells is known to lead to phosphoinositol hydrolysis and noradrenaline release. In this study, the effect of carbachol on tyrosine phosphorylation and mitogen-activated protein (MAP) kinase activity in SH-SY5Y cells was examined. Carbachol concentration-dependently induced tyrosine phosphorylation of several proteins, including one of 42 kDa. This tyrosine-phosphorylated 42 kDa protein co-eluted from a Mono Q anion-exchange column with MAP kinase activity and with immunologically detected MAP kinase. Stimulation of tyrosine phosphorylation and activation of MAP kinase were also observed after incubation of cells with phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF). Down-regulation or inhibition of protein kinase C (PKC) abolished the stimulatory effects of both carbachol and PMA on MAP kinase activity, whereas EGF-stimulated MAP kinase activity remained unaffected. Thus carbachol acting through the muscarinic (M3) receptor PKC-dependently induced tyrosine phosphorylation and activation of a 42 kDa MAP kinase in SH-SY5Y cells, whereas EGF-induced MAP kinase activation occurred independently of PKC.

Topics: Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Enzyme Activation; Epidermal Growth Factor; Humans; Neuroblastoma; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein Kinases; Receptors, Muscarinic; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine

Lithium ion, which inhibits hydrolytic degradation of inositol monophosphates, is the most common therapeutic agent used in the control of bipolar disorder. There exists evidence that elevated elemental vanadium levels may play an etiological role in at least some forms of manic-depression. Here we demonstrate that vanadate treatment of intact cells from several different clonal lines synergistically induces substantial augmentation in neurotransmitter receptor-mediated or growth factor receptor-triggered inositol trisphosphate accumulation in situ. Furthermore, studies done using cellular extracts indicate that effects of vanadate treatment in situ may be due to its ability to inhibit hydrolysis of inositol 1,4,5-trisphosphate inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in vitro. These results suggest that vanadate treatment may facilitate characterization of inositol phosphate metabolism and intracellular signaling.

Topics: Animals; Carbachol; Cell Line; Clone Cells; Epidermal Growth Factor; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Inositol Polyphosphate 5-Phosphatases; Models, Biological; Nerve Growth Factors; Neuroblastoma; PC12 Cells; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Receptors, Neurotransmitter; Vanadates

Neuroblastoma cell lines and tumors are characterized by low HLA class I expression. The majority of neuroblastoma cell lines and a high percentage of disseminated tumors display amplification of the nuclear protooncogene N-myc. An inverse correlation between HLA class I expression and N-myc amplification and overexpression has been recently described in neuroblastomas (NBs). In this study we have shown that cytokines (recombinant gamma-interferon, recombinant alpha-tumor necrosis factor), differentiation agents (dibutyryl cyclic AMP, phorbol myristate acetate) and growth factors (nerve growth factor, epithelial growth factor) were able to influence the growth rate and surface expression of HLA class I molecules as well as of a tumor-associated antigen on 2 representative NB cell lines. Induced decreased growth rate in NB cells was not always related to decreased N-myc expression. Analysis at the mRNA level revealed that both N-myc and HLA class I RNA steady-state levels could be modulated by several substances, including recombinant gamma-interferon, phorbol myristate acetate, dibutyryl cyclic AMP, and epithelial growth factor and were not necessarily linked. An inverse correlation between N-myc and HLA mRNA levels was observed only after exposure of NB cells to recombinant alpha-tumor necrosis factor. We conclude that N-myc and HLA class I RNA steady-state levels can be modulated independently and suggest that they are not necessarily inversely regulated.

Topics: Blotting, Northern; Bucladesine; Epidermal Growth Factor; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, myc; Histocompatibility Antigens Class I; Humans; In Vitro Techniques; Interferon-gamma; Nerve Growth Factors; Neuroblastoma; Oncogene Protein p55(v-myc); RNA; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

A human neuroblastoma cell line, CA-2E, has been established from a bone-marrow aspirate of a 16-month-old boy with progressive disease. The karyotype and antigen phenotype of the cells correspond to those of a neuroblastoma. This cell line grows well in liquid cultures supplemented with 5% fetal calf serum; conversely, colony formation in semi-solid medium by cells from early passages is dependent upon exogenous EGF. With time in continuous culture, the cloning efficiency in the absence of EGF increases, but the line remains sensitive to EGF, as evidenced by an enhancement of the number and size of colonies. A relative dependence upon EGF in liquid cultures has also been clearly demonstrated by limiting the concentration of serum. Long-term (over 2 weeks) treatment with EGF results in a decreased rate of proliferation, a decreased proportion of clonogenic cells, and the appearance of flat, epithelial-type cells. In some experiments, EGF also has a remarkable effect in inducing neurite outgrowth and process branching. Our results suggest that EGF may have both proliferation- and differentiation-inducing effects on this neuroblastoma cell line. We have also shown that EGF induces increased proliferation in 7 out of 8 other human neuroblastoma cell lines. Functional response of neuroblastoma cells to EGF appears to be a general phenomenon which may be related to a block in the normal maturation pathway of the neural crest cells from which this tumor originates.

Topics: Animals; Antigens, Neoplasm; beta-N-Acetylhexosaminidases; Bone Marrow Diseases; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; Humans; Karyotyping; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Phenotype; Tumor Cells, Cultured; Tumor Stem Cell Assay

Insulin and various growth factors (epidermal growth factor (EGF), insulin-like growth factor, fibroblast growth factor, and transforming growth factor alpha), which fail to modify the resting [Ca2+]i in PC12 rat pheochromocytoma and SKNBE human neuroblastoma cells when administered alone, became capable of inducing [Ca2+]i increases when administered a few (4-20) min after another agent, bradykinin. The latter peptide, working through a B2 receptor, caused hydrolysis of polyphosphoinositides and a large, biphasic [Ca2+]i transient (an initial (1-2 min) spike, originated primarily from intracellular stores, followed by a steady-state elevation dependent on Ca2+ influx). Priming by bradykinin of the growth factor effects was quickly dissipated by the addition of a B2 blocker. Activation of other receptors coupled to polyphosphoinositide hydrolysis: muscarinic and purinergic (in PC12 and SKNBE cells); bombesin and vasopressin receptors (in Swiss 3T3 cells), was without effect in priming. Bradykinin-primed, growth factor-induced [Ca2+]i rises in PC12 cells appeared after a 20-30-s delay; they were relatively small, but persistent; their concentration dependence was similar to that of other effects of the factors; and they included both release of Ca2+ from intracellular stores and stimulation of Ca2+ influx, preceded (in PC12 cells) by a transient increase of polyphosphoinositide hydrolysis. Thus the effect of growth factors (possibly dependent on the tyrosine kinase activity of their receptors) consisted in the reinforcement of the transmembrane signaling at B2 receptors. This is the first direct demonstration of a [Ca2+]i rise induced by insulin and insulin-like growth factor-I, and of such an effect of EGF in cell types endowed with a small number of specific EGF receptors.

Topics: Adrenal Gland Neoplasms; Animals; Bradykinin; Calcium; Calcium Channels; Cell Division; Egtazic Acid; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insulin; Insulin-Like Growth Factor I; Kinetics; Neuroblastoma; Pheochromocytoma; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rats; Receptors, Bradykinin; Receptors, Neurotransmitter; Signal Transduction; Transforming Growth Factors; Tumor Cells, Cultured

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Insulin; Kinetics; Mice; Molecular Weight; Neuroblastoma; Phosphoproteins; Platelet-Derived Growth Factor; Receptor, Insulin; Receptors, Somatomedin; Somatomedins

Using a growth factor defined assay for anchorage-independent growth (van Zoelen, E.J.J., van Oostwaard, Th.M.J., van der Saag, P.T. and de Laat, S.W. (1985) J. Cell. Physiol. 123, 151- 160, we have studied the ability of polypeptide growth factors produced by Neuro-2A neuroblastoma cells to induce anchorage-independent growth of normal rat kidney cells. Neuro-2A cells produce and secrete a PDGF-like growth factor in addition to TGF beta, which can be fully separated from each other by means of reverse-phase HPLC. Using a new, very sensitive technique for detection of TGF beta in growth factor samples based on its additional ability to act as a growth inhibitory factor, it is shown that the PDGF-like growth factor does not contain any detectable TGF beta. Still this neuroblastoma derived PDGF-like growth factor is able to induce anchorage-independent growth of NRK cells, particularly in the additional presence of EGF. It is concluded that under growth factor defined assay conditions TGF beta is not essential for phenotypic transformation of NRK cells.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Growth Substances; Kidney; Mice; Neuroblastoma; Peptides; Platelet-Derived Growth Factor; Rats; Transforming Growth Factors

The induction of ornithine decarboxylase (EC 4.1.1.17) (ODC) by amino acids and by the peptide hormones nerve growth factor (NGF) and epidermal growth factor (EGF) in salts-glucose media has been studied. Only those neutral amino acids taken into the cell via one of the Na+ dependent transport systems stimulate ODC activity. Asparagine and the nonmetabolizable alpha-amino-isobutyric acid (AIB) were used as representatives of this class of inducing amino acids, and their intracellular concentrations were related to the levels of ODC induced. A threshold intracellular concentration of asparagine or AIB has to be attained before ODC can be induced. Further slight increases in intracellular concentrations of asparagine or AIB produce disproportionately large increases of ODC, resulting in a sigmoidal curve of ODC induction. These results, and the fact that the decrease in ODC levels caused by valine is associated with a concurrent decrease in the intracellular level of the inducing amino acid, suggest that the intracellular amino acid level is causally related to the induction of ornithine decarboxylase. Glutamic acid, EGF, and NGF do not induce ODC except in the presence of an inducing amino acid. They act synergistically with the inducing amino acid and produce higher ODC levels at the same intracellular concentration of the inducing amino acid.

Topics: Amino Acids; Aminoisobutyric Acids; Asparagine; Cells, Cultured; Enzyme Induction; Epidermal Growth Factor; Glutamates; Glutamic Acid; Nerve Growth Factors; Neuroblastoma; Ornithine Decarboxylase; Valine

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Mice; Neuroblastoma; Oncogene Proteins v-sis; Peptide Biosynthesis; Peptides; Platelet-Derived Growth Factor; Poly A; Rats; Retroviridae Proteins; RNA, Messenger; Transforming Growth Factors

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.

Topics: Agar; Animals; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Kidney; Mice; Neuroblastoma; Peptides; Phenotype; Rats; Receptors, Cell Surface; Transforming Growth Factors

Mouse neuroblastoma Neuro-2A cells have been cultured in a chemically defined serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 30 nM selenite and 10 micrograms of transferrin per ml. In this medium, which does not contain any externally added polypeptide growth factor, cells proliferate rapidly with a doubling time of approximately equal to 10 hr. During exponential growth in this serum-free medium, Neuro-2A cells secrete a 15- to 20-kDa transforming growth factor with strong mitogenic action and the ability to induce anchorage-independent growth on nontransformed cells. This neuroblastoma-derived transforming growth factor (ND-TGF) is acid and heat stable but is sensitive to treatment with trypsin or dithiothreitol. However, it does not compete with epidermal growth factor (EGF) for receptor binding and does not require EGF receptors for its mitogenic activity. Experiments on the effects of EGF on ND-TGF-induced soft agar growth of normal rat kidney cells indicate that Neuro-2A cells secrete an EGF-potentiated TGF in addition to ND-TGF. It is suggested that Neuro-2A cells can proliferate in the absence of externally added growth factors as a result of autocrine production of polypeptide growth factors.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Neoplasm Proteins; Neuroblastoma; Peptide Biosynthesis; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.

Topics: Animals; Biological Transport, Active; Cell Differentiation; Clone Cells; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Mice; Neoplasms, Experimental; Neuroblastoma; Potassium; Receptors, Cell Surface; Sodium