losartan-potassium and Neuroblastoma

To review evidence on the use of erythropoietic stimulating agents (erythropoietin or darbepoetin) in children with cancer.. A systematic review of the published literature was performed using MEDLINE (1966-July 2007) and references from a Cochrane systematic review (focusing mainly on adults) published in 2006.. The review identified 12 studies, comprising five randomized trials, six case control studies and one open-label, dose-escalation study. All the studies that used adequate doses of recombinant human erythropoietin (rhEPO) (usually 150 IU/kg three times per week) demonstrated benefits for rhEPO except for one study in which rhEPO was added to G-CSF in children with high-risk neuroblastoma. Despite the heterogeneity of the populations studied, in terms of age, tumour type and chemotherapy regimen, rhEPO use was associated with consistent benefits in terms of reduced transfusion requirements and improved haematological parameters. Only one case of darbepoietin use was reported.. While more studies are required, it appears that rhEPO is safe in this vulnerable patient group and can benefit children with cancer by preventing or ameliorating anaemia.

Topics: Anemia; Child; Erythropoietin; Granulocyte Colony-Stimulating Factor; Humans; Neuroblastoma; Recombinant Proteins

Eighty percent of children with cancer suffer from anemia at the time of diagnosis. The physiopathology of anemia is complex. Although anemia can be life threatening, its consequences on the physical, psychological and social state of the child are often minimized. Blood transfusion is the main treatment of anemia: its efficacy is immediate but shortlasting, and it involves infectious and hemolytic risks. The human recombinant erythropoietin has been used for more than 25-years, and is often prescribed to adults with cancer and anemia. The human recombinant erythropoietin rHuEPO is nowadays used when blood transfusion is contra-indicated because of religious or cultural considerations, although several promising studies have been conducted about rHuEPO and children with cancer since 1996: it might be soon the preferential alternative treatment to anemia in children with cancer.

Topics: Adolescent; Anemia; Blood Transfusion; Child; Child, Preschool; Erythropoietin; Female; Humans; Infant; Leukemia; Lymphoma; Male; Neoplasms; Neuroblastoma; Recombinant Proteins

To evaluate the efficacy of recombinant erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) in reducing blood transfusion requirements and stimulating hematopoiesis in children with high-risk neuroblastoma.. Thirty-eight patients given six cycles of intensive induction chemotherapy for high-risk neuroblastoma were randomized to receive G-CSF (n = 20) or G-CSF + EPO (n = 18). Cytokines were given subcutaneously each day, starting 24 hours after each chemotherapy cycle and continuing until 48 hours before the start of the next cycle. The primary end point was the effect of EPO on total red cell transfusion requirements during induction therapy.. Patients who received G-CSF + EPO had a higher red cell transfusion requirement (median, 161.0 mL/kg) than did those who received G-CSF alone (median, 106.6 mL/kg; P =.005). In addition, among patients given transfusions for hemoglobin < or = 8 g/dL, those in the G-CSF + EPO group received more red cell transfusions than did those given G-CSF alone (median per patient, 10 v 8, respectively; P =.044). The two treatment groups had similar cumulative durations of neutropenia, incidences of febrile neutropenia, platelet transfusion requirements, and numbers of platelet transfusions; they also received induction chemotherapy for similar durations and had similar probabilities of progression-free survival and overall survival.. The addition of EPO to the G-CSF regimen provides no benefit for patients receiving intensive induction chemotherapy for high-risk neuroblastoma.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Blood Transfusion; Child; Child, Preschool; Disease-Free Survival; Erythrocyte Count; Erythropoietin; Female; Granulocyte Colony-Stimulating Factor; Humans; Infant; Male; Neoplasm Staging; Neuroblastoma; Survival Analysis; Tennessee

Neuroblastoma (NB) has a low frequency of recurrent mutations compared to other cancers, which hinders the development of targeted therapies and novel risk stratification strategies. Multikinase inhibitors have shown potential in treating high-risk NB, but their efficacy is likely impaired by the cancer cells' ability to adapt to these drugs through the employment of alternative signaling pathways. Based on the expression of 48 growth factor-related genes in 1189 NB tumors, we have developed a model for NB patient survival prediction. This model discriminates between stage 4 NB tumors with favorable outcomes (>80% overall survival) and very poor outcomes (<10%) independently from MYCN-amplification status. Using signaling pathway analysis and gene set enrichment methods in 60 NB patients with known therapy response, we identified signaling pathways, including EPO, NGF, and HGF, upregulated in patients with no or partial response. In a therapeutic setting, we showed that among six selected growth factors, EPO, and NGF showed the most pronounced protective effects in vitro against several promising anti-NB multikinase inhibitors: imatinib, dasatinib, crizotinib, cabozantinib, and axitinib. Mechanistically kinase inhibitors potentiated NB cells to stronger ERK activation by EPO and NGF. The protective action of these growth factors strongly correlated with ERK activation and was ERK-dependent. ERK inhibitors combined with anticancer drugs, especially with dasatinib, showed a synergistic effect on NB cell death. Consideration of growth factor signaling activity benefits NB outcome prediction and tailoring therapy regimens to treat NB.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neoplasm Staging; Nerve Growth Factor; Neuroblastoma; Protein Kinase Inhibitors; Signal Transduction; Survival Analysis

Chemotherapy induces anaemia in neuroblastoma patients. Cancer-associated anaemia may be treated with recombinant erythropoietin. However, the potential effects of erythropoietin on neuroblastoma and kidney cells have not been extensively evaluated. The present study was designed to investigate the effect of erythropoietin on the proliferation, and protection against vincristine- and etoposide-induced cell death in neuroblastoma (MSN), and embryonic kidney (HEK 293) cells.. The expression of erythropoietin and its receptor in MSN and HEK 293 was analysed by RT-PCR, immunocytochemistry, and Western blotting. The effect of erythropoietin on cell viability and proliferation was evaluated by the MTT assay, and by the Click-iT EdU Alexa Fluor 647 kit, respectively. For the cyto-protective assays, cells were incubated with erythropoietin before etoposide and vincristine treatment. Activation of signalling pathways was studied by Western blotting.. MSN and HEK 293 cells expressed the erythropoietin receptor, but not erythropoietin. Erythropoietin induced proliferation and protection against vincristine and etoposide in MSN cells. HEK 293 cells were not affected by erythropoietin. Erythropoietin showed an anti-apoptotic effect which was dependent on the activation of ERK1/2 and AKT. HEK 293 cells presented constitutively phosphorylated AKT, and showed no activation of ERK1/2 upon erythropoietin stimulation.. These results indicate that erythropoietin induces proliferation of MSN cells, and that it can ameliorate vincristine- and etoposide-induced apoptosis of these cells. Erythropoietin-mediated neuroprotection was regulated by the combined effect of the ERK1/2 and AKT signalling pathways. Our findings provide further insights into the potential effect of erythropoietin on neuroblastoma cells.

Topics: Cell Death; Cell Line, Tumor; Cell Proliferation; Erythropoietin; Etoposide; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Kidney; Neuroblastoma; Protective Agents; Proto-Oncogene Proteins c-akt; Receptors, Erythropoietin; Signal Transduction; Vincristine

The final aim of this study was to confirm the neuroprotective effects of recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles stabilized by sodium cholate (rhEPO-Ch-NP) and compare their effects with those of rhEPO using an in vitro model of cerebral ischemia. Glutamate-induced excitotoxic damage on SH-SY5Y cells, a human neuroblastoma cell line, with or without rhEPO-Ch-NPs was quantitatively evaluated. The rhEPO-Ch-NPs were carefully prepared using a water-in-oil-in-water (w/o/w) emulsion solvent evaporation technique with PLGA, sodium cholate hydrate, and ethyl acetate. The rhEPO-Ch-NPs were fully characterized by both transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). In addition, significant intracellular uptake of these particles was monitored by confocal microscopy. Notably, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and nuclear changes observed by 4',6-diamidino-2-phenylindole (DAPI) staining in SH-SY5Y cells demonstrated that rhEPO-Ch-NPs were safer at any concentration investigated and rescued more neuronal cells, while preserving normocytic features against glutamate-induced excitotoxic damages compared to rhEPO.

Topics: Cell Line, Tumor; Cell Survival; Drug Stability; Erythropoietin; Glutamic Acid; Humans; Lactic Acid; Nanoparticles; Neuroblastoma; Neurons; Neuroprotective Agents; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Recombinant Proteins; Sodium Cholate

The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits, comprising the "classical" Epo receptor signaling complex.

Topics: Animals; Apoptosis; Binding Sites; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Erythropoietin; Humans; Neuroblastoma; PC12 Cells; Rats; Receptors, Erythropoietin; RNA, Small Interfering; Staurosporine

Topics: Animals; Erythropoietin; Humans; Nervous System Neoplasms; Neuroblastoma; Schwann Cells

Previous studies have shown that increased vascularity is associated with tumour progression in human neuroblastoma (NB). The involvement of erythropoietin (Epo) in tumour angiogenesis has also been reported. The aim of this study was to correlate microvascular density and Epo/Epo-receptor (EpoR) expression in endothelial and tumour cells to the clinical stage of NB.. Specimens of NB obtained from 20 patients were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. The extent of angiogenesis was found to be up-regulated in advanced disease. In keeping with this observation, Epo/EpoR expression in tumour and endothelial cells, respectively, was also highly correlated with the extent of angiogenesis and higher clinical stage.. The correlation of Epo/EpoR expression with angiogenesis and tumour progression suggests the presence of a loop in the Epo-EpoR system. Epo is secreted by tumour cells and affects vascular endothelial cells via its receptor, promoting tumour angiogenesis in a paracrine manner. Data suggest that Epo represents an important mediator in NB angiogenesis. Understanding the mechanisms of NB angiogenesis provides the basis for a rational approach to the development of antiangiogenic therapy in patients affected by NB.

Topics: Adrenal Gland Neoplasms; Biomarkers, Tumor; Erythropoietin; Fluorescent Antibody Technique, Direct; Humans; Immunoenzyme Techniques; Microcirculation; Neoplasm Staging; Neovascularization, Pathologic; Neuroblastoma; Receptors, Erythropoietin; Spinal Neoplasms

Children with high-risk neuroblastomas (NB) potentially may benefit from treatment with recombinant human erythropoietin (Epo). Epo is a stimulator of erythropoiesis, acting through its receptor (EpoR). The objective of the current study was to evaluate expression levels of Epo and EpoR in NB and in normal tissues and their effects on the proliferation of tumor cells.. A tissue microarray study was performed with 101 primary tumors, 39 paired metastases, 56 paired control tissues, and 6 human NB cell lines. Immunohistochemical staining was performed using antibodies against Epo and EpoR. Immunostaining intensity was evaluated by using a semiquantitative score based on the percentage of positive cells. An in vitro analysis of cell proliferation and cell cycle in the presence of recombinant Epo was performed in the 6 cell lines.. The expression of EpoR was found to be significantly higher in tumors than in paired control tissues (P < .0001) compared with the expression of Epo (P = .06), and the expression of EpoR was significantly higher in lymph node metastases than in paired primary tumors (P = .02) compared with Epo (P = .99). Survival analysis demonstrated that the patients who had tumors with the highest expression of EpoR had a significantly better overall survival (P = .03). In the in vitro study, recombinant Epo did not modify proliferation and cell cycle in the cell lines regardless of the EpoR expression level.. Epo and EpoR were expressed in NB but did not modify tumor cell proliferation. The results of the current study suggested that Epo may be used safely for supportive care in children with NB.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Erythropoietin; Female; Humans; Immunohistochemistry; Infant; Infant, Newborn; Kaplan-Meier Estimate; Male; Neoplasm Staging; Neuroblastoma; Receptors, Erythropoietin; Recombinant Proteins

The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Line; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Humans; Mitogen-Activated Protein Kinases; Neuroblastoma; Prolactin; Proto-Oncogene Proteins c-akt; Receptors, Erythropoietin; Receptors, Prolactin; Recombinant Fusion Proteins; Signal Transduction; STAT5 Transcription Factor; Staurosporine; Transcription Factor RelA

Since apoptosis appeared to be related to neurodegenerative processes, neuroprotection has been involved in investigation of therapeutic approaches focused upon pharmacological agents to prevent neuronal programmed cell death. In this regard, erythropoietin (Epo) seems to play a critical role. The present work was focused on the study of the Epo protective effect upon human neuroblastoma SH-SY5Y cells subjected to differentiation by staurosporine. Under this condition, profuse neurite outgrowth was accompanied by programmed cell death (35% of apoptotic cells by Hoechst assay, showing characteristic DNA ladder pattern). A previous treatment with recombinant human Epo (rHuEpo) increased the expression of the specific receptor for Epo while prevented apoptosis. Simultaneously, morphological changes in neurite elongation and interconnection induced by staurosporine were blocked by Epo. These Epo effects proved to be associated to the induction of Bcl-xL at the mRNA and protein levels (RT-PCR and Western blot after immunoprecipitation) and were mediated by activation of pathways inhibited by wortmannin. In conclusion, the fact that both events induced by staurosporine, cell apoptosis and differentiation, were prevented in SH-SY5Y cells previously exposed to rHuEpo suggests interrelated signaling pathways triggered by the Epo/EpoR interaction.

Topics: Apoptosis; bcl-X Protein; Blotting, Western; Cell Differentiation; Dose-Response Relationship, Drug; Electrophoresis; Enzyme Inhibitors; Erythropoietin; Humans; Kinetics; Microscopy, Fluorescence; Neuroblastoma; Precipitin Tests; Receptors, Erythropoietin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staurosporine

We found that the Cu(II) and Zn(II)-specific chelator Clioquinol (10-50 microM) increased functional hypoxia-inducible factor 1alpha (HIF-1alpha) protein, leading to increased expression of its target genes, vascular endothelial growth factors and erythropoietin, in SH-SY5Y cells and HepG2 cells. Clioquinol inhibited ubiquitination of HIF-1alpha in a Cu(II)- and Zn(II)-dependent manner. It prevents FIH-1 from hydroxylating the asparagine residue (803) of HIF-1alpha in a Cu(II)- and Zn(II)-independent fashion. Therefore, it leads to the accumulation of HIF-1alpha that is prolyl but not asparaginyl hydroxylated. Consistent with this, co-immunoprecipitation assays showed that Clioquinol-induced HIF-1alpha interacted with cAMP-responsive element-binding protein in normoxic cells, implying that Clioquinol stabilizes the trans-active form of HIF-1alpha. Our results indicate that Clioquinol could be useful as an inducer of HIF-1alpha and its target genes in ischemic diseases.

Topics: Asparagine; Carcinoma, Hepatocellular; Cell Hypoxia; Chelating Agents; Clioquinol; Copper; Cyclic AMP Response Element-Binding Protein; Erythropoietin; Humans; Hydroxylation; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Immunoprecipitation; Liver Neoplasms; Neuroblastoma; Oxygen; Peptide Fragments; Procollagen-Proline Dioxygenase; Protein Binding; Protein Processing, Post-Translational; Tumor Cells, Cultured; Ubiquitin; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein; Zinc

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

Topics: Binding Sites; Blotting, Western; Cell Differentiation; CpG Islands; DNA Methylation; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Regulation; Helix-Loop-Helix Motifs; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Neuroblastoma; Nuclear Proteins; Peripheral Nervous System Neoplasms; Receptors, Erythropoietin; Transcription Factors; Tumor Cells, Cultured

Topics: Antineoplastic Combined Chemotherapy Protocols; Blood Transfusion; Clinical Trials as Topic; Disease-Free Survival; Erythropoietin; Humans; Neuroblastoma; Survival Rate

We report the detection of erythropoietin mRNA in the tumor from a patient with neuroblastoma and erythrocytosis. A 2-year-old girl with neuroblastoma presented with hemoglobin 21.3 g/dL, red blood cells 8.03 x 1012/L, and hematocrit 0.641. The serum erythropoietin level was 26.54 mU/mL. The hemoglobin, hematocrit, and serum erythropoietin level were within normal ranges 3 months after surgical excision of the tumor. RT-PCR analysis showed that erythropoietin mRNA expression in the tumor tissue was as high as that of normal renal tissue. These results conclusively demonstrated that the tumor was the site of the ectopic erythropoietin production.

Topics: Child, Preschool; DNA, Neoplasm; Erythropoietin; Female; Humans; Neuroblastoma; Polycythemia; Reverse Transcriptase Polymerase Chain Reaction; RNA

Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were found to express the gene for erythropoietin (EPO) in an oxygen (O(2))-dependent manner. However, NB cells had maximal production of EPO with lower partial pressure of O(2) values than the well-characterized hepatoma cell line HepG2. This maximal EPO expression was preceded by accumulation of the O(2)-sensitive alpha subunit of the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that the amount of the beta subunit of HIF-1, identical to aryl hydrocarbon receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2 increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1alpha, generating a functional HIF-1 complex. Using the hypoxia response element of the human EPO enhancer, we conducted electrophoretic mobility shift assays that showed accumulation and binding of HIF-1 complexes containing both ARNT1 and ARNT2 in NB cells. In addition to the HIF-1 complex, hepatocyte nuclear factor 4alpha (HNF4alpha) was found to be indispensable for hypoxia-induced EPO gene expression in hepatoma cells. Western blot analysis and polymerase chain reaction assessment showed that NB cells express neither HNF4alpha nor the splicing variant HNF4alpha7 and thus express EPO in an HNF4alpha-independent manner. Together, SH-SY5Y and Kelly cells may provide a new in vitro model for studying the mechanism of tissue-specific, hypoxia-inducible EPO gene expression.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; DNA Primers; DNA, Complementary; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Neuroblastoma; Oxygen Consumption; Tumor Cells, Cultured

1. Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. 2. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. 3. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. 4. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. 5. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. 6. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. 7. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels.

Topics: Blotting, Western; Brain Neoplasms; Calcium; Calcium Channels; Cell Line; Electrophysiology; Erythropoietin; Fluorescent Antibody Technique, Direct; Humans; Microscopy, Confocal; Neuroblastoma; Patch-Clamp Techniques; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Nucleus; Dinucleoside Phosphates; DNA Methylation; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, Reporter; HeLa Cells; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; L Cells; Leukemia; Liver; Liver Neoplasms; Luciferases; Mice; Neuroblastoma; Nuclear Proteins; Organ Specificity; Recombinant Fusion Proteins; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Erythropoietin is a growth factor. Cancer can be described as disturbance of the fine balance of positive and negative growth control mechanisms. The effect of human recombinant erythropoietin (EPO) was studied on the cell growth and differentiation of a human neuroblastoma cell line (h-NMB). Cell growth curves, trypan blue staining and thymidine uptake were used to assess cell proliferation and death. To assess cell differentiation, neutral endopeptidase (cell membrane enzyme marker), creatine kinase (cytosolic enzyme marker), dopamine uptake (dopamine transporter marker) and cell morphology were determined. Specific EPO receptor mRNA, by RT-PCR technique, was demonstrated. The incubation of erythropoietin with the tumor cell line resulted in inhibition of cell proliferation as evidenced in a diminished cell growth. EPO was shown to have induced a differentiation process as seen from the two different enzymatic markers, membranal and cytosolic, and from the cells dopamine uptake studies. However, the morphological changes did not document a full differentiation effect. EPO specific antibodies blocked the effects of EPO on cell proliferation and creatine kinase activity. In this study, EPO did not produce any sign of proliferation in the nervous tumor cell line used.

Topics: Base Sequence; Cell Differentiation; Cell Division; Cell Line; Creatine Kinase; DNA Primers; Dopamine; Erythropoietin; Humans; Kinetics; Molecular Sequence Data; Neprilysin; Neuroblastoma; Polymerase Chain Reaction; Receptors, Erythropoietin; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

A 4 year-old child being treated for neuroblastoma developed erythroblastopenia. We used specific erythroid lineage markers (hemoglobin and spectrin) and in vitro erythroid colony assays to characterize this hematologic picture and discuss its relationships with the disease and its treatment.

Topics: Bone Marrow; Cells, Cultured; Erythroblasts; Erythropoietin; Fluorescent Antibody Technique; Humans; Infant; Male; Neuroblastoma; Thalassemia