losartan-potassium and Carcinoma--Hepatocellular

Erythropoietin-producing hepatocellular carcinoma A4 (EphA4) is a transmembrane receptor protein which is a part of the most prominent family of receptor tyrosine kinases (RTKs). It serves a crucial role in both physiological, biological, and functional states binding with their ligand like Ephrins. Its abundance in the majority of the body's systems has been reported. Moreover, it draws much attention in the CNS since it influences axonal and vascular guidance. Also, it has a widespread role at the pathological state of various CNS disorders. Reports suggest it obstructs axonal regeneration in various neurodegenerative diseases and neurological disorders. Although, neuro-regeneration is still an open challenge to the modern drug discovery community.  Hence, in this review, we will provide information about the role of EphA4 in neurological diseases by which it may emerge as a therapeutic target for CNS disease. We will also provide a glance at numerous signaling pathways that activate or inhibit the EphA4-associated biological processes contributing to the course of neurodegenerative diseases. Thus, this work might serve as a basis for futuristic studies that are related to the target-based drug discovery in the field of neuro-regeneration. Pathological and physiological events associated with EphA4 and Ephrin upregulation and interaction.

Topics: Carcinoma, Hepatocellular; Erythropoietin; Humans; Liver Neoplasms; Nervous System Diseases; Protein Binding

The plasma level of erythropoietin (Epo) in anemic patients suffering from inflammation is often low in relation to the blood hemoglobin concentration. Various proinflammatory cytokines have been tested for their action on the synthesis of Epo. Interleukin 1 (IL-1) and tumor necrosis factor-alpha(TNF-alpha) suppress Epo gene expression in isolated perfused rat kidneys and in human hepatoma cell cultures. IL-6 inhibits in the kidney, and conflicting results have been reported for its effect on Epo synthesis in hepatic cells. Several other cytokines tested were without effect. Thus, mainly IL-1 and TNF-alpha seem to be responsible for the defect in Epo production in severe systemic and renal inflammatory diseases.

Topics: Animals; Carcinoma, Hepatocellular; Cytokines; Erythropoietin; Humans; Inflammation; Kidney; Liver Neoplasms; Tumor Cells, Cultured

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Enhancer Elements, Genetic; Erythropoietin; Humans; Liver Neoplasms; Mice; Molecular Sequence Data; Regulatory Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Erythropoietin; Gene Expression; Gene Expression Regulation; Humans; Liver Neoplasms; Mice; Mice, Transgenic; Molecular Sequence Data; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Tumor Cells, Cultured

Topics: Adult; Budd-Chiari Syndrome; Carcinoma, Hepatocellular; Erythropoiesis; Erythropoietin; Female; Humans; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Polycythemia

The triad of hemochromatosis, hepatoma and erythrocytosis is a rare combination. Hemochromatosis is often not recognized until the patient presents with the symptoms of hepatocellular carcinoma and erythrocytosis, and the development of erythrocytosis is an important clue to the under-lying hepatoma. The high serum iron concentration and the high saturation of the iron-binding protein, as well as the typical bone marrow hemosiderin pattern, are important aids in the recognition of hemochromatosis. To date, all patients with this triad have been elderly males. The clinical course is usually one of rapid deterioration and death. The seven previously reported cases have been reviewed and the relationship of the erythrocytosis to the increased production of erythropoietin is discussed.

Topics: Aged; Carcinoma, Hepatocellular; Erythropoietin; Hemochromatosis; Humans; Liver Neoplasms; Male; Middle Aged; Polycythemia

Topics: Adenocarcinoma; Adrenal Gland Neoplasms; Animals; Brain Neoplasms; Carcinoma; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cysts; Erythropoietin; Esophageal Neoplasms; Female; Growth Substances; Haplorhini; Humans; In Vitro Techniques; Kidney Neoplasms; Leiomyoma; Leukemia; Leukemia, Experimental; Liver Neoplasms; Lymphoma; Male; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Pheochromocytoma; Polycythemia; Rats; Sarcoma, Experimental; Skin Neoplasms; Time Factors; Wilms Tumor

Topics: Adolescent; Adrenocorticotropic Hormone; Adult; Brain Neoplasms; Carcinoma; Carcinoma, Bronchogenic; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cerebellar Neoplasms; Child; Child, Preschool; Choriocarcinoma; Cushing Syndrome; Diagnosis, Differential; Erythropoietin; Female; Gonadotropins; Hemangiosarcoma; Hormones, Ectopic; Humans; Hypercalcemia; Hypoglycemia; Infant; Infant, Newborn; Insulin; Insulin Secretion; Kidney Neoplasms; Liver Neoplasms; Lung Neoplasms; Male; Neoplasms; Parathyroid Hormone; Pheochromocytoma; Polycythemia; Pregnancy; Teratoma; Testicular Neoplasms; Thymus Neoplasms

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron-deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady-state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf-mediated iron uptake with anti-Tf receptor antibody 42/6, stimulated EPO production in serum-deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES-DFO) and in patients with advanced malignancy treated with anti-Tf receptor antibody 42/6, in a time- and dose-dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.

Topics: Adult; Antibodies, Monoclonal; Blood Cell Count; Carcinoma, Hepatocellular; Cell Hypoxia; Deferoxamine; Dose-Response Relationship, Drug; Double-Blind Method; Erythropoietin; Ferritins; Humans; Iron; Iron Deficiencies; Liver Neoplasms; Male; Neoplasms; Receptors, Transferrin; Tumor Cells, Cultured

We evaluated the benefit of autologous blood transfusion and the effect of recombinant human erythropoietin (rh-EPO) on preoperative autologous blood donation for hepatectomy in patients with cirrhosis.. Forty-two patients with cirrhosis and hepatocellular carcinoma underwent hepatectomy, 21 of whom (group A) donated autologous blood before operation. Eleven of these patients (group A1) were administered rh-EPO before operation, and ten patients (group A2) were untreated. Twenty-one patients (group B) did not donate autologous blood.. The frequency of homologous blood transfusion was 24% in group A and 62% in group B (p < 0.05). Preoperative erythropoiesis increased markedly in group A1, and postoperative erythropoietin production was not suppressed in this group. Postoperative hematocrits recovered significantly more rapidly in patients transfused with only autologous blood. Postoperative serum total bilirubin concentrations were significantly higher in patients with transfused homologous blood.. Autologous blood transfusion yields clinically superior results for hepatectomy in patients with cirrhosis when compared with homologous transfusion. Preoperative rh-EPO administration minimizes presurgical decreases in hematocrit caused by autologous blood donation.

Topics: Adult; Aged; Bilirubin; Blood Transfusion, Autologous; Carcinoma, Hepatocellular; Erythropoietin; Female; Hematocrit; Hepatectomy; Humans; Liver Cirrhosis; Liver Function Tests; Liver Neoplasms; Male; Middle Aged; Postoperative Complications; Recombinant Proteins

Epstein-Barr virus (EBV) is a human tumor-associated virus that encodes various microRNAs. EBV infection causes a variety of malignant tumors, including nasopharyngeal carcinoma and gastric cancer, etc. EBV-associated gastric cancer (EBVaGC) has unique molecular characteristics from other gastric cancers, but its pathogenic mechanism remains unclear. In recent years, erythropoietin-producing human hepatocellular 2 (EphA2) has been reported to be highly expressed in various cancers and promote tumor growth and metastasis. As an important cancer oncogene, EphA2 is a potential therapeutic target. However, whether EBV is involved in the regulation of EphA2 and thus affects the progression of EBVaGC remains unclear. In this study, we found that the expression of EphA2 in EBVaGC cells was significantly lower than that in EBV-negative gastric cancer (EBVnGC) cells. Additionally, overexpression of EphA2 in EBVaGC cells promoted migration and proliferation, and inhibited autophagy. EBV-miR-BART1-3p and BART18-5p were found to target the 3'-UTR of EphA2 and down-regulate its expression. Our results suggest that EBV may be involved in gastric cancer progression by targeting EphA2 through BART1-3p and BART18-5p.

Topics: Autophagy; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Epstein-Barr Virus Infections; Erythropoietin; Herpesvirus 4, Human; Humans; Liver Neoplasms; MicroRNAs; Stomach Neoplasms

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO-EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal-regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR-hepatocyte nuclear factor 4 alpha-EPO axis in human liver tumorigenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Constitutive Androstane Receptor; Erythropoietin; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver; Liver Neoplasms; Mice; Mice, Nude

EphA2 is a key factor underlying invasive propensity of gliomas, and is associated with poor prognosis of tumors. We aimed to develop a radiomics-based imaging index for predicting EphA2 expression in diffuse gliomas, and further estimating its value for grading of tumors.. A total of 182 patients with diffuse gliomas were included. All subjects underwent pre-operative MRI and post-operative pathological diagnosis. EphA2 expression of tumors was scored on pathological sections with immunohistochemical staining using monoclonal EphA2 antibody. MRI radiomics features were extracted from three-dimensional contrast-enhanced T1-weighted imaging and diffusion kurtosis imaging. Predictive models were constructed using machine learning-based radiomics features selection and three classifiers for predicting EphA2 expression and tumor grade. Features of best EphA2 expression model were subsequently used to construct another model of tumor grading. For each model, 146 cases (80%) were randomly picked as training and the rest 36 (20%) were testing cohorts. EphA2 expression was further correlated to the radiomics features in both grade models using Spearman's correlation.. Logistic regression model presented highest performance for predicting EphA2 expression (AUC: 0.836/0.724 in training/validation set). Tumor gradings model guided by features from EphA2 expression model demonstrated comparable performance (AUC: 0.930/0.983) to that constructed directly using imaging radiomics features (AUC: 0.960/0.977). Two radiomics features which included in both LR-grade models showed strong correlation (P < 0.05) with EphA2 expression.. The expression of EphA2 in gliomas could be predicted by radiomics features extracted from diffusion kurtosis MRI, which could also be used to assist tumor grading.

Topics: Brain; Brain Neoplasms; Carcinoma, Hepatocellular; Erythropoietin; Glioma; Humans; Liver Neoplasms; Magnetic Resonance Imaging; Receptors, Erythropoietin; Retrospective Studies

miR-10b-5p was lowly expressed in HCC, while EphA2 was highly expressed. Cell experiments revealed that miR-10b-5p overexpression or EphA2 knockdown could reduce cell proliferation, accelerate apoptosis, strongly upregulate Bax and Caspase-3, and downregulate Bcl-2. In contrast, miR-10b-5p knockdown or EphA2 overexpression gave rise to reverse biological phenotypes. Furthermore, dual luciferase reporter assay verified that miR-10b-5p was a target of EphA2, and the rescue experiment implied that transfection of pCMV-EphA2 or Si-EphA2 could reverse EphA2 expression and cell biological functions caused by miR-10b-5p overexpression or knockdown.. miR-10b-5p reduced HCC cell proliferation but accelerate apoptosis by regulating EphA2, suggesting it has the potential to be a clinical target for HCC.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Erythropoietin; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Invasiveness; Receptor, EphA2; Transfection; Up-Regulation

The development of endometriotic lesions is crucially dependent on the formation of new blood vessels. In the present study, we analysed whether this process is regulated by erythropoietin-producing hepatoma receptor B4 (EphB4) signalling.. We first assessed the anti-angiogenic action of the EphB4 inhibitor NVP-BHG712 in different in vitro angiogenesis assays. Then, endometriotic lesions were surgically induced in the dorsal skinfold chamber and peritoneal cavity of NVP-BHG712- or vehicle-treated BALB/c mice. This allowed to study the effect of EphB4 inhibition on their vascularisation and growth by means of intravital fluorescence microscopy, high-resolution ultrasound imaging, histology and immunohistochemistry.. Non-cytotoxic doses of NVP-BHG712 suppressed the migration, tube formation and sprouting activity of both human dermal microvascular endothelial cells (HDMEC) and mouse aortic rings. Accordingly, we also detected a lower blood vessel density in NVP-BHG712-treated endometriotic lesions. This was associated with a reduced lesion growth due to a significantly lower number of proliferating stromal cells when compared to vehicle-treated controls.. Inhibition of EphB4 signalling suppresses the vascularisation and growth of endometriotic lesions. Hence, EphB4 represents a promising pharmacological target for the treatment of endometriosis.

Topics: Animals; Carcinoma, Hepatocellular; Endometriosis; Endothelial Cells; Erythropoietin; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Receptor, EphB4

Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

Topics: Amyloid Neuropathies, Familial; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Survival; Dynamic Light Scattering; Erythropoietin; Humans; Liver Neoplasms; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; RNA, Messenger

To evaluate erythropoietin (Epo) and erythropoietin receptor (EpoR) expression, its relationship with vasculogenic mimicry (VM) and its prognostic value in human hepatocellular carcinoma (HCC), we examined Epo/EpoR expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-Schiff) double staining on 92 HCC specimens. The correlation between Epo/EpoR expression and VM formation was analyzed using two-tailed Chi-square test and Spearman correlation analysis. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between Epo/EpoR expression and VM formation (P < 0.05). Patients with Epo or EpoR expression exhibited poorer overall survival (OS) than Epo-negative or EpoR-negative patients (P < 0.05). Epo-positive/VM-positive and EpoR-positive/VM-positive patients had the worst OS (P < 0.05). In multivariate survival analysis, age, Epo and EpoR were independent prognostic factors related to OS. These results will provide evidence for further research on HCC microcirculation patterns and also will provide new possible targets for HCC diagnosis and treatment.

Topics: Age Factors; Biomarkers, Tumor; Carcinoma, Hepatocellular; Chi-Square Distribution; Erythropoietin; Female; Hepatectomy; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Male; Middle Aged; Molecular Mimicry; Multivariate Analysis; Platelet Endothelial Cell Adhesion Molecule-1; Proportional Hazards Models; Receptors, Erythropoietin; Risk Factors; Time Factors; Treatment Outcome

Increased red blood cell count (Erythrocytosis) is an important paraneoplastic syndrome of hepatocellular carcinoma (HCC) and is a significant risk factor for lethal lung artery thromboembolism. HCC-associated erythrocytosis is partially caused by the ability of several HCC cells to produce erythropoietin (EPO). Prolyl-4-hydroxylase 2 (PHD2) is an enzyme encoded by the gene EGLN1. The best-known function of PHD2 is to mediate the oxygen-dependent degradation of the labile α-subunit of hypoxia-inducible factor (HIF). However, there is increasing evidence that PHD2 also regulates HIF-independent pathways by interacting with other substrates.. In the EPO-producing human HCC cell line HepG2, the expression of PHD2 gene was silenced with siRNA. EPO production was estimated using quantitative PCR and ELISA.. In HepG2 cells, PHD2 suppresses the activity of TGF-β1 pathway and consequently maintains the expression of hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor promoting the EPO expression in hepatocytes. PHD2 knockdown caused a marked reduction of EPO production. HIF seemed not to be involved in this biology.. Our findings show that PHD2 represents a potential contributing factor for HCC-associated erythrocytosis. Selective inhibition of PHD2 in HCC cells might be considered as a new way to manage erythrocytosis in HCC patients.

Topics: Carcinoma, Hepatocellular; Erythropoietin; Hep G2 Cells; Hepatocyte Nuclear Factor 4; Humans; Liver Neoplasms; Polycythemia; Prolyl Hydroxylases; Signal Transduction; Transforming Growth Factor beta1

NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Tumor; Erythropoietin; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; HEK293 Cells; Humans; Liver Neoplasms; NADPH-Ferrihemoprotein Reductase; Protein Binding; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Scavenger Receptors, Class A; Two-Hybrid System Techniques

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy.

Topics: Acrylic Resins; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Dendrimers; DNA; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Viral; Genetic Therapy; HEK293 Cells; Humans; Hypoxia; Luciferases; Plasmids; Promoter Regions, Genetic; Simplexvirus; Thymidine Kinase; Transfection

Transcription factor GATA-4 is expressed in early fetal liver and essential for organogenesis. It is also implicated in carcinogenesis in several endoderm-derived organs. Hepatoblastoma (HB), the most common malignant pediatric liver tumor, has features of fetal liver including extramedullary hematopoiesis. We investigated the expression of GATA-4 and its purported target gene erythropoietin (Epo) in liver tumors and the role of GATA-4 in HB pathogenesis.. Immunohistochemistry, Western blotting, and reverse transcription-polymerase chain reaction were used for liver samples from patients with HB or hepatocellular carcinoma. To further investigate the role of GATA-4 in pediatric liver tumors, we used adenoviral transfections of wild-type or dominant negative GATA-4 constructs in the human HB cell line, HUH6.. We found abundant GATA-4 expression in both types of liver tumors in children, whereas it was absent in adult hepatocellular carcinoma. A close family member GATA-6 was expressed in a minority of childhood but not adult liver tumors. Epo, present in the fetal liver, was also expressed in childhood liver tumors. Moreover, cell line HUH6 was GATA-4 positive and produced Epo. We found that altering the amount of functional GATA-4 in HUH6 cells did not significantly affect either proliferation or apoptosis.. GATA-4 is abundant in pediatric liver tumors, but unraveling its exact role in these neoplasms requires further investigation.

Topics: Adenoviridae; Adult; Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Child; Erythropoietin; Fetus; GATA4 Transcription Factor; GATA6 Transcription Factor; Hepatoblastoma; Humans; Liver; Liver Neoplasms; Mice; Mice, Inbred C57BL; Transfection

Topics: Adult; Anemia; Antiviral Agents; Carcinoma, Hepatocellular; Cohort Studies; Drug Therapy, Combination; Erythropoietin; Female; Hepatitis C, Chronic; Humans; Liver Neoplasms; Male; Middle Aged; Thromboembolism

Hypoxia-inducible factor-1alpha (HIF-1alpha) is destabilized via the ubiquitin-proteasome system. Thus HIF-1alpha expression is robustly upregulated by proteasome inhibition, but paradoxically its activity is reduced. In the present study, we investigated the mechanism underlying the paradoxical response of HIF-1alpha to proteasome inhibition. In both Hep3B and HEK293 cells, a proteasome inhibitor MG132 noticeably attenuated hypoxic induction of erythropoietin and VEGF mRNAs. MG132 inactivated HIF-1alpha C-terminal transactivation domain (CAD), independently of factor inhibiting HIF-1 (FIH) and inhibited p300 recruitment by HIF-1alpha. We next tested the possibility that CITED2 is involved in the HIF-1 inactivation. CITED2 was found to be degraded via the ubiquitin-proteasome system and thus was stabilized by proteasome inhibition. Both the activity and the p300 binding of HIF-1alpha were inhibited by CITED2 expression and recovered by CITED2 siRNA in the presence of MG132. These results suggest that CITED2 is stabilized by proteasome inhibition and inactivates HIF-1 by interfering with the HIF-1alpha-p300 interaction. This may be an important mode-of-action for proteasome inhibition-based cancer therapy.

Topics: Carcinoma, Hepatocellular; Cell Hypoxia; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E1A-Associated p300 Protein; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoprecipitation; Kidney; Leupeptins; Liver Neoplasms; Mixed Function Oxygenases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Repressor Proteins; RNA, Messenger; Trans-Activators; Transcription Factors; Ubiquitin; Vascular Endothelial Growth Factor A

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPalpha showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPalpha binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

Topics: Animals; Antibodies; Antimicrobial Cationic Peptides; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-alpha; Cell Line, Tumor; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Gene Expression; Hepatocytes; Hepcidins; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Promoter Regions, Genetic; Receptors, Erythropoietin; RNA, Messenger; Signal Transduction

Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins.

Topics: Carcinoma, Hepatocellular; Cell Hypoxia; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Phenotype; Procollagen-Proline Dioxygenase; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

To correlate microvascular density and erythropoietin (Epo)/Epo-receptor (EpoR) expression in endothelial and tumour cells with histopathological type in hepatocellular carcinoma (HCC).. Specimens of primary HCC obtained from 50 patients who had undergone curative hepatectomy were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. Poorly differentiated HCC had a higher degree of vascularization than other stages and Epo/EpoR expression in both tumour and endothelial cells increased in parallel with grade of malignancy and was highly correlated with the extent of angiogenesis.. Epo/EpoR levels correlate with angiogenesis and progression of patients with HCC and these findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. It is thus suggested that Epo is an important factor in hepatic tumour angiogenesis. Understanding the mechanisms of HCC angiogenesis provides a basis for a rational approach to the development of antiangiogenic therapy in patients with hepatic cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Hepatocellular; Disease Progression; Erythropoietin; Female; Hepatectomy; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Microcirculation; Middle Aged; Neovascularization, Pathologic; Receptors, Erythropoietin

The hypoxia-inducible factor (HIF) pathway is crucial in mitigating the deleterious effects of oxygen deprivation. HIF-alpha is an essential component of the oxygen-sensing mechanisms and under normoxic conditions is targeted for degradation via hydroxylation by HIF-prolyl hydroxylases. Several HIF-prolyl hydroxylase inhibitors (PHIs) induced erythropoietin (epo) expression in vitro and in mice, with peak epo expression ranging from 5.6- to 207-fold above control animals. Furthermore, several PHIs induced fetal hemoglobin (HbF) expression in primary human erythroid cells in vitro, as determined by flow cytometry. One PHI, FG-2216, was further tested in a nonhuman primate model without and with chronic phlebotomy. FG-2216 was orally bioavailable and induced significant and reversible Epo induction in vivo (82- to 309-fold at 60 mg/kg). Chronic oral dosing in male rhesus macaques was well tolerated, significantly increased erythropoiesis, and prevented anemia induced by weekly phlebotomy. Furthermore, modest increases in HbF-containing red cells and reticulocytes were demonstrated by flow cytometry, though significant increases in HbF were not demonstrated by high-pressure liquid chromatography (HPLC). HIF PHIs represent a novel class of molecules with broad potential clinical application for congenital and acquired anemias.

Topics: Administration, Oral; Animals; Carcinoma, Hepatocellular; Chronic Disease; Enzyme Inhibitors; Erythrocytes; Erythroid Cells; Erythroid Precursor Cells; Erythropoietin; Fetal Hemoglobin; Flow Cytometry; Humans; Hypoxia; Liver Neoplasms; Macaca mulatta; Male; Mice; Oxygen; Phlebotomy; Polycythemia; Procollagen-Proline Dioxygenase; Reticulocytes

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

Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.

Topics: Animals; Anti-Bacterial Agents; Aryl Hydrocarbon Receptor Nuclear Translocator; Carcinoma, Hepatocellular; Cell Hypoxia; Colonic Neoplasms; Disulfides; DNA-Binding Proteins; E1A-Associated p300 Protein; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Indole Alkaloids; Liver Neoplasms; Luciferases; Male; Mice; Mice, Nude; Nuclear Proteins; Prostatic Neoplasms; Protein Binding; Receptors, Aryl Hydrocarbon; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Transcription Factors; Transcription, Genetic; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1beta or TNF-alpha treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1beta or TNF-alpha in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1beta or TNF-alpha. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.

Topics: Administration, Oral; Animals; Anisoles; Azepines; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; DNA Primers; DNA-Binding Proteins; Erythropoietin; GATA2 Transcription Factor; GATA3 Transcription Factor; Hemoglobins; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-1; Liver Neoplasms; Mice; Mice, Inbred ICR; Nuclear Proteins; Promoter Regions, Genetic; Trans-Activators; Transcription Factors; Transfection; Tumor Necrosis Factor-alpha

Quinolinic acid (QA) is a potent endogenous excitotoxin; elevation of its concentration in an organism has been implicated in the pathogenesis of various disorders. The purpose of this study was the assessment of QA impact on the process of erythropoiesis. Marked increase of QA concentration was observed in plasma and peripheral tissues of uremic rats. These changes were proportional to the amount of the removed renal tissue and positively correlated with the concentration of creatinine but negatively correlated with hematological parameters, i.e., hematocrit and Hb red blood cells count. The changes were accompanied by a slight decrease in the concentration of endogenic erythropoietin (EPO) in the plasma of animals with uremia. Chronic treatment with QA diminished the increase in EPO concentration after introduction of cobalt in rats. These changes were associated with the decrease in all hematological parameters after QA administration. The in vitro study in the conditions of hypoxia showed that QA inhibited the EPO release from HepG2 cells to the culture base. Additionally, in HepG2 cells QA had a dose-dependent inhibitory effect on hypoxia- and cobalt-induced EPO gene expression without any cell toxicity. In conclusion, the erythropoiesis in chronic renal failure could be attributed to the influence of QA on EPO synthesis. Thus we propose that QA can be a uremic toxin responsible for anemia in animals or patients with renal failure.

Topics: Anemia; Animals; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Survival; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Humans; Kidney Failure, Chronic; Male; Neurotoxins; Organ Specificity; Quinolinic Acid; Rats; Rats, Wistar; RNA, Messenger; Tumor Cells, Cultured

Long-term exposure of rats to cadmium (Cd) resulted in a marked suppression of erythropoietin (Epo) mRNA expression in the kidneys and the development of severe anemia. A recent report revealed that Cd inhibited hypoxia-inducible factor 1 (HIF-1) binding activity and Epo mRNA expression and protein production. However, Epo gene expression is also regulated by transcription factor GATA-2, which binds to the GATA binding site of the Epo promoter. To elucidate the mechanism of suppression of Epo by Cd, the effect of Cd on GATA-2 function was studied. Epo promoter/enhancer luciferase constructs, one with the wild-type promoter and another with a promoter with a mutant GATA site, were transfected into Hep3B cells. No significant difference in Epo promoter activity in these two types of cells was observed in the presence of Cd. The binding activity of GATA-2 was not affected by Cd. This study showed that Cd inhibited HIF-1 binding activity and Epo promoter activity, and then suppressed Epo protein production. Inhibition of Epo gene expression by Cd depends on suppression of HIF-1 binding activity, not on alteration of GATA function.

Topics: Cadmium; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; Dose-Response Relationship, Drug; Erythropoietin; GATA2 Transcription Factor; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Nuclear Proteins; RNA, Messenger; Transcription Factors; Transfection

Topics: Aged; Anemia, Refractory; Anemia, Refractory, with Excess of Blasts; Carcinoma, Hepatocellular; Cell Differentiation; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Humans; Hypersplenism; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Myelodysplastic Syndromes; Pancytopenia; Reticulocyte Count; Reticulocytes

Angiopoietin 1 (Ang-1) and its antagonist, angiopoietin 2 (Ang-2), are novel ligands that regulate the Tie2 receptor. The Ang-2 gene is upregulated in the hypervascular type of human hepatocellular carcinoma (HCC). To gain a better understanding of the role of the Ang-Tie2 system in HCC the expression of these genes was investigated in a series of human HCCs.. The expression of the angiopoietin and Tie2 proteins was investigated in nine normal liver tissues and 52 surgically resected HCCs. In addition, the effects of hypoxic stimuli on Ang-1, Ang-2, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) expression was investigated in Hep3B cells.. Ang-1, rather than Ang-2, was more frequently expressed in the normal liver. Ang-1 was expressed in 68% of HCCs, whereas Ang-2 was expressed in 81%, and was significantly higher in poorly differentiated HCCs characterised by high vascularity (p = 0.02), and in tumours with a peliotic change (p = 0.02). Strong expression of Tie2 was seen in tumour vessels in accordance with Ang-2 expression. In Hep3B cells, hypoxic stimuli upregulated VEGF and EPO, but not Ang-1 or Ang-2.. These data support the evidence that the reversal of Ang-1 and Ang-2 expression plays an important role in the angiogenic and dedifferentiation processes in HCC. The hypoxic stimuli were not responsible for Ang-2 upregulation, unlike that of VEGF, in human HCC cells.

Topics: Aged; Aged, 80 and over; Angiopoietin-1; Angiopoietin-2; Angiopoietins; Carcinoma, Hepatocellular; Cell Hypoxia; Disease Progression; Erythropoietin; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Neoplasm Proteins; Neovascularization, Pathologic; Receptor, TIE-2; Retrospective Studies; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Up-Regulation; Vascular Endothelial Growth Factor A

We have already reported that serum levels of soluble Fas (sFas) and Fas-positive mononuclear cells increased concomitantly with deterioration in renal function and the increases were statistically significant. Moreover, the severity of renal anemia in renal failure patients was significantly correlated with serum levels of sFas. Therefore, we investigated whether or not Fas and Fas ligand (FasL) influenced the production of erythropoietin (EPO). Hep G2 cells, an EPO productive human hepatocellular carcinoma cell line, were cultured in MEM medium with 10% of FCS containing 1, 10 or 100 ng/ml of sFas, or sFasL. The EPO concentrations of the supernatants were measured by the ELISA method, Annexin V positive cells were calculated by flow cytometry, H3 leucin uptake was measured by a liquid scintillation counter, an MTT assay was performed using the light absorption method, fragmented nuclei were stained by the TUNEL method and DNA laddering was observed by agarose gel electrophoresis. Their characteristics evaluated at 0, 24, 48 and 72 hrs. Both EPO production and H3 leucin uptake were suppressed in culture with sFas or sFasL, dose-dependently and declines in MTT activities accompanied these changes at 24 hrs. In addition, nuclear fragmentation and DNA laddering were found to be stimulated in culture with sFas or sFasL at 48 hrs. These data suggest that sFas induced apoptosis and had a cytotoxic effect on Hep G2 cells. In conclusion, hyper-sFas-emia observed in chronic renal failure may regulate the production of EPO, which indicates that sFas acts as a uremic toxin.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Erythropoietin; fas Receptor; Humans; Liver Neoplasms

Hypoxia-inducible factor 1 (HIF-1) is a phosphorylated protein and its phosphorylation is involved in HIF-1alpha subunit stabilization as well as in the regulation of HIF-1 transcriptional activity. In a variety of cell lines, the phosphorylation of HIF-1alpha is dependent on ERK or p38, two members of the mitogen-activated protein kinase (MAPK) superfamily. In addition, active MAPK could be inactivated through dephosphorylation by mitogen-activated protein kinase phosphatase-1 (MKP-1). MKP-1 has been identified as a hypoxia responsive gene, but its role in the response of cells to hypoxia is poorly understood. Here we found that hypoxia induces MKP-1 expression in human hepatoma cells HepG2 in a time-dependent manner. Inhibition of MKP-1 expression using siRNA technique could enhance HIF-1alpha phosphorylation, accompanied by an increase in transcriptionally active HIF-1 as well as a rise in the levels of HIF-1-induced erythropoietin expression.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; DNA-Binding Proteins; Down-Regulation; Dual Specificity Phosphatase 1; Enzyme Activation; Erythropoietin; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Liver Neoplasms; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Sensitivity and Specificity; Transcription Factors; Transcriptional Activation

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

We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin (Epo) production using hepatocellular carcinoma (Hep3B) cells in culture and in vivo in normal mice under normoxic and hypoxic conditions. CGS-21680, a selective adenosine A(2A) agonist, significantly increased levels of Epo in normoxic Hep3B cell cultures and in serum of normal mice under both normoxic and hypoxic conditions. CGS-21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures. SCH-58261, a selective adenosine A(2A) receptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O(2)). Enprofylline, a selective adenosine A(2B) receptor antagonist, significantly inhibited the increase in plasma levels of Epo in normal mice exposed to hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of Epo in normal mice. A model is presented for adenosine in hypoxic regulation of Epo production that involves kinases A and C and phospholipase A(2) pathways.

Topics: Adenosine; Alkaloids; Benzophenanthridines; Carcinoma, Hepatocellular; Enzyme Activation; Erythropoietin; Humans; Liver Neoplasms; Phenanthridines; Phenethylamines; Protein Kinase C; Purinergic P1 Receptor Antagonists; Pyrimidines; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Receptors, Purinergic P1; RNA, Messenger; Triazoles; Tumor Cells, Cultured; Xanthines

Homologous blood transfusion (HBT) has the risk of an immunosuppressive effect and may adversely affect the prognosis of patients with carcinomas. Autologous blood transfusion (ABT) has not yet become a standard procedure in gastroenteric cancer surgery. We investigated the usefulness and problems of ABT combined with the use of recombinant human erythropoietin (rh-EPO).. An evaluation of autologous blood transfusion (ABT) combined with recombinant human erythropoietin (rh-EPO) treatment was conducted in 46 patients with hepatocellular carcinoma undergoing hepatectomy. Preoperative autologous blood donation (ABD) was accomplished for 25 of the 46 patients. The preoperative changes in hemoglobin and hematocrit in relation to route of administration of erythropoietin were studied. In addition, intraoperative blood requirements and the postoperative complications for patients who predonated were compared with those of patients who underwent surgery without autologous predonation.. The proportion of patients not requiring additional homologous blood transfusions (HBT) during operation was significantly higher in the ABD group than in the non-ABD group (88% versus 38%). The incidence of postoperative complications was significantly higher in patients receiving HBT than in nontransfused patients and in those receiving ABT.. Preoperative autologous blood donation in combination with rh-EPO therapy markedly reduced the requirement for homologous blood transfusion during surgery in patients with hepatocellular carcinoma having hepatectomy.

Topics: Blood Transfusion, Autologous; Carcinoma, Hepatocellular; Case-Control Studies; Erythropoietin; Female; Hepatectomy; Humans; Incidence; Liver Neoplasms; Male; Middle Aged; Postoperative Complications; Recombinant Proteins

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

Topics: Anemia; Anemia, Iron-Deficiency; Animals; Base Sequence; Carcinoma, Hepatocellular; Ceruloplasmin; Cloning, Molecular; Consensus Sequence; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Female; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Mice; Molecular Sequence Data; Nuclear Proteins; Pregnancy; Promoter Regions, Genetic; Recombinant Proteins; Sequence Alignment; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Treatment of human hepatoma cells (HepG2) with NO-donors for 24 h inhibited hypoxia-induced erythropoietin (EPO) gene activation. NO was found to increase the production of reactive oxygen species (ROS), the putative signaling molecules between a cellular O2-sensor and hypoxia inducible factor 1 (HIF-1). HIF-1 is the prime regulator of O2-dependent genes such as EPO. NO-treatment for more than 20 h reduced HIF-1-driven reporter gene activity. In contrast, immediately after the addition of NO, ROS levels in HepG2 cells decreased below control values for as long as 4 h. Corresponding to these lowered ROS-levels, HIF-1 reporter gene activity and EPO gene expression transiently increased but were reduced when ROS levels rose thereafter. Our findings of a bimodal effect of NO on ROS production shed new light on the involvement of ROS in the mechanism of O2-sensing and may explain earlier conflicting data about the effect of NO on O2-dependent gene expression.

Topics: Acridines; Anaerobiosis; Carcinoma, Hepatocellular; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hydrogen Peroxide; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; NADH, NADPH Oxidoreductases; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nuclear Proteins; Onium Compounds; Oxygen; Penicillamine; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Spermine; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured

A case of erythrocytosis caused by a hepatocellular carcinoma (HCC) that produced erythropoietin (Epo) is described. A 64-year-old man, with a huge HCC tumor in the right lobe of the liver, showed a high concentration of hemoglobin and increased levels of serum Epo, alpha-fetoprotein (AFP), and protein induced by vitamin K absence II (PIVKA-II). Right lobectomy of the liver was performed. Histological findings of the specimen showed a moderately differentiated HCC. The existence of Epo was confirmed immunohistochemically only in the tumor tissue and not in the normal liver tissue. Erythrocytosis disappeared and the serum levels of Epo, AFP, and PIVKA-II returned to the normal range after the operation. Within 2 months after the operation, recurrent tumors appeared in the remnant liver, and the patient died 13 months after the operation.

Topics: alpha-Fetoproteins; Biomarkers; Carcinoma, Hepatocellular; Erythropoietin; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Polycythemia; Protein Precursors; Prothrombin

Erythropoietin (Epo), a glycoprotein hormone produced principally in the fetal kidney and in the adult liver in response to hypoxia, is the prime regulator of growth and differentiation in erythroid progenitor cells. The regulation of Epo gene expression is not fully understood, but two mechanisms have been proposed. One involves the participation of a heme protein capable of reversible oxygenation and the other depends on the intracellular concentration of reactive oxygen species (ROS), assumed to be a function of pO2. We have investigated the production of Epo in response to three stimuli, hypoxia, cobalt chloride, and the iron chelator desferrioxamine, in Hep3B cells. As expected, hypoxia caused a marked rise in Epo production. When the cells were exposed to the paired stimuli of hypoxia and cobalt no further increase was found. In contrast, chelation of iron under hypoxic conditions markedly enhanced Epo production, suggesting that the two stimuli act by separate pathways. The addition of carbon monoxide inhibited hypoxia-induced Epo production, independent of desferrioxamine concentration. Taken together these data support the concept that pO2 and ROS are sensed independently.

Topics: Carcinoma, Hepatocellular; Cell Hypoxia; Cycloheximide; Deferoxamine; Erythropoietin; Gene Expression Regulation; Humans; Oxygen; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

1. The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia-related gene expression. Cytosolic free calcium concentration ([Ca2+]i) has been implicated in cellular oxygen-sensing processes. We investigated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2. We found that the calcium ionophore ionomycin induced a rapid and sustained increase of [Ca2+]i while thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, only caused a 20 % elevation of [Ca2+]i within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3. Variations in [Ca2+]o did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production was not statistically significant. In the presence of extracellular Ca2+ the membrane permeant calcium chelator BAPTA-AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA-AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimulated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca2+ concentration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.

Topics: Blotting, Northern; Calcium; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cell Hypoxia; Chelating Agents; Egtazic Acid; Endothelial Growth Factors; Enzyme Inhibitors; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Kinetics; Liver Neoplasms; Lymphokines; Thapsigargin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Adult; Carcinoma, Hepatocellular; Erythropoietin; Humans; Liver Neoplasms; Male; Polycythemia

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Topics: Adult; Antigens, CD34; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cells, Cultured; Cobalt; Culture Media, Serum-Free; Enzyme-Linked Immunosorbent Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Liver Neoplasms; Molecular Sequence Data; Peptides; Receptors, Erythropoietin; RNA, Messenger; Tumor Cells, Cultured

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

We report the case of a patient with alcoholic liver cirrhosis and generalized atherosclerosis who rapidly developed erythrocytosis. Concomitantly we documented a significative and progressive increase of serum Erythropoietin (Epo) and a small focus of hepatocellular carcinoma (HCC) never diagnosed before. Even in absence of immunohistochemical and/or biomolecular evidence of Epo production in the neoplastic tissue we think the hypothesis of the paraneoplastic syndrome may be the most likely both for the strict temporal relationship between the observation of the neoplastic lesion and the appearance of polycythemia and for the absence of all other known causes of erythrocytosis. Objection to this hypothesis: 1) ectopic production of Epo during HCC has been usually described in large neoplastic lesions 2) liver cirrhosis by itself may be accompanied by increased Epo levels 3) an intratumoral hypoxia with compensatory production of Epo may have occurred 4) generalized vasculopathy could have determined renal hypoxia with greater local production of Epo.

Topics: Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Erythropoietin; Fatal Outcome; Humans; Liver Cirrhosis, Alcoholic; Liver Neoplasms; Male; Neoplasm Proteins; Polycythemia

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.

Topics: Alkaloids; Benzophenanthridines; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Humans; Ionophores; Liver Neoplasms; Models, Chemical; Oleic Acid; Phenanthridines; Phospholipases A; Phospholipases A2; Protein Kinase C; Quinacrine; Thapsigargin; Tumor Cells, Cultured

Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 microM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 microM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three-dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The .OH levels decreased in cobalt-treated (to 81%) as well as in nickel-treated (to 67%) HepG2 cells, accompanied by an increase of EPO expression to 167% and 150%, respectively. Our results underline the importance of .OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins.

Topics: Carcinoma, Hepatocellular; Cell Compartmentation; Cobalt; Drug Interactions; Electron Probe Microanalysis; Erythropoietin; Free Radical Scavengers; Hemeproteins; Humans; Hydrogen Peroxide; Iron; Liver; Metals, Heavy; Microscopy, Confocal; Nickel; Oxygen; Oxygen Consumption; Reactive Oxygen Species; Signal Transduction; Spectrophotometry; Tumor Cells, Cultured

Hypoxia is thought to be a common precursor of coronary artery disease and malignant tumors, both diseases representing the leading causes of death in industrial nations. So far, investigations of oxygen-regulated erythropoietin (EPO) gene expression in the human hepatoma cell lines Hep3B and HepG2 allowed many important insights into the mechanisms of oxygen-sensing, signalling and regulation of an increasing number of oxygen-responsive genes. To differentiate the various signalling pathways involved in EPO production by these two cell lines, we examined several factors that positively influenced EPO expression. The results demonstrate a keen differential effect of cell density and oxygen concentration on EPO induction in Hep3B compared to HepG2 cells. Using optimized cell culture conditions, EPO production rates as high as 1 U EPO per 10(6) Hep3B cells in 24 h could be achieved. We also found a moderate but reproducible positive effect of CoCl2 on hypoxia-induced EPO expression in Hep3B but a negative CoCl2 effect on hypoxic induction in HepG2 cells. CoCl2 inhibited cell growth in a concentration-dependent manner. Interleukin-6 was synergistic with hypoxia on EPO induction in Hep3B as well as HepG2 cells, and dexamethasone enhanced this effect in Hep3B but not in HepG2 cells. The moderate CoCl2-dependent increase of EPO production observed in hypoxic Hep3B cells might indicate that CoCl2 and hypoxia do not necessarily act via, identical signalling pathways.

Topics: Carcinoma, Hepatocellular; Cell Count; Cell Hypoxia; Cobalt; Dexamethasone; Erythropoietin; Humans; Interleukin-6; Liver Neoplasms; Oxygen; Signal Transduction

Erythropoietin (Epo) synthesis is suppressed in normoxia and stimulated in hypoxia. To test the hypothesis that the cellular H2O2 level is important in the control of Epo synthesis, we have studied effects of modulators of H2O2 generation and degradation on Epo production in human hepatic cell cultures (hepatoma lines HepG2 and Hep3B). In addition, we measured the activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) in cultures following hypoxia exposure or H2O2 treatment. The results show that the formation of immunoreactive Epo was stimulated in normoxic cultures by treatment with exogenous catalase thus mimicking the effect of hypoxia (24 h incubation periods). Epo production was also stimulated when scavengers of reactive O2 species (tetramethylthiourea, dihydrorhodamine) were added to the cells. On the other hand, stimulators of H2O2 generation (xanthine oxidase, glucose oxidase, NADH, NADPH) lowered Epo production in hypoxic cultures. Hypoxia exposure decreased superoxide dismutase activity and H2O2 treatment reduced catalase activity thus influencing the endogenous antioxidant defense system. These findings support the concept that reactive O2 species, primarily H2O2, act as messengers in the O2-dependent control of the hepatic production of Epo. Changes in the cellular activities of antioxidant enzymes appear to play only a minor role in this process.

Topics: Carcinoma, Hepatocellular; Catalase; Erythropoietin; Free Radical Scavengers; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Hypoxia; Reactive Oxygen Species; Superoxide Dismutase; Tumor Cells, Cultured

The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.

Topics: Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Cell Hypoxia; Cobalt; DNA; DNA Footprinting; DNA Methylation; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Genes; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Polymerase Chain Reaction; Trans-Activators; Tumor Cells, Cultured

We examined regulation of the human erythropoietin (Epo) gene through the GATA sequence in the Epo promoter and showed that Hep3B and HepG2 cells express human GATA-2 (hGATA-2) mRNA and protein. Nuclear extracts of QT6 cells transfected with hGATA-1, 2, or 3 transcription factors showed specific binding to the GATA element in the human Epo gene promoter by gel mobility shift assay. Transient transfection of Hep3B cells with hGATA-1, 2, or 3 showed that each of these transcription factors significantly decreased the level of expression of Epo mRNA as assessed by a competitive polymerase chain reaction. Transient transfection of Hep3B cells with hGATA-1, 2, and 3 and an Epo-reporter gene (growth hormone [GH]) construct showed significant inhibition of the Epo promoter. Antisense oligonucleotide for hGATA-2 transcription factor significantly increased the Epo protein in Hep3B cells under 1% O2 for 24 hours incubation. Furthermore, transient transfection of Hep3B cells with hGATA-1, 2, and 3 and an Epo-reporter gene (luciferase) construct also showed significant inhibition of the Epo promoter. However, transfection of the mutated GATA sequence of the Epo-luciferase gene with hGATA-1, 2, and 3 interfere with the inhibition of the Epo promoter. We conclude that the hGATA-1, 2, and 3 transcription factors specifically bind to the GATA element in the human Epo gene promoter and negatively regulate Epo gene expression.

Topics: Base Sequence; Carcinoma, Hepatocellular; DNA-Binding Proteins; DNA, Neoplasm; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA2 Transcription Factor; GATA3 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; Trans-Activators; Transcription Factors; Transfection; Tumor Cells, Cultured

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cyclic GMP; DNA Primers; Enzyme Inhibitors; Erythropoietin; Humans; Interferon-gamma; Liver Neoplasms; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

We have previously reported that a marked increase in erythropoietin (Epo) production can be demonstrated in Hep3B cell cultures in response to hypoxia (1). In order to determine whether this increase involves an autocrine mechanism, we have investigated the effects of purified human recombinant Epo (rHuEpo) on Epo production. Purified rHuEpo (5-80 mU/ml) produced a significant increase above control levels of Epo in Hep3B cell cultures under normoxic (20% O2) conditions. Hypoxic (1% o2) incubation of Hep3B cells with rHuEpo caused an increase over control levels of EpomRNA. Hep3B cells also expressed Epo receptor (Epo-R) transcripts. Binding studies [125I]Epo revealed that Hep3B cells contain a single class of binding site (kd=2.9 nmol/L and Bmax=1760 sites/cell). Antierythropoietin receptor monoclonal antibody inhibited the rHuEpo induced elevation in medium levels of Epo and blocked [125I]-Epo binding to Hep3B cell membranes. These results demonstrate that the expression of EpomRNA may be controlled, at least in part, by an autocrine mechanism.

Topics: Aerobiosis; Carcinoma, Hepatocellular; Cell Hypoxia; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Liver Neoplasms; Receptors, Erythropoietin; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured

The present studies were designed and carried out to determine if hydrogen peroxide (H2O2) is involved in the regulation of erythropoietin (Epo) gene expression and stimulation of Epo production in the hepatocellular (Hep 3B) cells. Hep 3B cells were incubated with varying concentrations of H2O2 for periods of 6 hours or 24 hours. In other experiments Hep 3B cells were incubated for 24 hours with or without increasing concentrations of catalase and in the presence of H2O2. Culture medium levels of Epo were determined and quantitation of Epo mRNA was also made. The results indicate that H2O2 increases the levels of Epo mRNA and Epo hormone production in Hep 3B cells, and that catalase, the specific scavenger of hydrogen peroxide, inhibits Epo production in these cells. Based on these findings, it is concluded that H2O2 takes part in the signal transduction mechanisms in Epo production. It is recommended that further studies be undertaken to find out the source of the hydrogen peroxide in the hepatocellular carcinoma cells.

Topics: Carcinoma, Hepatocellular; Catalase; Cell Line; Drug Evaluation, Preclinical; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Liver Neoplasms; RNA, Messenger; RNA, Neoplasm; Signal Transduction

Many inflammatory diseases are associated with a hypoproliferative anaemia. Patients with this anaemia often present with serum erythropoietin (EPO) concentrations that are too low for the degree of their anaemia. Proinflammatory cytokines, in addition to their inhibitory effects on proliferation of erythroid progenitors, could contribute to the pathogenesis of this anaemia by reducing EPO production. Because several cytokines stimulate nitric oxide (NO) synthase we propose that nitric oxide might mediate the suppression of EPO production during inflammation. In order to test this hypothesis we investigated the effects of NO donors on 24-h hypoxia-induced EPO production in the hepatocellular carcinoma cell line HepG2. Following application of the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), EPO production was dose-dependently reduced: compared to the untreated control EPO production was lowered by 89% with SNP (1000 microM), by 66% with SIN-1 (1000 microM), and by 72% with SNAP (500 microM). In contrast, 8-bromo-cGMP did not inhibit EPO formation. Since pyrogallol (300 microM) and H2O2 (250 microM) showed a comparable suppression of EPO synthesis, we propose that NO might affect EPO production either by a similar direct influence on the cellular redox state or via increasing the cellular content of reactive oxygen species.

Topics: Carcinoma, Hepatocellular; Cyclic GMP; Cytokines; Erythropoietin; Humans; Hydrogen Peroxide; Liver Neoplasms; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Penicillamine; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Tumor Cells, Cultured

p300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. They participate in numerous biological processes, including cell cycle arrest, differentiation, and transcription activation. p300 and/or CBP (p300/CBP) also coactivate CREB. How they participate in these processes is not yet known. In a search for specific p300 binding proteins, we have cloned the intact cDNA for HIF-1 alpha. This transcription factor mediates hypoxic induction of genes encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular endothelial growth factor. Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1 alpha and p300/CBP. Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP. Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by E1A. Hence, p300/CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including one (vascular endothelial growth factor) that plays a major role in tumor angiogenesis. Paradoxically, these data, to our knowledge for the first time, suggest that p300/ CBP are active in both transformation suppression and tumor development.

Topics: Adenovirus E1A Proteins; Carcinoma, Hepatocellular; Carrier Proteins; Cell Hypoxia; Cell Line; Cytomegalovirus; DNA Probes; Endothelial Growth Factors; Enhancer Elements, Genetic; Erythropoietin; Genes, Reporter; Genetic Vectors; Glutathione Transferase; Humans; Liver Neoplasms; Luciferases; Lymphokines; Nuclear Proteins; Osteosarcoma; Protein Binding; Recombinant Fusion Proteins; RNA, Messenger; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transcription of the erythropoietin (epo) gene is regulated in response to tissue hypoxia. In this study we show that constructs containing 117 bp of the epo promoter sequence cloned upstream of a luciferase reporter, respond to hypoxia when transfected into the human hepatoma cell line, Hep3B. The sequence -61 to -45 (EP17) relative to the transcription start of the murine epo gene imparted an approximately 4-fold induction of reporter gene expression due to hypoxia. Internal deletion of EP17 resulted in loss of induction by hypoxia without altering basal expression of the 117 bp epo promoter reporter construct. Mutagenesis studies showed that the bases at positions -53, -59, from -49 to -51 and from -55 to -57 are essential for hypoxic induction. The EP17 sequence is required for the 3' enhancer element of the epo gene to be maximally functional. Gel shift and UV cross-linking experiments showed the presence in Hep3B nuclear extracts, of two protein factors with approximate molecular weights of 52 kDa and 25 kDa that bind to EP17. Introduction of specific mutations in the EP17 region that abolish induction by hypoxia, also eliminated the binding of one or both of these factors. These experiments demonstrate a role for the proximal region of the epo promoter in hypoxic induction of the epo gene.

Topics: Animals; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Enhancer Elements, Genetic; Erythropoietin; Gene Expression; Genes, Reporter; Humans; Liver Neoplasms; Luciferases; Mice; Mutagenesis; Nuclear Proteins; Oxygen; Promoter Regions, Genetic; Transfection; Tumor Cells, Cultured

Hemopoietic alterations may occur during tumoral diseases, determining anemia. In most cases, serum EPO levels were lower than normal values. Hepatocellular carcinoma (HCC), one of the most frequent malignancies world-wide, is often characterized by mild anemia and increased serum EPO levels. We studied 30 HCC patients and 20 healthy subjects. We found that HCC patients presented higher serum EPO levels than healthy controls. In HCC patients, there was a significant inverse correlation between serum EPO levels and red blood cell count or hemoglobin levels. We postulated that the elevated serum EPO levels observed in these patients may be due to reduced hepatic clearance of EPO, and to the influence of cytokine-mediated inflammatory factors.

Topics: Aged; Carcinoma, Hepatocellular; Erythrocyte Count; Erythropoietin; Female; Hemoglobins; Humans; Liver Neoplasms; Male; Middle Aged; Polycythemia

Transcriptional regulation of gene expression by hypoxia is an important, but yet only marginally characterized mechanism by which organisms adapt to low oxygen concentrations. The human hepatoma cell line HepG2 is a widely used model for studying hypoxic induction of the hematopoietic growth factor erythropoietin. In an attempt to identify additional genes expressed in HepG2 cells during hypoxia, we differentially screened a cDNA library derived from hypoxic (1% O2) HepG2 cells using probes isolated from either normoxic (21% O2) or hypoxic cells. Two genes were identified, one encoding aldolase, a member of the glycolytic enzymes, and the other encoding alpha 1-antitrypsin which belongs to the family of the acute phase (AP) responsive proteins. Whereas hypoxic induction of glycolytic enzymes is well established, oxygen-dependent regulation of AP genes has not been reported so far. AP proteins are liver-derived plasma proteins whose production during inflammation is either up-regulated (positive AP reactants) or down-regulated (negative AP reactants). In the present study, we demonstrate that on the mRNA level hypoxic stimulation of HepG2 cells led to (i) an induction of the positive AP reactants alpha 1-antitrypsin, alpha 1-antichymotrypsin, complement C3, haptoglobin, and alpha 1-acid glycoprotein; (ii) a down-regulation of the negative AP reactant albumin; (iii) an up-regulation of the negative AP reactant transferrin; and (iv) unchanged levels of the positive AP reactants alpha- and beta-fibrinogen as well as hemopexin. Cycloheximide inhibited hypoxic up-regulation of AP mRNAs demonstrating that de novo protein synthesis is required for hypoxic induction. Nuclear run-on assays indicate that the hypoxic increase in AP mRNAs is mainly due to transcriptional regulation. The hypoxic response was compared to AP stimulation by interleukin 6. The results suggest that the adaptive response to hypoxia overlaps with, but is not identical with, the AP response mediated by interleukin 6.

Topics: Acute-Phase Proteins; alpha 1-Antitrypsin; Animals; Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Cycloheximide; Dexamethasone; DNA Primers; DNA, Complementary; Endothelial Growth Factors; Erythropoietin; Fructose-Bisphosphate Aldolase; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Library; Glycolysis; Humans; Interleukin-6; Liver Neoplasms; Lymphokines; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Transcription, Genetic; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.

Topics: Angiogenesis Inducing Agents; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Chelating Agents; Cobalt; Deferoxamine; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Liver Neoplasms; Lymphokines; Molecular Sequence Data; Neovascularization, Pathologic; Placenta Growth Factor; Platelet-Derived Growth Factor; Pregnancy Proteins; RNA, Messenger; RNA, Small Nuclear; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The precise cause of the anaemia that is commonly associated with severe pulmonary tuberculosis (PTB) has not been elucidated. The role of erythropoietin (Epo), the central hormone regulating red cell formation, still awaits clarification. We therefore determined serum Epo levels in patients with PTB; group 1, haemoglobin less than 110 g/L, group 2, haemoglobin greater than 110 g/L; group 3, controls, consisted of matched individuals with uncomplicated iron deficiency; group 4, healthy volunteers. Peripheral blood monocytes were obtained from patients with PTB and the controls, cultured, and the supernatant fluid (SNF) harvested. Tumour necrosis factor alpha (TNF alpha) levels were determined in the SNF, which were then added in various dilutions to a hepatocellular carcinoma cell line (HepG2) capable of regulated EPO synthesis in vitro. The influence of this cytokine was defined by the addition of specific neutralising anti-TNF alpha antibodies in this assay system. Patients in group 1 had significantly lower Epo levels (54 + 11 mU/mL) compared with those in group 3 (142 +/- 41 mU/mL) (p < 0.01). Monocyte supernatants from patients in the anaemic PTB group had markedly elevated TNF alpha levels and significantly suppressed Epo output by HepG2 cells in vitro (p < 0.01). This inhibition was consistently abrogated by anti-TNF alpha antibodies. Serum Epo levels were inappropriately low in untreated PTB patients when compared with corresponding haemoglobin levels in iron deficient controls. This blunted response could be ascribed to release of TNF alpha or other cytokines by activated monocytes.

Topics: Anemia; Biological Assay; Carcinoma, Hepatocellular; Cells, Cultured; Erythropoietin; Hemoglobins; Humans; Liver Neoplasms; Lymphocytes; Reference Values; Tuberculosis, Pulmonary; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

It has been shown previously that erythropoietin expression in vitro by hepatoma cells increases in response to hypoxia. To verify whether hypoxia of the tumor might result in hepatic release of erythropoietin in vivo, serum erythropoietin concentrations were measured immunoenzymatically in 12 patients (5 women, 7 men) who underwent transarterial chemoembolization for hepatocellular carcinoma. Peripheral blood samples were collected at baseline, and after 6 hours and 1, 2, 3, and 7 days after the procedure. In a second set of experiments, performed in three male patients also undergoing chemoembolization for hepatocellular carcinoma, paired blood samples were collected after catheterization of the hepatic veins and of the right antecubital vein. None of the patients had erythrocytosis. In comparison with a baseline mean value +/- SEM of 100.6 +/- 12.6 micrograms/L, serum erythropoietin concentrations were the following; +6 hours, 55.4 +/- 18.0 (P < .001); +1 day, 102.4 +/- 24.7 (P = NS), +2 days, 183.0 +/- 31.1 (P < .05); +3 days, 155.0 +/- 26.0 (P < .05); +7 days, 153.3 +/- 27.4 (P < .05) (matched Student's t-test). The ratio of hepatic vein/antecubital vein serum erythropoietin concentrations increased from 0.85 at baseline to 1.30 at +2 days, paralleling the increase of aspartate transaminase (r = .914, P < .005). After chemoembolization, no correlation was found between serum erythropoietin and alpha-1-fetoprotein concentrations. The concentration of the latter, stable initially, decreased 7 days after the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Erythropoietin; Female; Humans; Injections, Intra-Arterial; Liver; Liver Neoplasms; Male; Middle Aged; Osmolar Concentration

The purpose of this study was to characterize the effects of two new adenosine A2 agonists, 2-(p-(2-carboxyethyl)phenethyl amino)-5'-N-ethylcarboxamidoadenosine (CGS-21680) and N6-(2(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), on erythropoietin (EPO) production in vivo and in vitro. Intravenous injections of CGS-21680 (100 to 500 nmol/kg mouse/day) and DPMA (50 to 500 nmol/kg mouse/day) for 4 days produced significant increases in serum levels of EPO in exhypoxic polycythemic mice. CGS-21680 (10(-7) to 10(-6) mol/L) and DPMA (10(-8) to 10(-5) mol/L) also produced significant increases in medium levels of EPO in a cloned EPO-producing Hep3B hepatocellular carcinoma cell line after 18 hours of incubation in 1% O2. Both compounds also increased cellular cAMP levels significantly in a dose-dependent manner after 1 hour of incubation. A2 receptor binding assays with tritiated CGS-21680 revealed a single type of adenosine receptor binding site on Hep3B cell membranes with a dissociation constant of 132.9 nmol/L and a binding capacity of 270.6 fmol/mg protein. The Ki competition binding values versus tritiated CGS-21680 were 217 nmol/L for CGS-21680 and 86.8 nmol/L for DPMA. These results indicate that adenosine A2 receptor activation amplifies EPO production in response to hypoxia, both in vivo and in vitro.

Topics: Adenosine; Animals; Binding, Competitive; Carcinoma, Hepatocellular; Cyclic AMP; Erythropoietin; Female; Humans; Hypoxia; Kinetics; Liver Neoplasms; Mice; Phenethylamines; Polycythemia; Purinergic P1 Receptor Agonists; Tumor Cells, Cultured

Extramedullary hematopoiesis is a characteristic feature of hepatoblastoma (HB). We investigated 15 HB to characterize intratumoral hematopoietic foci and to find clues to the pathophysiology of their formation. By conventional histology and immunohistochemistry, we found erythroblasts in all and megakaryocytes in 10 of the HB, whereas granulocyte and monocyte precursor cells could not be identified in hematopoietic foci of any tumor. Only a minority of erythropoietic cells in these foci contained fetal Hb (HbF). We recently found that HB cells produce IL-1 beta and thus stimulate stromal cells to secrete IL-6. We therefore searched for other hematopoietic cytokines in HB. Supernatants of primary HB cultures were subjected to ELISA, bioassayed, and immunoblotted. We detected erythropoietin (EPO) in 11 of 15, stem cell factor (SCF) in 7 of 11, granulocyte colony-stimulating factor (G-CSF) in 4 of 15, granulocyte/macrophage colony-stimulating factor (GM-CSF) in 6 of 15, IL-3 in 1 of 12, leukemia inhibitory factor (LIF) in 1 of 9, and macrophage colony-stimulating factor (M-CSF) in 1 of 8 conditioned media. With immunoenzymatic labeling we localized EPO and SCF to the cytoplasm of epithelial HB cells, whereas stromal cells and cells of immature fibrous tissue of mixed HB expressed SCF, G-CSF, GM-CSF, LIF, and M-CSF. EPO and SCF could also be detected in extracts of epithelial HB cells. We conclude that, in HB, erythropoiesis and megakaryopoiesis but not the granulocyte-macrophage lineage is induced by fetal and embryonal tumor cells in cooperation with stromal cells by locally secreted cytokines.

Topics: Carcinoma, Hepatocellular; Child, Preschool; Cytokines; Erythroblasts; Erythropoiesis; Erythropoietin; Hematopoiesis; Hematopoietic Stem Cells; Humans; Immunohistochemistry; Infant; Liver Neoplasms; Megakaryocytes; Stem Cell Factor

Fanconi anemia (FA) is a recessively inherited disease characterized by bone marrow failure, congenital anomalies, chromosomal instability and hypersensitivity to crosslinking agents. Some of the cellular defects of FA are known to be responsive to the ambient oxygen concentration. We examined the responsiveness of the FA complementation group C (FAC) gene to changes in oxygen concentration using two types of human cell lines, hypoxia-responsive Hep3B hepatoma cells and Epstein-Barr virus-immortalized lymphoblasts (normal and FA complementation groups B and C). Although the expression of erythropoietin in Hep3B cells was induced in response to the hypoxia-mimicking agent CoCl₂, there was no concomitant induction in FAC expression as assessed by mRNA levels and immunoprecipitable protein, and no detectable change in the cytoplasmic location of the FAC polypeptide as determined by indirect immunofluorescence. In human lymphoblasts we examined the effect of oxygen (0.1% -95% O₂) on cell proliferation and FAC expression. FA lymphoblasts had a normal sensitivity to the cytostatic effect of hyperoxia, while in both control and FA lymphoblasts FAC mRNA levels were unaffected by oxygen. Our results indicate that ambient oxygen is not a regulator of the FAC gene.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Hypoxia; Cell Line, Transformed; Cobalt; DNA-Binding Proteins; Erythropoietin; Fanconi Anemia; Fanconi Anemia Complementation Group C Protein; Fanconi Anemia Complementation Group Proteins; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Liver Neoplasms; Lymphocytes; Nuclear Proteins; Oxygen; Protein Biosynthesis; Proteins; RNA, Messenger; Tumor Cells, Cultured

Effects of thyroid hormones on the production of erythropoietin (Epo) were investigated in isolated perfused rat kidneys and in the human hepatoma cell line, HepG2. Epo protein was measured by radioimmunoassay. L-triiodothyronine and L-thyroxine stimulated hypoxia-induced Epo formation both in the kidney and in HepG2 cells in a dose-dependent fashion. Quantitation of Epo mRNA by competitive polymerase chain reaction (PCR) showed that hypoxic HepG2 cells had three-fold higher Epo messenger RNA levels when treated with thyroid hormones for 3 hours. Measurements of oxygen consumption revealed that this effect was not due to an increase in the degree of hypoxia. Thus, apart from the known direct effect on erythroid precursors, thyroid hormones appear to stimulate erythropoiesis by a noncalorigenic increase in Epo production.

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Erythropoietin; Humans; Hypoxia; Kidney; Liver Neoplasms; Male; Oxygen Consumption; Polymerase Chain Reaction; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thyroxine; Triiodothyronine; Tumor Cells, Cultured

Hepatocellular carcinoma (HCC) is a common malignancy in southeast Asia and sub-Saharan Africa. During its clinical course, patients may manifest a variety of paraneoplastic syndromes, including erythrocytosis. However, there are few reports on the clinical and biochemical characteristics of HCC patients who manifest erythrocytosis. The purpose of this study is to evaluate the incidence of erythrocytosis in a large series of the Chinese patients with HCC, and to investigate the association of erythrocytosis with tumor volume and with serum levels of alpha-fetoprotein (AFP) and erythropoietin.. Among 792 Chinese HCC patients who were seen during a 3-year period, we identified HCC patients with erythrocytosis as those with hemoglobin levels greater than 16.7 gm/dL (two standard deviations above the mean hemoglobin level of matched normal controls). The tumor size and serum levels of AFP and erythropoietin were evaluated in HCC patients with erythrocytosis to compare with HCC patients without erythrocytosis.. 20 (2.5%) of 792 Chinese HCC patients presented with erythrocytosis. Nineteen of these 20 HCC patients were found to have either bi-lobar tumor involvement or a large tumor mass confined to one lobe of the liver. The estimated mean tumor volume of HCC patients with erythrocytosis was 50% of whole liver. When compared with HCC patients without erythrocytosis, the 20 HCC patients with high hemoglobin levels had significantly higher serum levels of AFP and erythropoietin (356,343 +/- 145,807 vs. 16,881 +/- 10,425 ng/mL, 135 +/- 45 vs. 25 +/- 4 mU/mL, respectively, p < 0.01).. Base on our findings, detection of erythrocytosis in a patient with HCC would indicate the presence of a large tumor burden, and high serum levels of both AFP and erythropoietin should be associated with this paraneoplastic syndrome.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Erythropoietin; Female; Humans; Liver Neoplasms; Male; Middle Aged; Paraneoplastic Syndromes; Polycythemia

A 68-year-old man with hepatocellular carcinoma complicated by erythrocytosis showed an increased plasma level of immunoreactive erythropoietin (EPO). Northern blot analysis and RT-PCR (reverse transcriptase and polymerase chain reaction) of EPO mRNA extracted from a surgical specimen indicated high expression of EPO mRNA in the tumor tissue. Histological and immunocytochemical examination showed that the tumor was a hepatocellular carcinoma with predominant immunostaining for EPO. The erythrocytosis improved and the high serum EPO level decreased after resection of the tumor. This is the first demonstration of EPO mRNA expression in hepatocellular carcinoma tissue by RT-PCR.

Topics: Aged; alpha-Fetoproteins; Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular; Erythropoietin; Gene Expression; Humans; Immunohistochemistry; Liver Neoplasms; Male; Molecular Sequence Data; Polycythemia; Polymerase Chain Reaction

Hypoxia-induced erythropoietin (Epo) production in vitro is suppressed by interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF) and phorbol esters. Herein, the Epo-synthesizing human hepatoma cell line HepG2 was used to investigate whether protein kinase C (PKC) is involved in the inhibitory action of the cytokines. Within 1 h after the onset of hypoxia, Epo mRNA levels were markedly increased in untreated HepG2 cells as quantitated by competitive reverse transcription PCR. The cytokines IL-1 beta and TNF prevented this hypoxia-induced increase in Epo mRNA levels. In phorbol-ester-treated cells first inhibitory effects on Epo mRNA levels were observed only after 3 h. Western blot analyses revealed the presence of four isoenzymes of PKC in HepG2 cells. None of these isoenzymes was translocated in response to TNF or IL-1 beta, suggesting that the cytokines do not activate PKC in HepG2 cells. In contrast, phorbol esters translocated and, upon prolonged exposure, down-regulated PKC isoenzymes alpha and epsilon. Activation of protein kinase A by dibutyryl-cAMP partially antagonized the cytokine-dependent inhibition of Epo production but did not influence the inhibitory effect of phorbol esters. Endogenous cAMP levels in HepG2 cells were unchanged by cytokine treatment. Obviously, at least two signaling pathways exist that can confer inhibition of Epo production in HepG2 cells. One of these may be mediated by down-regulation of the PKC alpha or epsilon isoenzyme. The other pathway, however, which is triggered by IL-1 beta and TNF, is independent of PKC.

Topics: 1-Methyl-3-isobutylxanthine; Bucladesine; Carcinoma, Hepatocellular; Cell Hypoxia; Cyclic AMP; Cytokines; Erythropoietin; Gene Expression; Humans; Interleukin-1; Isoenzymes; Kinetics; Liver Neoplasms; Protein Kinase C; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Aged; Carcinoma, Hepatocellular; Erythrocyte Count; Erythropoietin; Hepatorenal Syndrome; Humans; Liver Cirrhosis, Alcoholic; Liver Neoplasms; Male; Paraneoplastic Syndromes; Polycythemia

The addition of exogenous H2O2 inhibited hypoxia-induced erythropoietin (Epo) production in the human hepatoma cell line HepG2. Likewise, elevation of endogenous H2O2 levels by the addition of menadione or the catalase inhibitor, aminotriazole, dose-dependently lowered Epo production. The inhibitory effect of exogenous H2O2 on Epo formation could be completely overcome by co-incubation with catalase. When GSH levels in HepG2 cells were lowered, Epo production was more susceptible to H2O2-induced inhibition, indicating that H2O2 might affect thiol groups in regulatory proteins. Endogenous production of H2O2 in HepG2 cells was dependent on the pericellular O2 tension, being lowest under conditions of hypoxia. Our results support the hypothesis that an H2O2-generating haem protein might be part of the O2 sensor that controls Epo production. High H2O2 levels under conditions of normoxia suppress, whereas lower levels in hypoxic cells allow epo gene expression.

Topics: Amitrole; Base Sequence; Carcinoma, Hepatocellular; Catalase; DNA Primers; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation; Glutathione; Humans; Hydrogen Peroxide; Hypoxia; Liver Neoplasms; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Vitamin K

Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Cloning, Molecular; Cobalt; Cyanides; Cycloheximide; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Isoenzymes; L Cells; L-Lactate Dehydrogenase; Liver Neoplasms; Mice; Molecular Sequence Data; Oxygen; Phosphoglycerate Kinase; Promoter Regions, Genetic; Sequence Deletion; Sequence Homology, Nucleic Acid; TATA Box; Transfection; Tumor Cells, Cultured

Laboratory experiments have demonstrated that tetra- and triiodothyronine (T4, T3) enhance hypoxia-induced erythropoietin (Epo) production. In the present study serum immunoreactive Epo was measured in 29 patients with hyperthyroidism and in 10 patients with hypothyroidism. Epo levels were inversely correlated to the blood haemoglobin concentration [Hb] in both groups of patients. However, Epo levels at given [Hb] were significantly higher in the hyperthyroid state. In vitro studies confirmed that T4 and T3 stimulate Epo synthesis in the human liver cell line HepG2. This stimulating effect persisted for at least 1 day after the removal of T4 and T3 from the cultures. Thus, while thyroidal disorders affect steady-state levels of circulating Epo, it seems unlikely that thyroid hormones play a major role in abrupt adjustments of Epo production, such as the diurnal changes.

Topics: Carcinoma, Hepatocellular; Erythropoietin; Hemoglobins; Humans; Hyperthyroidism; Hypothyroidism; Liver; Liver Neoplasms; Thyroxine; Triiodothyronine; Tumor Cells, Cultured

Topics: Anemia; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Hypoxia; Cytokines; Erythropoietin; Humans; Interleukin-1; Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The present studies were undertaken using a cloned erythropoietin (Ep) producing hepatocellular carcinoma cell line (Hep3B) to attempt to correlate the receptor binding properties and biological activities of 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA) and N6-cyclopentyldenosine (CPA). Ep and cAMP levels in cultures of Hep3B cells in response to these adenosine analogues were measured by radioimmunoassay. Receptor binding affinities of the adenosine analogues were determined by measuring inhibition of binding of [3H] NECA to Hep3B cell membranes. Scatchard analysis of [3H]NECA to Hep3B cell membranes. Scatchard analysis of [3H]NECA binding to Hep3B cell membranes indicates a single class of binding sites with a dissociation constant of 431 nM and a binding capacity of 573 fmol/mg protein. The adenosine analogues tested produced a significant increase in Ep secretion and cAMP accumulation (ED50 for cAMP accumulation, NECA = 3.3 x 10(-7) M, CHA = 4.2 x 10(-6) M, CPA = 2.2 x 10(-6) M). In addition, NECA showed a higher binding affinity (Ki, 3.8 x 10(-7) M) for Hep3B cell membranes in comparison with CHA (Ki, 6.3 x 10(-6) M) and CPA (Ki, 3.9 x 10(-6) M). These results indicate that the rank order for potency for NECA, CHA and CPA in their binding to Hep3B cell membrane receptors correlates very well with their biological effects on Ep secretion and cAMP accumulation in Hep3B cells.

Topics: Adenosine; Carcinoma, Hepatocellular; Cyclic AMP; Erythropoietin; Humans; Receptors, Purinergic P1; Tumor Cells, Cultured

In patients with the anemia of chronic diseases, the plasma level of EPO is often low in relation to the blood hemoglobin concentration. Because infectious and inflammatory processes cause activation of cytokine-producing macrophages and lymphocytes, we investigated whether isolated inflammatory cytokines influence the synthesis of EPO in vitro. IL-1 and TNF-alpha were shown to inhibit EPO mRNA levels and EPO formation in the human hepatoma cell cultures HepG2 and Hep3B, and to lower EPO formation in isolated perfused rat kidneys. IFN-alpha and IFN-beta also induced some inhibition of EPO production in HepG2 cultures. IL-3, TGF-beta 2, and IFN-gamma did not inhibit. IL-6 stimulated the production of EPO in Hep3B cells but was ineffective in HepG2 cells and lowered EPO production in isolated perfused rat kidneys. IL-1, TNF-alpha, and possibly other cytokines could contribute to defective EPO production in renal and nonrenal immune responses.

Topics: Anemia; Animals; Carcinoma, Hepatocellular; Cell Line; Chronic Disease; Culture Media, Conditioned; Cytokines; Erythropoietin; Hemoglobins; Humans; Inflammation; Interferon-alpha; Interferon-beta; Kidney; Kinetics; Liver Neoplasms; Macrophages, Peritoneal; Male; Mice; Monokines; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Catalase; Cell Line; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression; Hemeproteins; Humans; Hydrogen Peroxide; Kinetics; Liver Neoplasms; Tumor Cells, Cultured

The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.

Topics: Blotting, Northern; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; DNA; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Leucine; Liver Neoplasms; Protein Kinase C; Radioimmunoassay; RNA; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured; Uridine

Erythrocytosis is occasionally observed in patients with hepatocellular carcinoma (HCC). The pathogenesis of the phenomenon remains uncertain. It has been speculated that tumors produce erythropoietin (Epo), and several studies on the Epo in tumor tissues have been reported. Using a sensitive enzyme linked immunosorbent assay, we measured the serum Epo concentration in 92 HCC patients and 30 liver cirrhosis (LC) patients. The levels of Epo in normal subjects, HCC patients and LC patients were 10.5 +/- 4.1 (mean +/- SD, mU/ml), 55.6 +/- 218.0 and 18.4 +/- 19.4, respectively. Some patients with high Epo values had low levels of hemoglobin (Hb), and a scatter-gram of the two parameters was similar to that in iron deficiency anemia. In patients whose Hb levels were more than 12 g/dl, we found Epo levels of 15.0 +/- 8.8 (mean +/- SD mU/ml) and 10.3 +/- 7.7 in HCC and LC, respectively. Epo values in HCC were significantly higher than those of normal subjects (P < 0.001) and LC patients (P < 0.05), and 18.2% (10/55) had concentrations above the upper limit of the normal range. The increase was not, however, a marked one. In conclusion, as the incidence of erythrocytosis was low (2.2%) in HCC patients, the high Epo values in some patients could be related to the abnormal production of Epo by HCC.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Hemoglobins; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged

Patients with hepatocellular carcinoma sometimes have erythrocytosis and high plasma erythropoietin levels. However, previous studies have not revealed direct evidence that the carcinoma cells produce the erythropoietin. To address this question, we carried out light and electron microscopic immunohistochemical studies, using a human erythropoietin antibody to the liver in three male patients with hepatocellular carcinoma and erythrocytosis. alpha-Feto-protein localization was also examined in serial liver sections by light microscopic immunohistochemistry with an antibody to alpha-fetoprotein. All three patients demonstrated high hemoglobin levels (16.7, 17.6 and 18.1 gm/dl) and high plasma erythropoietin levels (227, 266 and 280 mU/ml). In one patient the plasma erythropoietin level in the hepatic vein was significantly higher than that in the hepatic artery. The levels of plasma erythropoietin, as well as such tumor markers for hepatocellular carcinoma as serum alpha-fetoprotein and plasma des-gamma-carboxyprothrombin, were significantly reduced after treatment with an anticancer drug, cisplatin. Light microscopic immunohistochemistry showed that erythropoietin was definitely present in the cytoplasm of the hepatocellular carcinoma cells, but not in normal hepatocytes around the carcinoma lesion or in other nonparenchymal cells such as vascular endothelial cells and Kupffer cells. In electron microscopic immunohistochemistry, reaction products for erythropoietin were revealed in the cisternae of the endoplasmic reticulum in the carcinoma cells, suggesting the production of erythropoietin by these cells. Light microscopic immunohistochemistry showed that alpha-fetoprotein was localized in the hepatocellular carcinoma cells that were erythropoietin positive in the serial sections. These findings indicated that hepatocellular carcinoma cells produced erythropoietin as well as alpha-fetoprotein in these cases, leading to the complication of erythrocytosis.

Topics: Aged; alpha-Fetoproteins; Carcinoma, Hepatocellular; Endoplasmic Reticulum; Erythropoietin; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Male; Microscopy, Electron; Middle Aged; Polycythemia

N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, in a concentration range of 10(-9) to 10(-7) M, produced a significant decrease in erythropoietin (EPO) levels in a human hepatocellular carcinoma (Hep G2) cell culture (medium levels of EPO, 91.81 +/- 1.61 and 94.36 +/- 0.97% of control, respectively) after 24 h incubation in a hypoxic atmosphere. CPA, at a concentration of 10(-9) M, also produced a significant decrease in Hep G2 cell levels of adenosine 3',5'-cyclic monophosphate (cAMP; 78.13 +/- 3.89% of control) after 2 h incubation. CPA (10(-9) M) also significantly inhibited forskolin-stimulated increases in EPO production and cAMP accumulation in Hep G2 cells. On the other hand, 2-[p-(2-carboxyethyl)phenethyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2-receptor agonist, produced no significant change in EPO production in a dose range of 10(-10) to 10(-6) M but increased cAMP accumulation at 10(-6) M. A1-receptor binding assays using N6-[3H]cyclohexyladenosine revealed a single type of adenosine receptor binding site on Hep G2 cell membranes with a dissociation constant of 71.4 nM and a binding capacity of 1,530 fmol/mg protein. These results indicate that Hep G2 cells contain high-affinity adenosine A1 receptors that are linked to decreased cAMP accumulation and EPO production.

Topics: Adenosine; Carcinoma, Hepatocellular; Colforsin; Cyclic AMP; Erythropoietin; Humans; Liver Neoplasms; Receptors, Purinergic P1; Tumor Cells, Cultured

On the basis of Fick's law of gas diffusion, it has been proposed that cells in conventional monolayer cultures may be severely hypoxic. Because knowledge of the cellular O2 availability is important for the interpretation of biochemical and toxicological cell culture work, microelectrode measurements of the pericellular PO2 were carried out using the erythropoietin (Epo)-producing human hepatoma cell lines Hep G2 and Hep 3B as an in vitro model. In confluent hepatoma cultures grown in polystyrene dishes and incubated in air with 5% CO2, the pericellular steady-state PO2 was < 1 mmHg. The rates of the production of immunoreactive Epo and lactate were high due to a misproportion between O2 supply and O2 requirements. Epo production decreased when shaken instead of static cultures were studied, or when the O2 concentration in the gas atmosphere was increased gradually up to 95%. In cultures grown on gas-permeable supports, pericellular and gas PO2 values were very similar, with increased Epo production at lowered PO2. In agreement with mathematical models, our experimental data make PO2 measurements desirable for studies of O2-dependent biological functions in cell cultures.

Topics: Carcinoma, Hepatocellular; Cell Line; Culture Techniques; Erythropoietin; Extracellular Space; Glucose; Humans; Kinetics; Lactates; Liver Neoplasms; Microelectrodes; Oxygen; Oxygen Consumption; Partial Pressure; Time Factors; Tumor Cells, Cultured

The present studies were undertaken to assess the effects of 5'-N-ethylcarboxamideadenosine (NECA), an adenosine analogue, on erythropoietin (Epo) production. NECA (0.05 and 0.1 mumol/kg i.v.) produced significant increases in serum Epo levels (368.8 +/- 56.1 and 384.6 +/- 45.9 mU/ml, respectively) in exhypoxic polycythemic mice after a four hour exposure to hypoxia when compared with hypoxia controls (133.2 +/- 18.2 mU/ml). The hypoxic kidney Epo levels were 46.4 +/- 13.4 mU/kg kidney which were significantly higher than normoxic kidney Ep levels (< 1.24 mU/kg kidney). Theophylline (20 mg/kg i.p.), an adenosine receptor antagonist, significantly inhibited the stimulatory effects of NECA on serum Epo levels. In vitro cultures of an Epo producing hepatocellular carcinoma (Hep3B) cell line with NECA (> or = 10(-6) M) for 20 hours under hypoxic conditions (1% O2) produced significant increases in medium levels of Epo when compared with hypoxia controls. Hepatocellular carcinoma cells treated with NECA at a concentration range of 10(-7) M to 5 x 10(-5) M for one hour in a hypoxic atmosphere also had significantly higher cAMP levels than that of hypoxia controls. Scatchard analyses of [3H]NECA binding to membrane preparations of hepatocellular carcinoma cells showed low affinity binding sites with a dissociation-constant (Kd) of 0.44 microM and a binding capacity of 863 fmol/mg protein. These findings suggest that the increase in Epo production in response to NECA under hypoxic conditions can be attributed, at least in part, to stimulation of adenosine A2 receptors which is coupled to adenylyl cyclase activation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Erythropoietin; Female; Hypoxia; Liver Neoplasms; Mice; Mice, Inbred Strains; Osmolar Concentration; Polycythemia; Theophylline; Tumor Cells, Cultured

The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogenous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus nonhypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Hypoxia; Erythropoietin; Fibroblasts; Humans; Immunoenzyme Techniques; Immunohistochemistry; Intermediate Filament Proteins; Kidney; Liver Neoplasms; Oxygen Consumption; Polymerase Chain Reaction; RNA, Messenger; Swine; Tumor Cells, Cultured

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15-0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Topics: Animals; Carcinoma, Hepatocellular; Erythropoietin; Exons; Gene Expression Regulation; Humans; Kidney; Liver Neoplasms; Male; Organ Specificity; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Restriction Mapping; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Erythropoietin; Hemeproteins; Humans; Hydrogen Peroxide; Liver Neoplasms; NADH, NADPH Oxidoreductases; NADPH Oxidases; Oxygen; Potassium Cyanide; Potentiometry; Spectrophotometry; Tumor Cells, Cultured

The present studies were undertaken to characterize erythropoietin (Ep) production in an Ep-producing hepatocellular carcinoma (Hep3B) cell line. Hep3B cells which had been maintained in culture were transplanted under the renal capsule and subcutaneously in nude mice. The Hep3B xenograft doubling time is approximately 7 days. The mean hematocrit value of the Hep3B tumor-bearing nude mice was 33.2 +/- 1.1% (n = 8), which was significantly lower than that of control nongrafted nude mice (40.8 +/- 1.7%, n = 5). The Hep3B tumor-bearing nude mice showed significantly higher Ep levels in the sera (37.5 +/- 5.5 munits/ml, n = 8) than control nude mice (13.5 +/- 2.7 munits/ml, n = 5). Ep levels in the sera were correlated (R = 0.714) with the total Ep in the tumor extracts, whereas no Ep was detectable in any of the kidney extracts. On the other hand, an inverse linear relationship (R = -0.811) between the hematocrit values and Ep levels in the sera was demonstrated in the Hep3B tumor-bearing nude mice. The Hep3B cells recultured after growing in the nude mice were capable of enhancing Ep production in response to hypoxia, very similar to the original Hep3B cells which had been maintained in culture during the same time period. In addition, 15-methyl-prostaglandin E1 at a concentration range of 4-400 ng/ml produced significant increases in Ep secretion and cAMP accumulation in Hep3B cultures under hypoxic conditions (1% O2). The Ep produced by Hep3B cells expressed 3.7 times higher in vitro bioactivity than immunoactivity. The bioactivity of Hep3B Ep was completely neutralized by an antibody to highly purified human recombinant Ep. In contrast, the in vivo bioactivity of the Hep3B Ep was less than one tenth of its immunoactivity. These results indicate that the Hep3B tumor-bearing nude mice and the in vitro Hep3B culture system may provide a reproducible model system which should be useful for studies of the mechanism of Ep production.

Topics: Animals; Biological Assay; Carcinoma, Hepatocellular; Cell Division; Cyclic AMP; Erythropoietin; Female; Humans; Kidney; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Radioimmunoassay; Transplantation, Heterologous; Tumor Cells, Cultured

Erythropoietin (Epo) synthesis increases in response to hypoxia. The hepatoma cell line Hep 3B produces low basal levels of Epo mRNA which increase markedly with hypoxia. To define the sequences necessary for this response, we linked fragments of the human Epo gene to a luciferase vector, introduced these plasmids into Hep 3B cells and assayed for luciferase activity after growth in 1% or 21% oxygen. A 621-bp Epo promoter fragment resulted in a 2.4-fold increase in luciferase activity with hypoxia. We tested several Epo gene fragments upstream of this Epo promoter fragment and found that a 613-bp Bgl II-Pvu II 3' fragment had a 10-fold increase in activity with hypoxia regardless of orientation. This fragment had a similar level of activity when linked to a simian virus 40 promoter. Portions of this fragment retained activity, including a 38-bp Apa I-Taq I fragment that had a 17-fold increase in activity with hypoxia. Deletion of nt 4-13 or 19-28 from this 38-bp fragment resulted in a loss of activity. The 24-bp upstream portion of the 38-bp fragment showed an 8-fold increase in activity with hypoxia. However, deletion of nt 19-24 or mutagenesis of nt 21 or 22 in this 24-bp fragment resulted in loss of activity. Our studies indicate that the transcriptional response of the human Epo gene to hypoxia is mediated in part by promoter sequences and to a greater degree by an enhancer element located in a 24-bp portion of the 3' flanking sequence of the gene.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Enhancer Elements, Genetic; Erythropoietin; Humans; Liver Neoplasms; Luciferases; Molecular Sequence Data; Plasmids; Recombinant Fusion Proteins; Restriction Mapping; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.

Topics: Animals; Base Sequence; beta-Galactosidase; Binding Sites; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Nucleus; Chloramphenicol O-Acetyltransferase; CHO Cells; Cricetinae; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Macromolecular Substances; Molecular Sequence Data; Nuclear Proteins; Oligonucleotide Probes; Recombinant Fusion Proteins; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Ultraviolet Rays

Exposure of Hep3B cells to metalloporphyrins (tinprotoporphyrin and heme) or cobalt chloride resulted in the production of a significant number of heme oxygenase transcripts, erythropoietin transcripts or both, as indicated by in situ hybridization. Exposure to heme 10 mumol/L resulted in a 30-fold to 40-fold increase in cells expressing erythropoietin messenger RNA (erythropoietin-positive cells) by 6 hr; this increased level remained elevated for 24 hr. Tin-protoporphyrin (10 mumol/L) produced an eightfold to 10-fold increase in erythropoietin RNA within 40 min. This value then returned to control levels by 60 min. Exposure to cobalt chloride (100 mumol/L) resulted in a 20-fold to 30-fold increase in erythropoietin expression for 5 to 20 min, returning to control by 40 min. Additionally, nuclear runoff assays demonstrated that the increase in heme oxygenase or erythropoietin messenger RNA accumulation by cobalt chloride appeared to be a result of stimulated transcription of the heme oxygenase and erythropoietin genes. However, the pattern for heme oxygenase messenger RNA induction was different from that for erythropoietin expression. Heme produced an immediate expression of heme oxygenase RNA (50-fold within 5 min) and a second sustained response during the next 24 hr. Tin-protoporphyrin also produced an immediate response (40-fold within 5 min) and remained elevated (20-fold) for 6 hr. Cobalt chloride produced a 22-fold increase within 20 min and returned to the control value by 1 hr. Thus both erythropoietin and heme oxygenase genes appear to be expressed after treatment with tin-protoporphyrin, heme or cobalt chloride; however, the time and patterns of expression are different.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma, Hepatocellular; Cobalt; Erythropoietin; Gene Expression; Heme; Heme Oxygenase (Decyclizing); Humans; Liver Neoplasms; Metalloporphyrins; Protoporphyrins; RNA, Messenger; Tumor Cells, Cultured

Serum erythropoietin (Epo) levels are depressed in anemic AIDS patients relative to controls. The basis for abnormal regulation of Epo has not been defined. The hepatoma cell line Hep3B produces Epo in response to hypoxia and serves as a model for the study of Epo regulation. Hep3B cells are infectible with human immunodeficiency virus-1 (HIV-1) and were used as a model for evaluating potential direct effects of HIV on Epo expression. HIV-1 infected or transfected Hep3B cells produced Epo at significantly lower levels than uninfected Hep3B cells under low O2 conditions or following exposure to cobalt chloride. Epo production induced by hypoxia of HIV-1 infected Hep3B cells was depressed compared with non-HIV containing Hep3B cells when normalized for cell number, total cellular protein or albumin, but not depressed when normalized for alpha-fetoprotein production. The cellular levels of Epo mRNA were not diminished in the HIV-1+ Hep3B cells, indicating a probable posttranscriptional effect of HIV-1 on Epo production. Cellular protein production or secretion rates as measured by precipitable 3H-leucine were not affected by the presence of HIV-1. HIV-1 appeared to depress the production of Epo and some, but not all, other cellular proteins. These results suggest that impaired production of Epo may be a direct effect of HIV-1 infection possibly contributing to anemia in AIDS.

Topics: Carcinoma, Hepatocellular; Erythropoietin; Gene Expression; HIV-1; Humans; Liver Neoplasms; RNA, Messenger; Tumor Cells, Cultured

There is evidence that the inadequate erythropoietin (Epo) production observed in patients undergoing allogeneic bone marrow transplantation (BMT) might be ascribed to an inhibitory effect caused by the immunosuppressive drug cyclosporin A (CsA). In this in vitro study, we have evaluated the effects of CsA on the release of Epo in the culture medium by the human Hep3B hepatoma cell line. In cultures incubated with both CsA and the nonimmunosuppressive CsA analog MeAla-6, but not with the CsA-unrelated immunosuppressive agent FK-506, the levels of Epo in the medium were significantly reduced in comparison with controls, at concentrations (0.01 to 1.6 mumol/L) not affecting total protein synthetic rate nor the constitutive secretion of alpha-fetoprotein. Hep3B cells were found to contain a CsA-binding molecule, with an M(r) of 18 Kd, as assessed by high performance liquid chromatography (HPLC) and ligand-blotting analysis. CsA did not affect the expression of the Epo gene, as judged by Northern blot analysis, but caused a significant amount of Epo to remain unsecreted within the cells; almost all (97% of total) of the intracellular Epo was associated with the plasma membrane subcellular fraction. We conclude that: (1) CsA is able to inhibit Epo release in vitro by Hep3B cells, further supporting the hypothesis that the drug might have a role in the inappropriately low Epo levels observed in BMT patients; (2) the inhibitory effect appears to be specific and not caused by a general impairment of protein synthesis and/or secretion; and (3) the reduced Epo levels found in the medium of CsA-treated Hep3B cultures are supposed to be the consequence of an inability of the cells to correctly process Epo molecules for the secretory pathway.

Topics: Amino Acid Isomerases; Carcinoma, Hepatocellular; Carrier Proteins; Cyclosporine; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression; Humans; In Vitro Techniques; Liver; Liver Neoplasms; Peptidylprolyl Isomerase; Protein Biosynthesis; RNA, Messenger; Tumor Cells, Cultured

The present study was undertaken to assess the adenosine receptor regulation of erythropoietin (Ep) secretion in hepatocellular carcinoma cells (Hep3B) in response to hypoxia. In vitro cultures of Hep3B cells with N6-cyclohexyladenosine (CHA) at a concentration of 10(-5)M produced significant increases in cyclic AMP accumulation (142.43 +/- 13.31 pmole/10(6) cells) after 1 hr and Ep secretion (29.83 +/- 1.69 mU/ml) after 20 hr when compared with their respective hypoxia controls (cAMP: 3.05 +/- 0.26 pmole/10(6) cells, Ep: 19.41 +/- 1.41 mU/ml). The stimulatory effects of CHA on both Ep secretion and cyclic AMP accumulation were significantly inhibited by 8-phenyltheophylline (8-PT) at a concentration of 5 x 10(-7). No significant change in cell growth was observed at the CHA and 8-PT concentrations used in these experiments employing a spectrophotometric method. Incubation with 8-bromo cyclic AMP (10(-4)M) in response to hypoxia also produced a significant enhancement of Ep secretion (30.74 +/- 0.50 mU/ml) when compared with hypoxia controls (22.69 +/- 0.23 mU/ml), whereas no significant increase occurred in a normoxic atmosphere. CHA inhibited specific binding of [3H]5'-N-ethylcarboxamideadenosine (100 nM) to Hep3B cell membrane preparations in a dose-dependent manner in the displacement experiments and IC50 was 7.72 x 10(-6)M. These results indicate that Ep secretion is stimulated by adenosine membrane A2 receptors which are linked to adenylyl cyclase activation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Carcinoma, Hepatocellular; Cell Hypoxia; Cyclic AMP; Erythropoietin; Humans; Liver Neoplasms; Receptors, Purinergic; Theophylline; Tumor Cells, Cultured

A case of hepatocellular carcinoma complicated by erythrocytosis showed an increased level of serum immunoreactive erythropoietin (EPO) and EPO bioactivity. RT-PCR (reverse transcriptase and polymerase chain reaction) of EPO mRNA extracted from a surgical specimen indicated high expression of EPO mRNA in the tumor tissue. The nucleotide sequences of PCR amplified regions of the EPO precursor mRNA in tumor tissue showed three differences to those of normal EPO cDNA. The deduced amino acid sequence of the coding region also showed three differences from that of normal EPO. The erythrocytosis improved and the high serum EPO immunoreactive and bioactive level decreased after resection of the tumor. This is the first demonstration of mutant EPO mRNA expression and bioactive mutant EPO protein in hepatocellular carcinoma tissue.

Topics: Aged; Base Sequence; Carcinoma, Hepatocellular; Erythropoietin; Gene Expression; Humans; Liver Neoplasms; Male; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Oligonucleotides, Antisense; Polycythemia; Polymerase Chain Reaction; Reference Values

The blood level of erythropoietin (Epo) is often anomalously low in anemic patients with inflammatory or malignant diseases. Therefore, we studied effects of pure recombinant immunomodulatory peptides on Epo formation in cultures of the human hepatoma cell line, HepG2. Interleukin (IL)-1 beta, IL-1 alpha, and tumor necrosis factor alpha lowered Epo production with half-maximal inhibition at 2, 5, and 20 U/ml, respectively. IL-6, transforming growth factor beta 2 and interferon gamma did not inhibit. Furthermore, IL-1 beta (10 U/ml) proved to block Epo formation in isolated serum-free perfused rat kidneys. Proposedly, monokines play a role in the pathogenesis of Epo deficiency in various diseases.

Topics: Animals; Carcinoma, Hepatocellular; Erythropoietin; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-1; Kidney; Liver Neoplasms; Monokines; Rats; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

This study reports the effects of cyclic adenosine 3'-5' monophosphate (cAMP) and hypoxia on erythropoietin biosynthesis in an erythropoietin-producing hepatocellular carcinoma cell line (Hep3B). Erythropoietin levels in the medium and cell extracts of low-density Hep3B cells after 20-hour incubation under hypoxic conditions (1% O2) were 25.33 +/- 1.50 mU/ml/10(7) cells and 3.60 +/- 0.50 mU/10(7) cells, respectively. These levels were significantly higher than in the respective normoxic controls (medium, 2.51 +/- 0.31 mU/ml/10(7) cells; cell extracts, undetectable [less than 0.31 mU/10(7) cells]). Cobalt also produced a significant increase in medium and cell erythropoietin levels. However, hypoxia and cobalt alone failed to produce an increase in cAMP accumulation in the cell cultures. Erythropoietin levels in the medium and cell extracts from cells exposed to 8-bromo cAMP (1 x 10(-4) mol/L) and forskolin (4 x 10(-6) mol/L) in a hypoxic atmosphere were significantly (p less than 0.05) higher than in the respective hypoxic controls. In addition, forskolin produced a significant (p less than 0.05) increase in cAMP accumulation (180 +/- 11.5 pmol/10(6) cells) under hypoxic conditions compared with the hypoxic controls (cAMP, 2.27 +/- 0.33 pmol/10(6) cells). These results suggest that cAMP elevation is not required in vitro in Hep3B cells for the increase in medium and cell levels of erythropoietin after hypoxia, but may be involved indirectly in erythropoietin biosynthesis, secretion, or both in vivo through some synergistic action with hypoxia.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Carcinoma, Hepatocellular; Cobalt; Colforsin; Cyclic AMP; Erythropoietin; Humans; Hypoxia; Liver Neoplasms; Tumor Cells, Cultured

Using the human hepatoma cell line Hep G2, we have studied a possible role of protein kinase C (PKC) activity for regulation of erythropoietin (EPO) production. During a 72-h incubation, EPO production by the cells was stimulated sevenfold by exposure to low oxygen tension (1%) and threefold by exposure to cobaltous chloride (100 microM). The phorbol ester phorbol 12-myristate-13 acetate (PMA) led to a concentration-dependent inhibition of basal and stimulated EPO formation (ED50 10 nM). This decrease of EPO production, which was apparent already after 1 h of incubation with PMA, reached its maximal effect after 24 h and held on for 72 h. It was paralleled by an inhibition of the increase of EPO mRNA levels in response to stimulation. A 24-h preincubation of the cells with PMA (100 nM) virtually blunted the effect of hypoxia on EPO formation. Recovery of EPO synthesis after removal of PMA took 48-72 h. The effect of PMA on EPO production was mimicked by phorbol 12,13-dibutyrate (ED50 1 microM) but not by 4 alpha-phorbol 12,13-didecanoate. The synthetic diacylglycerol analogues oleolyl-acetylglycerol and dioctanoylglycerol (2-200 microM) also had no effect on either basal or stimulated EPO production. Treatment with PMA caused a translocation of the alpha-isoenzyme of PKC from the cytosol to the membrane after 1 h and a disappearance of the membrane-bound form after 24 h of incubation. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two structurally different inhibitors of PKC activity, inhibited basal and stimulated EPO production with ED50 values of 9 nM and 50 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Carcinoma, Hepatocellular; Erythropoietin; Humans; Isoquinolines; Liver Neoplasms; Oxygen; Piperazines; Protein Kinase C; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate; Tissue Distribution; Tumor Cells, Cultured

The effect of zinc on erythropoietin (EPO) synthesis in HepG2 cells was investigated. The increase in EPO synthesis induced by Co2+ (50 microM), Ni2+ (300 microM) or oxygen (1% O2) was inhibited by the presence of ZnCl2 (50-150 microM) in the tissue-culture medium, whereas basal EPO synthesis was unaffected. The effect was reflected by corresponding changes in the EPO mRNA level. These effects of zinc on EPO synthesis could not be mimicked by CdCl2 (less than or equal to 2 microM). Addition of FeCl3 to the medium appeared to decrease the inhibitory effect of zinc on hypoxia-induced EPO synthesis, implying that zinc may interfere with an iron-dependent step in EPO regulation.

Topics: Blotting, Northern; Cadmium; Carcinoma, Hepatocellular; Cations, Divalent; Cell Hypoxia; Cobalt; Depression, Chemical; Erythropoietin; Nickel; RNA, Messenger; Tumor Cells, Cultured; Zinc

The mechanisms which control the production of erythropoietin (Epo) remain enigmatic. Recent data suggest that the half-time of Epo messenger RNA (mRNA) is increased by hypoxia in Hep 3B cells, a human hepatoma line. The post-transcriptional regulation of other rapidly degraded mRNAs is mediated by sequence-specific mRNA binding proteins. In order to determine if Epo mRNA specific binding proteins exist, we probed cytosolic lysates from Hep 3B cells and mouse tissues with radiolabeled Epo RNA. A cytosolic protein that binds specifically to Epo RNA was identified in the Epo-producing, hepatoblastoma Hep 3B cell line by gel mobility shift assay. This protein was identified in both normoxic and hypoxic cells and bound specifically to a 120-base fragment of the 3'-untranslated region (3'-UTR) of Epo mRNA. Binding was completed with unlabeled Epo RNA, but not with granulocyte-macrophage colony-stimulating factor RNA. Ultraviolet light cross-linked Epo RNA-protein complexes migrated as two bands of 70 and 135-140 kD on sodium dodecyl sulfate-polyacrylamide gels. Binding activity was markedly increased in brain and spleen lysates from mice subjected to 24 h of hypoxia. Therefore, the post-transcriptional regulation of Epo expression in response to hypoxia may in part be due to the interaction of Epo RNA with its specific binding protein.

Topics: Animals; Binding, Competitive; Carcinoma, Hepatocellular; Carrier Proteins; Cytosol; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Mice; Oxygen; Restriction Mapping; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation

The present studies were undertaken to assess the direct effects of N6-cyclohexyladenosine (CHA), a stable adenosine analogue, on erythropoietin (Ep) secretion in hepatocellular carcinoma cells (Hep 3B). Ep levels in the medium of low density Hep 3B cells treated with CHA in concentrations of 10(-5) and 5 x 10(-5) M for 20 h under hypoxic conditions (1% O2) were significantly higher than that of hypoxic controls. In addition, CHA at the same concentrations produced significant increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels in Hep 3B cells after 1-h incubation under hypoxic conditions when compared with hypoxic controls. Dibutyryl cAMP (10(-5), 10(-4) M) also caused significant increases in Ep secretion when compared with control hypoxic cells. On the other hand, 8-phenyltheophylline, an adenosine receptor antagonist, significantly inhibited the stimulatory effects of CHA on both Ep secretion and cAMP accumulation in the Hep 3B cell cultures in response to hypoxia. These data suggest that Ep secretion may be regulated by adenosine receptor-coupled activation of adenylyl cyclase and the generation of cAMP.

Topics: Adenosine; Anaerobiosis; Bucladesine; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cyclic AMP; Erythropoietin; Humans; Hypoxia; Kinetics; Liver Neoplasms; Theophylline

The role of protein kinase C (PKC) in the control of erythropoietin (Epo) production was studied using the human hepatoma cell line HepG2. Inhibition of PKC by staurosporine and the selective PKC inhibitor CGP 41251 significantly reduced Epo formation. No inhibition occurred with the inactive staurosporine derivative CGP 42700. Treatment with phorbol 12-myristate 13-acetate (PMA) for 24 h dose-dependently inhibited Epo formation, thus suggesting that down-regulation of PKC might be responsible for this inhibition. Immunoblotting experiments showed that incubation of HepG2 cells with PMA for 24 h resulted in a selective and almost complete down-regulation of PKC-alpha. Thus, PKC-alpha may play a permissive role in Epo synthesis in HepG2 cells.

Topics: Alkaloids; Carcinoma, Hepatocellular; Erythropoietin; Humans; Isoenzymes; Kinetics; Liver Neoplasms; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The effects of several immunomodulatory peptides (recombinant, human) on the in vitro production of erythropoietin (Epo) were studied in cultures of the human hepatoma cell line Hep G2. A dose-dependent decrease of up to 60% in Epo production was induced by interleukin-1 beta, interleukin-1 alpha, and tumor necrosis factor-alpha (in that order of potency). In contrast, moderately increased Epo levels resulted with interleukin-6 or interferon-gamma treatment at high concentrations. Concomitant measurements of the production of alpha-fetoprotein indicated that the observed effects were specific for Epo. Hence, we suspect a modulating role of the immune system in the in vivo control of Epo production and postulate that interleukin-1 and tumor necrosis factor-alpha are involved in some of the cases of lowered blood Epo levels in association with renal diseases, chronic inflammation, and malignancies.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Line; Dose-Response Relationship, Drug; Erythropoietin; Humans; Interferon-gamma; Interleukin-1; Interleukin-6; Kinetics; Liver Neoplasms; Recombinant Proteins; Tumor Necrosis Factor-alpha

We have measured the serum erythropoietin concentrations in 14 patients with liver cirrhosis and in 14 patients with a hepatocellular carcinoma. Among these patients, 2 with liver cirrhosis (14.3%) and 7 with a hepatocellular carcinoma (50.0%) were found to have raised serum erythropoietin concentrations, ranging up to 40 mU/ml. Negative correlations were found between erythropoietin and the RBC, and the Hb and Ht in the cases with liver cirrhosis. In contrast, a positive correlation which was not significant was found only between the erythropoietin and the RBC in cases involving a hepatocellular carcinoma. This has suggested that the relationship between the erythropoietin and the RBC in cases of a hepatocellular carcinoma differs from the relationship seen under the usual physiological circumstances of those with liver cirrhosis.

Topics: Aged; Carcinoma, Hepatocellular; Erythrocyte Count; Erythropoietin; Female; Hematocrit; Hemoglobins; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged

Erythropoietin (Epo) gene transcription is stimulated in Hep3B cells under hypoxic conditions. We have prepared transcriptionally active nuclear extracts from normal and hypoxia-induced Hep3B cells and shown that the hypoxic extracts produce a consistent increase in the level of Epo transcription in vitro, relative to control Hep3B cells. Hypoxic treated HeLa cells failed to express the endogenous Epo gene in vivo, and extracts prepared from them did not show increased Epo transcription in vitro. The Epo transcript which is induced in vitro is initiated at the same site as Epo RNA synthesized in intact Hep3B cells and in human kidney adenocarcinoma cells. This system will facilitate the purification and analysis of factors and sequences required for Epo gene transcription in response to changes in tissue oxygen tension.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Erythropoietin; Genes; Humans; Hypoxia; Liver Neoplasms; Restriction Mapping; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic

Knowledge of the endogenous blood level of erythropoietin (Epo) has gained recent interest in view of the advances in Epo replacement therapy in anemic patients. By radioimmunoassay, we have carried out comparative measurements of the serum Epo level in patients suffering from chronic enterocolitis or leukemia. In chronic enterocolitis, the Epo level showed an exponential increase with the degree of anemia (up to 250 U Epo/1 serum at 70 g hemoglobin/1 blood). Similarly anemic patients with leukemia and severe bone marrow insufficiency of erythropoiesis had much higher Epo levels (usually above 500 U/1). Our findings indicate that the level of Epo is not only dependent on the blood hemoglobin concentration but also on the type of anemia. In fact, additional in vitro studies showed that immunomodulatory peptides can significantly influence the production of Epo in the hepatoma cell culture HepG2.

Topics: Adult; Anemia, Hemolytic; Carcinoma, Hepatocellular; Enterocolitis; Erythropoietin; Humans; Interferon-gamma; Interleukin-1; Leukemia; Liver Neoplasms; Radioimmunoassay; Recombinant Proteins; Tumor Cells, Cultured

Effects of agents affecting cytochrome P450 were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P450, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T3 and T4 stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyldithiocarbamate or cysteamine chloride, which inhibit cytochrome P450. These findings support the idea that O2 sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.

Topics: Carcinoma, Hepatocellular; Cobalt; Cysteamine; Cytochrome P-450 Enzyme System; Ditiocarb; Erythropoietin; Humans; Liver Neoplasms; Methylcholanthrene; Metyrapone; Microsomes; Oxygen; Phenobarbital; Thyroxine; Triiodothyronine; Tumor Cells, Cultured

Erythropoietin (Ep) levels in spent culture media of a Hep G2 human hepatoblastoma cell line were measured by radioimmunoassay (RIA), fetal mouse liver erythroid colony formation (FMLC), and the exhypoxic polycythemic mouse assay (EHPCMA). The Hep G2 cells at high density produced approximately 700 mU/ml Ep when measured with the RIA. On the other hand, the Ep levels when assayed in EHPCMA and FMLC were 50 and 2,600 mU/ml, respectively. The bioactivity in FMLC was completely neutralized by an antibody to purified human recombinant Ep, indicating that the erythropoietic activity in the Hep G2 spent culture medium was immunologically equivalent to Ep. Ep levels in the medium from low-density Hep G2 cells in 5% O2 and 1% O2 were 2.5- and 4-fold greater, respectively, than that of 20% O2. In contrast, hyperoxia (40% O2) significantly inhibited Ep production. A significant increase in Ep secretion was also observed when the cells were incubated with cobaltous chloride (2 X 10(-6) -2.5 X 10(-4) M). Tunicamycin (0.5 micrograms/ml), which inhibits N-linked glycosylation, significantly reduced the enhancement of Ep secretion induced by hypoxia (1% O2) without affecting cell growth. Forskolin and cholera toxin, each of which increased the levels of cyclic AMP in the Hep G2 cells by 40-fold, produced a significant (P less than 0.05) further increase in Ep secretion in the presence of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Cholera Toxin; Colony-Forming Units Assay; Cyclic AMP; Erythropoietin; Fetus; Hematopoietic Stem Cells; Humans; Kinetics; Liver; Liver Neoplasms; Mice; Radioimmunoassay; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Erythropoietin; Gene Expression Regulation; Genes, Regulator; Hemeproteins; Humans; Hypoxia; Liver Neoplasms; Metals; Oxygen; Tumor Cells, Cultured

Topics: Animals; Biological Assay; Blotting, Northern; Carcinoma, Hepatocellular; Cell Hypoxia; Cobalt; Erythropoietin; Gene Expression Regulation, Neoplastic; Hemeproteins; Humans; Liver Neoplasms; Manganese; Mice; Neoplasm Proteins; Nickel; Oxygen; Radioimmunoassay; RNA, Messenger; Second Messenger Systems; Tumor Cells, Cultured

Topics: Animals; Carcinoma, Hepatocellular; Cations; Cell Hypoxia; Erythropoietin; Gene Expression Regulation; Genes; Hemeproteins; Humans; Liver; Liver Neoplasms; Mice; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

We have identified a monoclonal antibody (ERY-1), which reacts with erythrocytes, erythroid precursor cells, and with embryonal yolk sac, and normal liver and kidney. The antibody also decorates the neoplastic cells of hepatocellular, renal, and yolk sac carcinomas. No reactivity was seen in a variety of other epithelial or mesenchymal neoplasms. It is possible that ERY-1 recognizes an erythropoiesis-associated antigen present in yolk sac, kidney, liver, and bone marrow, all of which are involved in erythropoiesis in various stages of human development. Furthermore, ERY-1 has proved to be extremely useful in the histopathologic diagnosis of hepatocellular and renal cell carcinomas.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Enzyme-Linked Immunosorbent Assay; Erythroblasts; Erythropoiesis; Erythropoietin; Humans; Immunohistochemistry; Kidney Neoplasms; Liver Neoplasms

A case of hepatocellular carcinoma associated with polycythemia and chronic thyroiditis, is reported in a 76-year-old female. At autopsy, the liver tumor was shown to be co-existent with liver cirrhosis. A series of hematological studies including the determination of plasma erythropoietin levels, led to the conclusion that this patient's polycythemia was most likely due to an excessive production of erythropoietin by the liver tumor. Chronic thyroiditis, another combined disease, might be related to liver cirrhosis, if the formerly advocated "hepato-thyroidal syndrome" is accepted. The author reports on this patient, since prior to this case there has been no documented case report of hepatocellular carcinoma accompanying both polycythemia and chronic thyroiditis.

Topics: Aged; Carcinoma, Hepatocellular; Erythropoietin; Female; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Polycythemia; Thyroiditis, Autoimmune

Erythropoietin (Epo), the hormone that stimulates red blood cell production, is synthesized in the kidney and liver in response to hypoxia. The human hepatoma cell line Hep3B regulates its production of Epo in a physiologic manner. Either hypoxia or cobalt chloride markedly increases expression of Epo mRNA as well as production of biologically active and immunologically distinct Epo protein. New protein synthesis is required before the induction of increased levels of hypoxia- or cobalt-induced Epo mRNA. Hypoxia, cobalt chloride, and nickel chloride appear to stimulate Epo production through a common pathway. The inhibition of Epo production at low partial pressures of oxygen by carbon monoxide provides evidence that a heme protein is integrally involved in the oxygen-sensing mechanism. This hypothesis is further supported by the finding that when heme synthesis is blocked, hypoxia-, cobalt-, and nickel-induced Epo production are all markedly inhibited. A model is proposed in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate the production of Epo.

Topics: Anaerobiosis; Carbon Monoxide; Carcinoma, Hepatocellular; Cell Line; Cobalt; Cycloheximide; Erythropoietin; Gene Expression Regulation; Genes; Hemeproteins; Humans; Iron; Liver Neoplasms; Manganese; Nickel; Transcription, Genetic

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cobalt; Cricetinae; Erythropoietin; Glycosylation; Humans; Hypoxia; Liver Neoplasms; Protein Processing, Post-Translational; RNA, Messenger; Transfection; Tumor Cells, Cultured; Tunicamycin

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

Topics: Carcinoma, Hepatocellular; Cell Line; Erythropoietin; Gene Expression Regulation; Humans; Liver Neoplasms; Neoplasm Proteins; RNA, Messenger; Tumor Cells, Cultured

Erythrocytosis (polycythemia) is a well-described paraneoplastic phenomenon in patients with hepatocellular carcinoma, but its pathogenesis remains uncertain. Using a radioimmunoassay, we have measured serum erythropoietin concentrations in 65 southern African blacks with this tumor and 61 matched controls. Four patients had an increased hemoglobin concentration and packed cell volume, and the remainder had normal values. Twenty-three percent of the patients with hepatocellular carcinoma (15/65) were found to have raised serum erythropoietin concentrations, the values ranging up to 344 mu/ml. Only one of these patients had an increased hemoglobin concentration and packed cell volume. This apparent anomaly could be explained if the erythrocytosis that would normally result from high serum erythropoietin values had been counteracted by the inhibition of erythropoiesis which occurs in advanced malignant disease. Alternatively, the erythropoietin produced by the tumor might not always be biologically active. Three patients had increased hemoglobin values and packed cell volumes in the presence of normal serum erythropoietin concentrations. One of these patients was hypoxic as a result of multiple pulmonary metastases, and the others may also have been. There was no correlation between serum erythropoietin and alpha-fetoprotein concentrations in individual patients.

Topics: Adolescent; Adult; Aged; alpha-Fetoproteins; Bilirubin; Carcinoma, Hepatocellular; Erythrocyte Volume; Erythropoietin; Female; Hemoglobins; Humans; Liver Neoplasms; Male; Middle Aged; Radioimmunoassay; Serum Albumin; South Africa

Topics: Animals; Carcinoma, Hepatocellular; Erythropoietin; Female; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Oocytes; Protein Biosynthesis; RNA, Messenger; Xenopus laevis

A case of hepatocellular carcinoma associated with erythrocytosis is described. The patient was successfully treated with a combination chemotherapy of 5-fluorouracil, mitomycin C, cyclophosphamide and chromomycin A3, which was followed by a left lateral hepatectomy, and has survived for more than 9 years after the operation without any signs of recurrence. With the regression of hepatic tumor by the anticancer chemotherapy, the remission of erythrocytosis was observed, indicating that the tumor was producing erythropoietin. The data suggest also that erythrocytosis could be a marker for evaluation of chemotherapeutic effects in some patients with hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Erythropoietin; Fluorouracil; Hepatectomy; Humans; Liver Neoplasms; Male; Middle Aged; Polycythemia

A Black patient with hepatocellular cancer and an increased red cell mass (erythrocytosis) is described. Assay of the patient's serum in ex-hypoxic polycythaemic mice showed the presence of erythropoietic activity. To determine the incidence of erythrocytosis in Southern African Blacks with hepatocellular cancer, haemoglobin and haematocrit values in 117 such patients were reviewed. Four patients (3,4%) had haemoglobin levels above the upper limit of normal and 2 (1,7%) of the patients had raised haematocrit values. Fifty-eight per cent of the patients were anaemic when they were first seen.

Topics: Adult; Altitude; Black or African American; Black People; Carcinoma, Hepatocellular; Erythropoietin; Hematocrit; Hemoglobins; Humans; Liver Neoplasms; Male; Polycythemia; South Africa

Topics: Carcinoma, Hepatocellular; Erythropoietin; Humans; Kidney; Liver; Liver Neoplasms; Male; Middle Aged; Polycythemia

Topics: Adrenal Gland Neoplasms; Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Erythropoietin; Heart Neoplasms; Hemochromatosis; Humans; Hypoglycemia; Iron; Liver; Liver Neoplasms; Lung Neoplasms; Male; Neoplasm Metastasis; Polycythemia; Precancerous Conditions

Among the malignant tumors of nonendocrine origin that are capable of producing polypeptide hormones and of manifesting as different endocrine syndromes discussed here are ectopic ACTH syndrome, SIADH, and ectopic gonadotropin-producing tumors.

Topics: Adrenocorticotropic Hormone; Carcinoma, Hepatocellular; Carcinoma, Small Cell; Chorionic Gonadotropin; Cushing Syndrome; Diagnosis, Differential; Erythropoietin; Follicle Stimulating Hormone; Gynecomastia; Hormones, Ectopic; Humans; Hyperthyroidism; Hypoglycemia; Hyponatremia; Liver Neoplasms; Lung Neoplasms; Luteinizing Hormone; Male; Paraneoplastic Endocrine Syndromes; Polycythemia; Puberty, Precocious; Thyrotropin; Vasopressins; Water Intoxication

Topics: Aged; Animals; Biopsy, Needle; Carcinoma, Hepatocellular; Chronic Disease; Erythropoietin; Female; Fluorouracil; Hemochromatosis; Humans; Liver Neoplasms; Male; Mice; Polycythemia

Topics: Adult; Bloodletting; Carcinoma, Hepatocellular; Chromium Radioisotopes; Circadian Rhythm; Erythropoietin; Hematocrit; Humans; Hypoxia; Iron Radioisotopes; Liver; Liver Neoplasms; Male; Middle Aged; Polycythemia

Topics: Carcinoma, Hepatocellular; Erythropoietin; Hemochromatosis; Humans; Liver Neoplasms; Male; Middle Aged; Polycythemia

Topics: Animals; Carcinoma, Hepatocellular; Erythropoietin; Hepatectomy; Humans; Kidney; Liver; Liver Neoplasms; Nephrectomy; Rats; Renal Artery Obstruction

Topics: Adult; Carcinoma, Hepatocellular; Erythropoietin; Humans; Liver Neoplasms; Male; Neoplasms

Topics: Adult; Animals; Autopsy; Carcinoma, Hepatocellular; Erythropoietin; Humans; Hypoxia; Iron Isotopes; Kidney; Liver; Liver Neoplasms; Male; Mitochondria; Polycythemia; Rats; Tissue Extracts

Topics: Adolescent; Adult; Animals; Carcinoma, Hepatocellular; Erythropoietin; Female; Hemangioma; Humans; Kidney Neoplasms; Liver Neoplasms; Male; Neoplasms; Polycythemia; Rats

Topics: Adult; Aged; Biological Assay; Carcinoma, Hepatocellular; Erythropoiesis; Erythropoietin; Humans; Liver Neoplasms; Male; Middle Aged; Polycythemia

Topics: Adult; Carcinoma, Hepatocellular; Cholesterol; Erythropoietin; Humans; Hyperlipidemias; Lipoproteins; Liver; Liver Function Tests; Liver Neoplasms; Male; Phospholipids; Polycythemia; Triglycerides

Topics: Adenocarcinoma; Animals; Blood; Carcinoma; Carcinoma, Hepatocellular; Epoetin Alfa; Erythropoietin; Growth Substances; Humans; Iron; Iron Isotopes; Liver Extracts; Liver Neoplasms; Liver Neoplasms, Experimental; Lymphoma; Neoplasms; Neoplasms, Experimental; Radiometry; Rats

Topics: Aged; Carcinoma, Hepatocellular; Erythropoietin; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Polycythemia; Urine