losartan-potassium and Cell-Transformation--Neoplastic

Tumor hypoxia, mostly resulting from poor perfusion and anemia, is one of the key factors in inducing the development of cell clones with an aggressive and treatment-resistant phenotype that leads to rapid progression and poor prognosis. Studies in patients with solid tumors suggest that there is a range of hemoglobin (Hb) concentrations that is optimum for tumor oxygenation. When used to achieve an Hb level within this range, erythropoiesis-stimulating agents (ESAs) can be expected to increase tumor oxygenation, and this may favorably influence sensitivity to treatment as well as quality of life. There is no robust evidence that ESAs, when used as indicated, have a negative effect on survival in patients with solid tumors. When used outside the indications recommended, the rise in Hb level that results may reduce tumor blood flow and tissue oxygenation because of a raised viscosity within the abnormal tumor microvasculature. In the current situation, it remains important to use ESAs within the approved indications and according to treatment guidelines such as those developed by the European Organization for Research and Treatment of Cancer.

Topics: Anemia; Cell Differentiation; Cell Hypoxia; Cell Proliferation; Cell Transformation, Neoplastic; Erythropoietin; Hematinics; Hemoglobins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neoplasms; Oxygen; Recombinant Proteins

Anabolic steroid and peptide hormones or growth factors are utilized to increase the performance of athletes of professional or amateur sports. Despite their well-documented adverse effects, the use of some of these agents has significantly grown and has been extended also to non-athletes with the aim to improve appearance or to counteract ageing. Pre-clinical studies and epidemiological observations in patients with an excess of hormone production or in patients chronically treated with hormones/growth factors for various pathologies have warned about the potential risk of cancer development and progression which may be also associated to the use of certain doping agents. Anabolic steroids have been described to provoke liver tumours; growth hormone or high levels of its mediator insulin-like growth factor-1 (IGF-1) have been associated with colon, breast, and prostate cancers. Actually, IGF-1 promotes cell cycle progression and inhibits apoptosis either by triggering other growth factors or by interacting with pathways which have an established role in carcinogenesis and cancer promotion. More recently, the finding that erythropoietin (Epo) may promote angiogenesis and inhibit apoptosis or modulate chemo- or radiosensitivity in cancer cells expressing the Epo receptor, raised the concern that the use of recombinant Epo to increase tissue oxygenation might favour tumour survival and aggressiveness. Cancer risk associated to doping might be higher than that of patients using hormones/growth factors as replacement therapy, since enormous doses are taken by the athletes often for a long period of time. Moreover, these substances are often used in combination with other licit or illicit drugs and this renders almost unpredictable all the possible adverse effects including cancer. Anyway, athletes should be made aware that long-term treatment with doping agents might increase the risk of developing cancer.

Topics: Anabolic Agents; Cell Transformation, Neoplastic; Doping in Sports; Erythropoietin; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Neoplasms; Recombinant Proteins; Risk Assessment; Steroids

The Friend spleen focus-forming virus has been a valuable tool for understanding the molecular events involved in the multiple stages of leukaemia. As summarized in Figure 3, the primary effect of SFFV, which occurs within days, is to cause a polyclonal proliferation of erythroid precursor cells that can proliferate in the absence of their normal regulator erythropoietin. This is the direct result of the unique envelope glycoprotein encoded by SFFV, which is transported to the cell surface and apparently interacts with the EpoR or another component of the multimeric EpoR complex, resulting in the constitutive activation of the Epo signal transduction pathway. Within this proliferating population of erythroid cells is a rare cell that has undergone several genetic changes due to the integration of the viral genome in specific sites in the mouse DNA. This leads to the activation of a gene encoding the PU.1 transcription factor, whose high expression in erythroid cells may be the cause of the block in differentiation that is characteristic of SFFV-transformed erythroid cells. SFFV integration can also lead to the inactivation of the p53 tumour supressor gene, giving these cells a growth advantage in the mouse. The disease induced by SFFV in mice is very similar to polycythaemia vera in humans (Golde et al, 1981). The major clinical feature of polycythaemia vera is the continuous expansion of the number of mature red blood cells in the presence of low serum Epo levels. Also, BFU-E and CFU-E from these patients can form in the absence of Epo like the analogous cells from SFFV-infected mice (Casadevall et al, 1982). It is possible that haematopoietic cells from individuals suffering from this disease express a protein similar to the envelope glycoprotein of SFFV that can interact with the EpoR and lead to its constitutive activation. Alternatively, these patients may contain a mutant EpoR gene that is constitutively activated like the mutant EpoR described earlier. As we understand more fully how the SFFV envelope protein constitutively activates te EpoR complex, we can begin to design therapies to counteract its action that can then be applied to treating patients with polycythaemia vera or other human diseases associated with uncontrolled erythropoiesis.

Topics: Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Defective Viruses; DNA-Binding Proteins; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Genes, env; Genome, Viral; Helper Viruses; Hyperplasia; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mutagenesis, Insertional; Receptors, Erythropoietin; Retroviridae Infections; Retroviridae Proteins, Oncogenic; Signal Transduction; Spleen Focus-Forming Viruses; Tumor Virus Infections; Viral Envelope Proteins; Virus Replication

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; Endoplasmic Reticulum; Erythropoietin; Friend murine leukemia virus; Gene Products, env; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Receptors, Erythropoietin; Viral Envelope Proteins

Topics: Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Colony-Stimulating Factors; Erythropoietin; Growth Substances; Hematopoietic Stem Cells; Humans; Interleukins; Leukemia; Oncogenes; Receptors, Cell Surface

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Erythropoietin; Friend murine leukemia virus; Humans; Leukemia, Erythroblastic, Acute; Lymphokines; Tissue Inhibitor of Metalloproteinases

Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.

Topics: Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chromatin; Dimethyl Sulfoxide; DNA; Erythropoiesis; Erythropoietin; Globins; Hemoglobins; RNA, Messenger

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

We report long-term results of treatment of myelodysplastic syndrome (MDS) with erythropoietin and granulocyte colony-stimulating factor (G-CSF). A total of 129 patients were followed up 45 months after last inclusion in the Nordic MDS Group studies. Erythroid response rate was 39% and median response duration 23 months (range, 3-116 months or more). Complete responders showed longer response duration than partial responders (29 versus 12 months, P = .006). The International Prognostic Scoring System (IPSS) groups Low/Intermediate-1 (Low/Int-1) had longer response duration than Int-2/High (25 versus 7 months, P = .002). The time until 25% developed acute myeloid leukemia (AML) was longer in the good and intermediate predictive groups for erythroid response compared with the poor predictive group (52 versus 13 months, P = .008). Only 1 of 20 long-term responders developed AML. We assessed the effect on long-term outcome by comparing treated patients with untreated patients selected from the IPSS database using multivariate Cox regression, adjusting for major prognostic variables. There was no difference in survival (odds ratio [OR], 0.9; 95% confidence interval [CI], 0.7-1.2; P = .55) or risk of AML evolution (OR, 1.3; 95% CI, 0.7-2.2; P = .40) between treated and untreated patients. Patients with high/intermediate probability of response and with IPSS Low/Int-1 show frequent and durable responses without adverse effects on outcome, while other patients should not be considered candidates for this treatment.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Analysis of Variance; Anemia; Cell Transformation, Neoplastic; Erythropoietin; Female; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Predictive Value of Tests; Prognosis; Survival Analysis; Treatment Outcome

The JAK2 V617F mutant-mediated aberrant signaling pathway is a hallmark of myeloproliferative neoplasms (MPNs). Although cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling (SOCS) are negative regulators of the JAK-STAT pathway, the functional role of CIS/SOCS family members in the JAK2 V617F mutant-induced oncogenic signaling pathway has not yet been elucidated. In this study, we found that the expression of CIS and SOCS1 was induced through the activation of signal transducer and activator of transcription 5 (STAT5) in not only the cells stimulated with Epo or IL-3 but also the cells transformed by the JAK2 V617F mutant. Cell proliferation and tumor formation in nude mice induced by the JAK2 V617F mutant were significantly enhanced when the expression of CIS was silenced using an RNA interference technique, whereas the knockdown of SOCS1 had no effect. The enforced expression of CIS caused apoptotic cell death in the transformed by JAK2 V617F mutant and drastically inhibited the JAK2 V617F mutant-induced tumor formation. CIS interacted with phosphorylated EpoR at Y401, which was critical for the activation of STAT5 and ERK. Whereas the activation of STAT5 and ERK in the transformed cells by JAK2 V617F mutant was increased by the knockdown of CIS, the enforced expression of CIS reduced the activation of these molecules. Furthermore, these anti-tumor effects of CIS required the function of SH2 domain and its tyrosine phosphorylation at Y253. We herein elucidated the mechanism by which CIS functions as a novel type of tumor suppressor in JAK2 V617F mutant-induced tumorigenesis.

Topics: Amino Acid Sequence; Animals; Carcinogenesis; Cell Proliferation; Cell Transformation, Neoplastic; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Female; Gene Knockdown Techniques; Humans; Janus Kinase 2; Mice, Inbred BALB C; Mice, Nude; Models, Biological; Mutation; Phosphorylation; Protein Binding; Receptors, Erythropoietin; STAT5 Transcription Factor; Suppressor of Cytokine Signaling Proteins

Angiogenesis is essential for tumor development. Accumulating evidence suggests that adenosine monophosphate-activated protein kinase (AMPK), an energy sensor and redox modulator, is associated with cancer development. However, the effect of AMPK on tumor development is controversial, and whether AMPK affects tumor angiogenesis has not been resolved. We show that deletion of AMPKα1, but not AMPKα2, upregulates non-canonical nuclear factor kappa B2 (NF-κB2)/p52-mediated cyclin-dependent kinase 2 (CDK2), which is responsible for the anchorage-independent cell growth of immortalized mouse embryo fibroblasts (MEFs). Co-culture with AMPKα1 knockout MEFs (or their conditioned medium) enhances the migration and network formation of human microvascular endothelial cells, which is dependent on p52-upregulated erythropoietin (Epo). AMPKα1 deletion stimulates cellular proliferation of allograft MEFs, angiogenesis, and tumor development in athymic nu/nu mice, which is partly ameliorated by antibody-mediated Epo neutralization. Therefore, the AMPKα1-p52-Epo pathway may be involved in stromal fibroblast-mediated angiogenesis and tumorigenesis.

Topics: AMP-Activated Protein Kinases; Animals; Cell Transformation, Neoplastic; Coculture Techniques; Cyclin-Dependent Kinase 2; Erythropoietin; Fibroblasts; Humans; Mice; Mice, Knockout; Mice, Nude; Neovascularization, Pathologic; NF-kappa B p52 Subunit

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Topics: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Erythropoietin; Female; Gene Expression Regulation, Neoplastic; Humans; Indans; Indoles; Kidney Neoplasms; Male; Mice; Mice, Inbred NOD; Mice, SCID; Molecular Targeted Therapy; Mutation; Pyrroles; Reproducibility of Results; Sulfones; Sunitinib; Xenograft Model Antitumor Assays

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Progression; Disease-Free Survival; Endothelial Cells; Erythropoietin; Female; Gene Expression Regulation; Humans; Hypoxia; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Neoplastic Stem Cells; Recurrence; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; DNA Fragmentation; Erythropoietin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Janus Kinase 2; Mice; Mice, Nude; Oligonucleotide Array Sequence Analysis; Ornithine Decarboxylase; Phosphorylation; Plasmids; Proto-Oncogene Proteins c-myc; Spermidine; STAT5 Transcription Factor

The putative presence of the erythropoietin receptor (EpoR) on human cancer cells has given rise to controversial discussion about the use of recombinant human erythropoietin (rhuEpo) for treatment of patients with chemotherapy-induced anemia. In vivo analysis of the EpoR status in tumors could help in elucidating the role of erythropoietin in cancer. Thus, the aim of this study was to develop a targeted EpoR probe for the investigation of EpoR expression in human lung cancer xenografts by fluorescence-mediated tomography.. Epo-Cy5.5 was generated by coupling Cy5.5 to rhuEpo. In vitro binding assays were performed using the EpoR-positive non-small cell lung cancer (NSCLC) cell lines A549 (lower EpoR expression) and H838 (higher EpoR expression), the EpoR-negative cell line H2030, and EpoR/EGFP-overexpressing HeLa cells. In vivo specificity of Epo-Cy5.5 was confirmed by competition analyses using micro-CT/fluorescence-mediated tomography fusion imaging. Biodistribution was analyzed over 50 h after injection. Binding of Epo-Cy5.5 was validated on tumor cryosections.. After intravenous injection, the probe was rapidly cleared from the circulation. An accumulation was observed in liver and kidneys, with a maximum at 7 h after injection followed by a decline, indicating renal excretion. Almost constant accumulation of Epo-Cy5.5 was found in bone marrow and tumors, indicating specific receptor binding. The probe allowed the discrimination between H838 with higher EpoR expression (89.54 ± 15.91 nM at 25 h) and A549 tumors with lower EpoR expression (60.45 ± 14.59 nM at 25 h, P < 0.05). Tumor accumulation of Epo-Cy5.5 could be significantly reduced by adding unlabeled rhuEpo (P < 0.05 at 4, 7, and 24 h). In vitro validation confirmed specific binding of Epo-Cy5.5 to the tumor cells, and this binding correlated with the EpoR expression level. Binding was also observed on endothelial cells. Vessel density and Epo-Cy5.5 binding on endothelial cells were comparable.. Epo-Cy5.5 allows the longitudinal analysis of EpoR expression in tumors and thereby can investigate the influence of erythropoietin on EpoR expression, tumor growth, and angiogenesis.

Topics: Animals; Bone Marrow; Carbocyanines; Cell Line, Tumor; Cell Transformation, Neoplastic; Endothelial Cells; Erythropoietin; Female; Gene Expression Regulation, Neoplastic; Humans; Infrared Rays; Lung Neoplasms; Mice; Molecular Imaging; Molecular Probes; Receptors, Erythropoietin; Substrate Specificity

Recent data suggest that erythropoietin (EPO) plays a substantial role in cancer development and clinical outcome by stimulating cell proliferation, invasion and angiogenesis. A functional polymorphism (rs1617640 G>T) in the promoter region of the EPO gene increases EPO protein expression. In the present study, we investigated the association of EPO rs1617640 G>T with susceptibility and clinical outcome of early-stage breast cancer. Genomic DNA of 539 female patients with histologically confirmed early-stage breast cancer and 804 controls was genotyped for EPO rs1617640 G>T. No association was found between EPO rs1617640 G>T and early-stage breast cancer susceptibility and clinical outcome (hazard ratio=1.24, 95% confidence interval=1.82-1.90, p=0.31). In conclusion, our findings suggest a lack of influence of EPO rs1617640 G>T on early-stage breast carcinogenesis and clinical outcome.

Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Erythropoietin; Female; Genetic Predisposition to Disease; Humans; Polymorphism, Genetic; Promoter Regions, Genetic; Treatment Outcome

Phospholipase C-γl (PLC-γl) is known to play a critical role in cell adhesion and migration and is highly expressed in metastatic tumors. In the current study, we found that cells transformed by PLC overexpression (PLC-γl cells) exhibited a marked decrease in expression of the Epo receptor (EpoR). Here, we assessed the role of EpoR-dependent signaling pathways in PLC-γl-dependent regulation of cell adhesion and migration.. Expression and phosphorylation of EpoR and its functional role in PLC-γl cells were evaluated by immunoblot analysis or cell adhesion assay. The mechanism for PLC-γ1-induced EpoR downregulation was analyzed by blockage of proteosomal degradation with MG132. EpoR expression was also confirmed in colorectal cancer tissues in which PLC-γl was highly expressed.. EpoR was present on rat fibroblasts, where it functionally active and capable of increasing cell adhesion and migratory activity. However, PLC-γl cells significantly decreased the Epo-dependent effects via ubiquitination-proteosomal degradation of EpoR. A marked decrease of EpoR expression was confirmed in colorectal cancer tissues that showed high-level of PLC-γl expression.. The Epo/EpoR complex plays a critical role in the adhesion and migration of rat fibroblasts, and its functional inactivation is associated with PLC-γl-dependent reduction of cell-matrix adhesion and this also affects cell migration.

Topics: Animals; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Colorectal Neoplasms; Down-Regulation; Erythropoietin; Fibroblasts; Focal Adhesions; Humans; Male; Mice; Middle Aged; Paxillin; PC12 Cells; Phospholipase C gamma; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rats; Receptors, Erythropoietin; Signal Transduction

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore, their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow (BM), liver, and spleen of mice exposed to a single dose (4 h) or seven daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased erythropoeitin signaling and erythropoiesis in the spleen and BM, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe) demonstrated that tissue hypoxia was present in the spleen and BM. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE-induced hemangiosarcoma in mice.

Topics: Animals; Bone Marrow; Cell Cycle; Cell Differentiation; Cell Hypoxia; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Endothelial Cells; Erythropoiesis; Erythropoietin; Ethylene Glycols; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genomic Instability; Hemangiosarcoma; Hematopoietic Stem Cells; Hemolysis; Hepatitis; Immunohistochemistry; Liver; Macrophage Activation; Male; Mice; Models, Biological; Signal Transduction; Spleen; Systems Biology; Time Factors

Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Topics: Animals; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Enzyme Inhibitors; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Janus Kinase 2; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; ras Proteins; Rats; Receptors, Erythropoietin; RNA Interference; Signal Transduction; STAT5 Transcription Factor; Transcriptional Activation; Up-Regulation

Preliminary findings of various types of globin expressed in the respiratory bronchiolar and alveolar epithelium prompted us to compare the expression of erythropoietin (Epo) and its receptor (EpoR) in normal (healthy) human lung tissues with that in malignant lung tissues. The expression of Epo and EpoR was examined at the transcriptional and protein levels in normal and malignant lung tissues by reverse transcription-PCR, western blot, and immunohistochemical analyses. EpoR mRNA, but not Epo mRNA, was detected in all samples. In normal tissues, EpoR was detected in the mesothelium, chondrocytes, alveolar cells, vascular endothelial cells, smooth muscle fibers, macrophages, and neutrophils, while in malignant foci, the cancer cells of five malignant types showed various intensities of EpoR immunoreactivity. The pattern of staining of EpoR protein was generally stronger in the malignant tissues than in the normal samples. Phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK-ERK1/2) was frequently seen in malignant cells, but not in the normal tissues, with the exception of macrophages. Based on the expression of Epo and EpoR mRNA with the EpoR in almost all cell components in normal tissues, we suggest that the normal lung may produce various types of globin through the autocrine and/or paracrine role of Epo. When the Epo signal is upregulated by hypoxic stress, the normal cells appear to transform into malignant cells and proliferate through activated MAPK signaling.

Topics: Autocrine Communication; Blotting, Western; Cell Transformation, Neoplastic; Erythropoietin; Globins; Humans; Hypoxia; Immunohistochemistry; Lung; Lung Neoplasms; MAP Kinase Signaling System; Paracrine Communication; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

To assess the effect of erythropoietin (EPO) plus granulocyte-colony stimulating factor (G-CSF) treatment on survival and leukemic transformation in myelodysplastic syndrome (MDS).. We compared the long-term outcome of patients with MDS treated with EPO plus G-CSF (n = 121) with untreated patients (n = 237) with MDS using multivariate Cox regression with delayed entry, for the first time adjusting for all major prognostic variables (WHO classification, karyotype, cytopenias, level of transfusion-need, age, and sex).. The erythroid response rate to EPO plus G-CSF was 39%, and the median response duration 23 months (range, 3 to 116+). In the multivariate analysis, treatment was associated with improved overall survival (hazard ratio, 0.61; 95% CI, 0.44 to 0.83; P = .002). Interestingly, this positive association was primarily observed in patients requiring fewer than 2 units of RBCs per month. Treatment was not linked to the rate of acute myeloid leukemia in any defined subgroup, including patients with an increase of marrow blasts or an unfavorable karyotype.. The inherent risk of leukemic evolution in MDS makes the current investigation highly relevant, in light of the recent reports of potential negative effects of EPO treatment on outcome in patients with cancer. We conclude that treatment of anemia in MDS with EPO plus G-CSF may have a positive impact on outcome in patients with no or low transfusion need, while not affecting the risk of leukemic transformation.

Topics: Aged; Anemia; Blood Transfusion; Cell Transformation, Neoplastic; Clinical Trials, Phase II as Topic; Erythropoietin; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Precancerous Conditions

Malignant phenotypic traits are caused by microenvironmental selection pressures during carcinogenesis. Hypoxia can drive a tumor toward a more aggressive malignant phenotype. The objective was to better understand the role of the hypoxia-regulated genes in cervical carcinogenesis.. We analyzed the expression of the hypoxia-regulated genes, including hypoxia-inducible factor-1alpha (HIF-1alpha), erythropoietin (Epo), vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), carbonic anhydrase IX (CAIX), and MET, in cervical cell lines and human tissue samples of cervical intraepithelial neoplasia (CIN I-III) and invasive squamous cell carcinoma (ISCC).. CAIX and MET were expressed in cervical carcinoma cell lines, but not in normal or human papillomavirus-immortalized cervical cells. In clinical tissue samples, Epo, VEGF, GLUT1, and CAIX were not detected in normal squamous epithelia. GLUT1 was expressed in nearly all cases of CIN and ISCC, however, CAIX was expressed only in CIN III and ISCC. HIF-1alpha and MET expression was confined to the basal cells in normal squamous epithelia and was detected in the dysplastic cells of CIN and ISCC.. The role of HIF-1alpha and MET changes from response to proliferation to tumor progression during cervical carcinogenesis. GLUT1 expression, a glycolytic phenotype adaptive to glycolysis, occurs early during cervical carcinogenesis and is a specific marker for dysplasia or carcinoma. MET and CAIX may contribute tumor progression in later stage. CAIX expression, an acid-resistant phenotype, may be a powerful adaptive advantage during carcinogenesis. Successful adaptation to the hypoxia-glycolysis-acidosis sequence in a microenvironment is crucial during carcinogenesis.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Line, Tumor; Cell Transformation, Neoplastic; Erythropoietin; Female; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Middle Aged; Papillomaviridae; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Activation; Erythropoietin; Gene Expression Regulation, Neoplastic; Hematopoiesis, Extramedullary; Humans; Janus Kinase 2; Liver; Mice; Mutation, Missense; Myeloproliferative Disorders; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-pim-1; Reactive Oxygen Species; Receptors, Erythropoietin; Signal Transduction; STAT5 Transcription Factor; Transduction, Genetic

Previous prognostic studies in primary myelofibrosis have focused on risk factors for overall survival and have resulted in the establishment of several prognostic scoring systems. However, to the authors' knowledge, information regarding risk factors for leukemic transformation in primary myelofibrosis is limited.. The current retrospective study examined clinical variables at the time of diagnosis and specific treatment modalities for their effect on leukemic transformation in 311 patients with primary myelofibrosis who were seen at the Mayo Clinic.. Univariate analysis of parameters at the time of diagnosis revealed a significant association between inferior leukemia-free survival and a peripheral blood blast percentage>or=3 (P<.0001), a platelet count<100x10(9)/L (P=.004), a monocyte count>or=1x10(9)/L (P=.02), the presence of hypercatabolic symptoms (P=.03), a low hemoglobin level (P=.04), and a high leukocyte count (P=.04). The first 2 parameters were found to maintain their statistical significance during multivariate analysis. Neither leukemia-free nor overall survival was found to be affected by the presence of <3% peripheral blood blasts or JAK2V617F mutation. The evaluation of treatment effect on leukemic transformation unexpectedly revealed a significant and independent association with previous therapy with either erythropoiesis-stimulating agents (P=.004) or danazol (P=.007), even when the aforementioned prognostic indicators at the time of diagnosis were added as covariates to the multivariate model. In contrast, leukemia-free survival was not found to be affected by a treatment history with hydroxyurea, thalidomide, or other drugs.. A peripheral blood blast percentage>or=3 and/or a platelet count<100x10(9)/L at the time of diagnosis were found to be strong and independent predictors of leukemic transformation in patients with primary myelofibrosis. The unexpected association between leukemic transformation and a history of treatment with erythropoiesis-stimulating agents or danazol requires validation by prospective studies.

Topics: Cell Transformation, Neoplastic; Danazol; Erythropoietin; Female; Humans; Leukemia; Male; Middle Aged; Primary Myelofibrosis; Risk Factors

Friend erythroleukemia virus has long served as a paradigm for the study of the multistage progression of leukemia. Friend virus infects erythroid progenitor cells, followed by an initial polyclonal expansion of infected cells, which is driven by the activation of a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). Subsequently, the accumulation of additional mutations in p53 and the activation of PU.1 result in full leukemic transformation. The early stages of transformation induced by Friend virus are characterized in vitro by the Epo-independent growth of infected erythroblasts. We have shown previously that this transforming event requires the kinase activity and Grb2 binding site of Sf-Stk and the recruitment of a Grb2/Gab2 complex to Sf-Stk. Here, we demonstrate that Stat3 is required for the Epo-independent growth of Friend virus-infected cells and that the activation of Stat3 by Sf-Stk is mediated by a novel Stat3 binding site in Gab2. These results underscore a central role for Stat3 in hematopoietic transformation and describe a previously unidentified role for Gab2 in the recruitment and activation of Stat3 in response to transforming signals generated by tyrosine kinases.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Binding Sites; Cell Transformation, Neoplastic; Disease Progression; Erythroid Precursor Cells; Erythropoietin; Friend murine leukemia virus; Humans; Leukemia, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Phosphoproteins; Receptor Protein-Tyrosine Kinases; Retroviridae Infections; Sequence Alignment; STAT3 Transcription Factor; Tumor Virus Infections

Autocrine/paracrine erythropoietin (EPO) action, promoting cell survival and mediated by its receptor (EPOR) in various solid tumors, including breast carcinoma, questions about the prognostic and therapeutic interest of this system. The expression of EPO/EPOR is steroid dependent in some tissues; however, a clear relationship of EPO/EPOR and steroid receptors in breast cancer has not been established thus far. Recently, the field of steroid receptors has expanded, including rapid effects mediated by membrane-associated receptors, regulating cell survival or apoptosis. The aim of this study was to evaluate EPO/EPOR and membrane-associated steroid receptor expression in breast carcinoma, in view of their prognostic significance, compared with other established markers [estrogen receptor (ER)-progesterone receptor (PR) status and Her2 expression] and hypoxia-induced factor 1 nuclear localization in 61 breast cancer specimens followed for

Topics: Adult; Aged; Breast; Breast Neoplasms; Cell Nucleus; Cell Transformation, Neoplastic; Disease-Free Survival; Erythropoietin; Female; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microscopy, Fluorescence; Middle Aged; Mitochondrial Proteins; Neoplasm Proteins; Prognosis; Receptor, ErbB-2; Receptors, Androgen; Receptors, Erythropoietin; Receptors, Estrogen; Receptors, Progesterone; Statistics as Topic

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistep process. At disease onset, preleukemic cells are arrested in differentiation at the proerythroblast stage (HS1 stage) and their survival and growth are under the tight control of erythropoietin (Epo). During disease progression, malignant proerythroblasts characterized by Epo autonomous growth and in vivo tumorigenicity can be isolated (HS2 stage). During analysis of transcriptional profiling representive of discrete stages of leukemic progression, we found that the phosphatidylinositol 4-phosphatase type II gene was turned off in malignant cells. PI-4-phosphatase II is an enzyme that hydrolyses the 4-phosphate position of phosphatidylinositol-3-4-bisphosphate (PtdIns(3,4)P(2)) to form PtdIns(3)P. Using malignant cells engineered to stably express PI-4-phosphatase II, we showed that PI-4-phosphatase II reduced Akt activation level. Moreover, stimulation of malignant cells with Epo-induced PI-4-phosphatase II transcription pointing this gene as an Epo-responsive gene. This study provides first insight for a physiological role of PI-4-phosphatase II in the proerythroblast by controlling Epo responsiveness through a negative regulation of the PI3K/Akt pathway.

Topics: Animals; Blotting, Northern; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Erythroblasts; Erythropoietin; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Leukemia, Erythroblastic, Acute; Mice; Mice, Transgenic; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel-Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4(-/-) perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1alpha to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1alpha in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients - presumably due to activated Wnt signaling - a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1alpha. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Cell Line; Cell Transformation, Neoplastic; Colon; Colonic Polyps; Colorectal Neoplasms; Disease Progression; Epithelial Cells; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intestinal Mucosa; Kidney; L Cells; Mice; Mice, Knockout; Neovascularization, Pathologic; Nerve Tissue Proteins; Precancerous Conditions; Promoter Regions, Genetic; Recombinant Fusion Proteins; Signal Transduction; TCF Transcription Factors; Transcription Factor 4; Transcription Factor 7-Like 2 Protein; Von Hippel-Lindau Tumor Suppressor Protein; Wnt Proteins; Wnt3 Protein

Treatment of anaemia with recombinant human erythropoietin beta (rHuEpo) in patients with squamous cell carcinoma of the head and neck (HNSCC) undergoing curative radiotherapy does not improve cancer control. In fact, incompletely resected patients with HNSCC receiving radiation in combination with rHuEpo showed poorer loco-regional progression-free survival than patients receiving radiation in combination with placebo. It could be hypothesized that the effects of rHuEpo on tumour cell growth might only be manifested in vivo and after cell trauma, and that treating anaemia with rHuEpo might contribute to poor outcome after incomplete surgical resection.. The aim of the present study was to examine the effect of rHuEpo alone and in combination with surgical trauma on the growth of human squamous cell carcinoma in vivo, xenografted onto nude mice.. The surgical trauma was inflicted through subcutaneous transection of the tumour with a needle. Immunohistochemical staining verified expression of the EPO receptor in tumour cells.. rHuEpo alone had no effect on the growth of xenografted HNSCC. However, a significant increase in tumour growth was observed after surgical trauma in combination with rHuEpo compared with surgery alone (p = 0.0008).

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cheek; Erythropoietin; Humans; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Recurrence, Local; Neoplasm Transplantation; Recombinant Proteins; Xenograft Model Antitumor Assays

Growth factor independence-1B (Gfi-1B) is a transcription factor with a highly conserved transcriptional repressor snail-Gfi-1 (SNAG) domain and 6 zinc-finger domains at the N- and C-terminus, respectively. Disruption of the Gfi-1B gene is lethal in the embryo with failure to produce definitive enucleated erythrocytes. In this study, we analyzed the role of Gfi-1B in human erythropoiesis. We observed an increase of Gfi-1B expression during erythroid maturation of human primary progenitor cells. We studied the consequences of variations in Gfi-1B expression in 2 transformed cell lines (K562 and UT7 cells), as well as in primary CD36(+)/GPA(-) progenitors. A knock-down of Gfi-1B delayed the terminal differentiation of K562 and primary cells. Forced expression of Gfi-1B in UT7 and K562 cells led to an arrest of proliferation and an induction of erythroid differentiation. Enforced expression of Gfi-1B in primary cells at the colony-forming units-erythroid (CFU-E) stage led to a partial glycophorin A (GPA) induction after erythropoietin (EPO) withdrawal but failed to protect cells from apoptosis. Deletion of the SNAG repressor domain abolished Gfi-1B-induced erythroid maturation, strongly suggesting that Gfi-1B acts in the late stage of erythroid differentiation as a transcriptional repressor.

Topics: CD36 Antigens; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Down-Regulation; Erythroid Precursor Cells; Erythropoietin; Gene Silencing; Humans; K562 Cells; Protein Structure, Tertiary; Proto-Oncogene Proteins; Repressor Proteins; RNA, Small Interfering; Transfection; Up-Regulation

Many leukemia and cancer cells exhibit constitutive activation of STAT5, which was suggested to provide an anti-apoptotic advantage. Transformation of cytokine-dependent hematopoietic cells, such as Ba/F3 cells to autonomous growth and tumorigenicity equally results in selection for constitutive activation of STAT5. We compared STAT5 signaling between erythropoietin(Epo)-dependent cells and cells that were transformed by oncogenic activation of the erythropoietin receptor (EpoR) by coexpression of the gp55-P envelope protein of the spleen focus forming virus or by expression of the R129C constitutively active EpoR mutant. In transformed cells it was mainly STAT5B that was constitutively activated. In contrast, Epo stimulation activated both STAT5A and STAT5B. In transformed cells, chromatin immunoprecipitation (ChIP) showed STAT5 to be physically bound to promoters of STAT5 target genes, such as Bcl(XL), and to be able to promote transactivation of the Bcl(XL) promoter in a constitutive fashion. Sequencing of native sequences after ChIP with anti-STAT5 antibodies in Epo-dependent and -transformed cells indicated that in gp55-transformed cells, STAT5B bound in the chromatin not only to N3 high affinity, but also to low affinity N4 GAS sites. Transactivation for N3 GAS sites in luciferase reporters was specific to gp55 transformation. Because we also found preferential constitutive STAT5B activation after transformation of cells by a truncated form of the G-CSF-R that produces severe neutropenia (Kostmann syndrome) and favors leukemia in humans, we discuss the potential role of STAT5B in oncogenic transformation of hematopoietic cells.

Topics: Active Transport, Cell Nucleus; Animals; bcl-X Protein; Cell Line; Cell Transformation, Neoplastic; DNA-Binding Proteins; Erythropoietin; Growth Substances; Hematopoietic Stem Cells; Humans; Ligands; Mice; Milk Proteins; Neutropenia; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytokine; Receptors, Erythropoietin; Receptors, Granulocyte Colony-Stimulating Factor; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Transcription, Genetic; Tumor Suppressor Proteins; Viral Envelope Proteins

Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl(2) treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Cobalt; Coloring Agents; Culture Media, Conditioned; Disease Progression; Dose-Response Relationship, Drug; Erythropoietin; Humans; Hypoxia; Immunohistochemistry; MAP Kinase Signaling System; Melanocytes; Melanoma; Membrane Potentials; Mitochondria; Phosphorylation; Receptors, Erythropoietin; Tetrazolium Salts; Thiazoles; Trypan Blue

FLI-1 is a transcriptional regulator of the ETS family of proteins. Insertional activation at the FLI-1 locus is an early event in F-murine leukemia virus-induced erythroleukemia. Consistent with its essential role in erythroid transformation, enforced expression of FLI-1 in primary erythroblasts strongly impairs the response of these cells to erythropoietin (Epo), a cytokine essential to erythropoiesis. We show here that point mutations in the ETS domain that abolished FLI-1 binding to specific DNA elements (ETS-binding sites) suppressed the ability of FLI-1 to transform erythroblasts. The exchange of the entire ETS domain (DNA binding domain) of FLI-1 for that of PU.1 changed the DNA binding specificity of FLI-1 for that of PU.1 and impaired FLI-1 transforming properties. In contrast, ETS domain swapping mutants that maintained the DNA binding specificity of FLI-1 did not affect the ability of FLI-1 to transform erythroblasts. Deletion and swapping mutants that failed to inhibit the DNA binding activity of FLI-1 but impaired its transcriptional activation properties were also transformation-defective. Taken together, these results show that both the ability of FLI-1 to inhibit Epo-induced differentiation of erythroblasts and to confer enhanced cell survival in the absence of Epo critically depend upon FLI-1 ETS-binding site-dependent transcriptional activation properties.

Topics: Animals; Cell Differentiation; Cell Transformation, Neoplastic; Chickens; DNA; DNA-Binding Proteins; Erythroblasts; Erythropoietin; Humans; Mice; Protein Binding; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; Trans-Activators; Transcriptional Activation

Erythropoietin (Epo), induced by hypoxia, controls the survival, proliferation, and differentiation of Epo receptor (EpoR)-bearing erythroid progenitors and plays a role in the protection of neurons from hypoxic damage. Hypoxia in malignant disease is associated with invasion, metastasis, resistance to therapy, and selection for cells with diminished apoptotic potential. The authors recently demonstrated the basal and hypoxia-stimulated expression of Epo and EpoR in human breast carcinoma cell lines and in breast carcinomas, suggesting a role for autocrine Epo signaling in the hypoxic adaptations of mammary neoplasms.. The authors characterized the expression of Epo and EpoR by immunohistochemistry in 184 invasive mammary carcinomas and 158 in situ mammary carcinomas and benign mammary epithelium. They analyzed the correlation of Epo and EpoR immunostaining with clinicopathologic tumor features and the patients' smoking history.. Benign mammary epithelial cells showed weak-to-moderate expression of Epo and EpoR. EpoR immunostaining was increased in carcinomas compared with benign epithelium both in nonsmokers and smokers, and Epo immunostaining was increased in carcinomas compared with benign epithelium in nonsmokers but not in smokers. Prominent Epo staining was seen in tumor cells adjacent to necrotic areas and at the infiltrating edge of tumors. EpoR staining, but not Epo staining, was significantly greater in tumors that showed high histologic grade, tumor necrosis, lymphovascular invasion, lymph node metastases, and loss of hormone receptor expression.. The current findings suggest that increased EpoR expression may play an important role in breast carcinogenesis. The induction of autocrine or paracrine Epo signaling may represent a novel mechanism by which hypoxia can promote breast carcinoma.

Topics: Breast Neoplasms; Carcinoma; Cell Transformation, Neoplastic; Erythropoietin; Female; Humans; Hypoxia; Immunohistochemistry; Receptors, Erythropoietin; Signal Transduction; Smoking

This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Enzyme Activation; Erythroid Precursor Cells; Erythropoietin; Hemoglobins; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Liver; Mice; Molecular Sequence Data; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Signal Transduction; src-Family Kinases

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Delta257 and Delta321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Delta321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Delta321 receptor, the Delta257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias. Oncogene (2000) 19, 953 - 960.

Topics: Animals; Cell Differentiation; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Erythropoietin; Genes, Dominant; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Mutation; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Tumor Cells, Cultured

Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (triangle upSH3), the SH2 domain was deleted (triangle upSH2), the C-terminal actin-binding domain was deleted (triangle upABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the triangle upSH3 mutant was equivalent, the triangle upSH2 mutant was moderately impaired, and the triangle upABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and triangle upSH3. However, time to death was prolonged by at least twofold for triangle upSH2 and greater than threefold for triangle upABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.

Topics: Amino Acid Substitution; Animals; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Erythropoietin; G1 Phase; Genes, abl; Hematopoietic Stem Cells; Humans; Mice; Mice, Nude; Phosphorylation; Point Mutation; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-abl; Receptors, Erythropoietin; Recombinant Fusion Proteins; Sequence Deletion; Signal Transduction; src Homology Domains; Structure-Activity Relationship

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Base Sequence; Cell Transformation, Neoplastic; Cloning, Molecular; DNA, Complementary; Erythropoietin; Fusion Proteins, bcr-abl; Genes; Granulocyte-Macrophage Colony-Stimulating Factor; GRB2 Adaptor Protein; Humans; Leukemia, Megakaryoblastic, Acute; Molecular Sequence Data; Neoplasm Proteins; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Recombinant Fusion Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; src Homology Domains; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

Growth factors or humoral agents can support haemopoiesis in various bone marrow disorders. They have the ability to act on multiple cell lineages and in myeloid cells, and the potential to act on the neoplastic equivalent of normal cells. Anaemia is a common feature of multiple myeloma seen in at least two-thirds of patients at presentation. Erythropoietin is increasingly being used with variable effect for the treatment of this anaemia, especially in cases associated with renal failure and in patients in whom blood transfusion may be undesirable or contraindicated. We describe a patient treated with recombinant erythropoietin who developed fulminating malignant transformation. The demonstration of erythropoietin receptors on a human myeloma cell line and the occurrence of the rare complication of plasma cell leukaemia in our patient stresses the need for caution and invites detailed clinical and laboratory studies before its general use.

Topics: Anemia; Cell Transformation, Neoplastic; Disease Progression; Erythropoietin; Fatal Outcome; Humans; Leukemia, Plasma Cell; Male; Middle Aged; Multiple Myeloma; Recombinant Proteins

The erythroleukaemic cell line TF-1, infected with either the pBabe neo retrovirus or the retrovirus bearing the human erythropoietin (hEpo) gene, developed three growth factor-independent clones. Erythropoietin (Epo), interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) accelerated the proliferation of these clones. Autonomous growth of the clones was independent of Epo because it was not altered by Epo anti-sense oligonucleotides, nor was Epo detectable in culture supernatants. Cells from the mutant clones could not be induced by Epo to express glycophorin A and haemoglobin synthesis was markedly reduced. Haemin reversed the block in Epo-induced haemoglobin synthesis. Acquisition of growth factor-independence appears to be linked with the selective loss of differentiation capacity. These cells may provide a useful model for the study of the mechanisms involved in leukaemic transformation.

Topics: Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; DNA Primers; Erythropoietin; Glycophorins; Granulocyte-Macrophage Colony-Stimulating Factor; Hemoglobins; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Oligonucleotides, Antisense; Polymerase Chain Reaction; Retroviridae; Tumor Cells, Cultured

HU-3 is a bipotential cell line derived from the bone marrow of a patient with megakaryoblastic leukemia. Continuously proliferating cells evolved from cultures supplemented with nutrient medium containing human serum and granulocyte-macrophage (GM) colony-stimulating factor (CSF). Growth and viability of the HU-3 cell line was strictly dependent on the presence of GM-CSF, interleukin-3, or thrombopoietin (Tpo). Independent of the cytokine, the cells constitutively expressed a well-defined megakaryocyte phenotype, with 70-95% of the cells positive for CD4, CD34, and platelet glycoproteins Ib, IIb, and IIIa. Fewer than 10% of the cells had detectable erythroid glycophorin A. Erythropoiesis was induced in HU-3 parental cells and five clones harvested from culture medium containing GM-CSF by replacement of the growth-promoting cytokine with stem cell factor (SCF) and erythropoietin (Epo). During the first week of induction, the proliferating cells slowly acquired erythroid markers. Concomitant with a maturational growth arrest during the second week, there was a rapid accumulation of gamma and beta globin chains and benzidine reactive hemoglobin, as well as a distinct erythroid morphology. The culture declined after 12 days because of the transient effect of SCF in maintaining viability. Parental and cloned cells cultured for 7 days in Tpo-supplemented medium responded to the synergistic growth effect of SCF and Epo but were markedly suppressed in their yield of hemoglobinized cells. Recycling of the cells in GM-CSF for 4 days did not reverse the suppressive effect of Tpo. These results suggest a role for Tpo in the lineage commitment of erythromegakaryocytic progenitors by suppressing the erythroid potential. With its constitutive megakaryocyte phenotype and inducible erythroid potential, the self-renewing bipotential HU-3 cell line may represent one of the earliest stages in megakaryocytopoiesis before irreversible lineage commitment. The suppressive effect of Tpo on the erythroid potential of cloned HU-3 cells enhances the value of this cell line for deciphering the molecular and cellular events during lineage commitment of progenitor cells.

Topics: Cell Division; Cell Transformation, Neoplastic; Cytogenetics; Cytokines; Erythropoiesis; Erythropoietin; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Megakaryoblastic, Acute; Stem Cell Factor; Thrombopoietin; Tumor Cells, Cultured

Ineffective hematopoiesis with associated cytopenias and potential evolution to acute myeloid leukemia (AML) characterize patients with myelodysplastic syndrome (MDS). We evaluated levels of apoptosis and of apoptosis-related oncoproteins (c-Myc, which enhances, and Bcl-2, which diminishes apoptosis) expressed within CD34+ and CD34- marrow cell populations of MDS patients (n = 24) to determine their potential roles in the abnormal hematopoiesis of this disorder. Marrow cells were permeabilized and CD34+ and CD34- cells were separately analyzed by FACS to detect: (1) a subdiploid (sub-G1) DNA population, and (2) expression of Bcl-2 and c-Myc oncoproteins. Within the CD34+ subset, a significantly increased percentage of cells demonstrated apoptotic/sub-G1 DNA content in early (ie. refractory anemia) MDS patients compared with normal individuals and AML patients (mean values: 9.1% > 2.1% > 1.2%). Correlated with these findings, the ratio of expression of c-Myc to Bcl-2 oncoproteins among CD34+ cells was significantly increased for MDS patients compared to those from normal and AML individuals (mean values: 1.6 > 1.2 > 0.9). Bcl-2 and c-Myc oncoprotein levels were maturation stage-dependent, with high levels expressed within CD34+ marrow cells, decreasing markedly with myeloid maturation. Treatment of seven MDS patients with the cytokines granulocyte colony-stimulating factor plus erythropoietin was associated with decreased levels of apoptosis within CD34+ marrow cells and may contribute to the enhanced hematopoiesis in vivo that was shown. These findings are consistent with the hypothesis that altered balance between cell-death (eg, c-Myc) and cell-survival (eg, Bcl-2) programs were associated with the increased degrees of apoptosis present in MDS hematopoietic precursors and may contribute to the ineffective hematopoiesis in this disorder, in contrast to decreased apoptosis and enhanced leukemic cell survival in AML.

Topics: Acute Disease; Adult; Aged; Apoptosis; Bone Marrow; Cell Cycle; Cell Transformation, Neoplastic; Disease Progression; DNA, Neoplasm; Erythropoietin; Female; Gene Expression Regulation; Genes, bcl-2; Genes, myc; Granulocyte Colony-Stimulating Factor; Hematopoiesis; Humans; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Recombinant Proteins

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.

Topics: Animals; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Clone Cells; Culture Media, Conditioned; Erythropoietin; Humans; Interleukin-3; Janus Kinase 2; Kinetics; Mice; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Signal Transduction; Spleen Focus-Forming Viruses; Transfection; Viral Envelope Proteins

A favorable prognosis and a low rate of leukemic transformation has been attributed to the 5q- syndrome, a myelodysplastic syndrome (MDS) characterized by macrocytic anemia, hypolobulated micromegakaryocytic hyperplasia, and an interstitial deletion of chromosome 5. We examined the characteristics and outcome of 43 consecutive patients in our institution strictly defined by morphologic criteria and a solitary 5q- cytogenetic defect. The median age at diagnosis was 68 years, with a clear female predominance (7:3). Eighty percent of the patients were red blood cell transfusion-dependent at diagnosis and all untransfused patients had macrocytic indexes. In contrast, significant neutropenia or thrombocytopenia was rare. The French-American-British (FAB) class distributions were RA (72%), RARS (7%), RAEB (16%), and RAEB-IT (5%). At a median follow-up of 31 months, 56% of the patients survive, with a projected median survival of 63 months. The incidence of acute leukemia was 16% and was uniformly fatal. Clinical hemosiderosis occurred in 28% of the patients, resulting in two deaths. Neither survival nor the risk of leukemic transformation was predictable from initial clinical parameters, including FAB classification, Bournemouth score, and degree of aneuploidy. The lack of significant neutropenia and thrombocytopenia seemed to account for a very low incidence of infection and bleeding resulting in a prognosis equal or superior to historical patients with MDS. Therapeutic endeavors, including the use of corticosteroids, androgens, cis-retinoic acid, pyridoxine, and danazol, were largely unsuccessful.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Blood Transfusion; Bone Marrow; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 5; Erythropoietin; Female; Gene Deletion; Humans; Leukemia; Male; Middle Aged; Myelodysplastic Syndromes; Prognosis

In this study, specific binding sites were examined for erythropoietin (EPO) on the mouse leukemic cell line, L8057. This cell line is megakaryoblastic in origin as evidenced by an enlargement of cell size, multinuclearity, intense activity of acetylcholinesterase, more expression of glycoprotein IIb and IIIa antigen, and higher ploidy distribution after the treatment with 12-o-tetradecanoylphorbor-13-acetate (TPA). The original undifferentiated cells possessed a single class of low affinity binding sites for recombinant human (rh) EPO with a Kd of 3.5 nM. Following the treatment with TPA, high affinity binding sites (Kd; 440 pM) were expressed in addition to the low affinity sites. EPO stimulated the incorporation of 3H-leucine into TPA-treated L8057 cells, and the maximal effect of EPO was observed at the same order as the Kd value of high affinity sites. The present data demonstrates that the expression of high affinity binding sites for EPO is associated with the differentiation of L8057 cells which have megakaryocytic characteristics. Furthermore, protein synthesis stimulated by EPO may be mediated through the high affinity sites.

Topics: Acetylcholinesterase; Animals; Antigens; Cell Differentiation; Cell Transformation, Neoplastic; DNA, Neoplasm; Erythropoietin; Iodine Radioisotopes; Leucine; Leukemia, Experimental; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Mice; Platelet Membrane Glycoproteins; Protein Binding; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Tetradecanoylphorbol Acetate; Thymidine; Tumor Cells, Cultured

Transplantation of spleen cells from primary reconstituted mice expressing the v-src oncogene to secondary and tertiary irradiated recipients resulted in the emergence of erythroid precursors with a transformed phenotype. When cultured in methyl cellulose, these precursors generated colonies of undifferentiated cells that could be expanded into continuously growing factor-dependent cell lines in liquid culture. All lines tested had a similar phenotype and expressed the v-src oncogene. In addition they responded to factors that regulate normal erythroid development, namely erythropoietin (Epo), interleukin-3 (IL-3), and mast cell growth factor (MGF), the ligand to the c-kit encoded receptor. When cells from one of the lines were maintained in the absence of factor, a "factor independent" subpopulation emerged that appeared to grow in an autocrine fashion. Conditioned medium from these cells stimulated their own growth as well as the growth of broad spectrum of normal precursors. Studies with neutralizing antibodies indicated that the predominant colony-stimulating factor produced by these cells is IL-3.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Division; Cell Line; Cell Separation; Cell Transformation, Neoplastic; Erythroid Precursor Cells; Erythropoietin; Gene Expression; Genes, src; Growth Substances; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Interleukin-3; Mice; Phenotype; Spleen; Stem Cell Factor

The Friend or Moloney mink cell focus-forming (MCF) virus encodes a recombinant-type envelope glycoprotein, gp70, that is closely related to the membrane glycoprotein, gp55, of Friend spleen focus-forming virus (SFFV). We have shown previously that gp55 has the ability to activate cell growth by binding to the cellular receptor for erythropoietin. Here we show that gp70 encoded by either the Friend or Moloney MCF virus also binds to the erythropoietin receptor and that coexpression of the receptor and gp70 in an interleukin-3 (IL-3)-dependent cell line can activate IL-3-independent growth. Furthermore, when the cDNA for the human IL-2 receptor beta chain, which is related by sequence to the erythropoietin receptor, was introduced into this cell line, it became growth factor independent after infection either with SFFV or with one of the two MCF viruses but not with an ecotropic virus. Based on these observations, we propose a mechanism for the early stage of leukemogenesis induced by the MCF-type murine leukemia viruses.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Friend murine leukemia virus; Humans; Interleukin-3; Leukemia; Mice; Mink Cell Focus-Inducing Viruses; Moloney murine leukemia virus; Receptors, Cell Surface; Receptors, Erythropoietin; Receptors, Interleukin-2; Retroviridae Proteins, Oncogenic; Viral Envelope Proteins

Friend erythroleukemia cells (FLC) passaged in mice are highly tumorigenic and multiply extensively in the livers of suckling DBA/2 mice without differentiating. In contrast, in vitro passaged FLCs injected intravenously were of low tumorigenicity, multiplied to a limited extent in the livers of suckling mice, and underwent marked differentiation from the proerythroblast to the orthochromatic erythroblast stage in the liver. The presence of characteristic C-type virions budding from the cell surface in various stages of erythroid differentiation served as a marker of the injected FLCs. When the same in vitro passaged FLCs that differentiated in the liver were injected subcutaneously in suckling mice, they formed large subcutaneous tumors consisting of sheets of undifferentiated tumor cells. It is concluded that the tumorigenicity of FLCs depended on the site of tumor growth and that there is an inverse correlation between the tumorigenic capacity and the capacity to differentiate.

Topics: Animals; Animals, Suckling; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Erythropoietin; Friend murine leukemia virus; Injections, Intravenous; Injections, Subcutaneous; Interleukin-3; Leukemia, Erythroblastic, Acute; Liver; Mice

We have compared the effects of the v-erb A oncogene on proliferation and differentiation of normal erythroid progenitors with those of tyrosine kinase oncogenes, e.g. v-sea. For this, a v-erb A retrovirus containing the neomycin resistance gene as a selectable marker or, alternatively, a v-erb A-ts v-sea retrovirus were used to infect normal bone marrow cells. V-erb A induced the outgrowth of immature, erythropoietin(EPO)-dependent erythroid cells from infected bone marrow which ceased to proliferate and disintegrated after 9 to 18 divisions. In contrast, ts-v-sea erythroblasts grew for the expected 25 to 40 population doublings in the absence of EPO. Transcription of the erythrocyte genes carbonic anhydrase II and erythrocyte anion transporter was significantly inhibited in v-erb A infected erythroblasts, indicating that v-erb A alone was sufficient for the repression of the erythrocyte-specific genes observed in AEV-transformed leukemic cells. A detailed analysis of the differentiation phenotype induced by v-erb A in erythroblasts (in the presence or absence of a temperature-inactivated ts sea oncogene) indicates that v-erb A-erythroblasts express a partially mature, aberrant phenotype characterized by the coexpression of mature and immature differentiation antigens. This phenotype clearly differs from that induced by tyrosine kinase oncogenes in erythroid cells.

Topics: Animals; Antigens, Surface; Carbonic Anhydrases; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chickens; Erythroblasts; Erythrocytes; Erythropoietin; Fibroblasts; Hematopoietic Stem Cells; Oncogene Proteins v-erbA; Oncogenes; Retroviridae Proteins, Oncogenic; Transcription Factors; Transcription, Genetic

The receptors for erythropoietin and other cytokines constitute a new superfamily. They have no tyrosine-kinase or other enzyme motif and their signal-transducing mechanism is unclear. Here we describe two classes of activating mutations in the erythropoietin receptor (EPOR). A single point mutation in the exoplasmic domain enables it to induce hormone-independent cell growth and tumorigenesis after expression in nontumorigenic, interleukin-3-dependent haematopoietic cells. A C-terminal truncation in the cytoplasmic domain of the EPOR renders the receptor hyperresponsive to erythropoietin, but is insufficient to induce hormone-independent growth or tumorigenicity. The activating point mutation retards intracellular transport and turnover of the receptor. These alterations in metabolism and tumorigenicity caused by the EPOR with activating point mutations are similar to those observed in erythropoietin-independent activation of the wild type EPOR by association with gp55, the Friend spleen focus-forming virus glycoprotein.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Erythropoietin; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotide Probes; Receptors, Cell Surface; Receptors, Erythropoietin; Transfection

The abilities of human recombinant IL-3, GM-CSF, G-CSF, M-CSF and Epo to induce maturation in human AML cells in vitro were investigated using cell specimens from 25 AML patients. The experiments were carried out under exactly defined serum-free culture conditions. In the absence of CSFs, monocytic and/or granulocytic maturation was detected in 14/25 cases. IL-3, GM-CSF, G-CSF and M-CSF elevated the proportions of monocyte/macrophages in 3/25, 2/25, 1/25 and 6/25 cases respectively, and increased the percentages of mature granulocytes in 2/25, 1/25, 1/25 and 0/25 cases, and if so only to a limited extent (values below 50%). The 3H-thymidine (3H-TdR) uptake studies revealed that IL-3, GM-CSF, G-CSF and M-CSF were efficient stimulators of DNA synthesis of AML cells in 19, 15, 13 and four of those cases, respectively. Thus, although the cells in most cases responded to CSFs by activation of DNA synthesis, they were unable to give rise to terminally differentiated stages. Provision of CSFs in combination was more frequently effective in enhancing maturation and also increased the magnitude of maturation response. Monocytic versus granulocytic maturation of AML cells after culture did not correlate with the FAB cytology nor with the type of CSF presented; but generally granulocytic maturation was an infrequent phenomenon. Epo stimulated erythroid differentiation and DNA synthesis only in the case of erythroleukaemia, but it had no effect on the cells of 10 other AML cases. Extrapolation of these in vitro findings would suggest that CSFs would have a limited therapeutic utility to induce AML cell maturation in vivo and that hazards of stimulating blast cell proliferation with these factors may be anticipated.

Topics: Cell Transformation, Neoplastic; Colony-Stimulating Factors; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Growth Substances; Humans; Interleukin-3; Leukemia, Myeloid, Acute; Macrophage Colony-Stimulating Factor; Macrophages; Monocytes; Tumor Cells, Cultured

We have established a novel cell line, designated as TF-1, from a patient with erythroleukemia, which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1, but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion, and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I, the morphological and cytochemical features, and the constitutive expression of globin genes, indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate, TF-1 entered two different pathways. Specifically, hemin and delta-aminolevulinic acid induced hemoglobin synthesis, whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary, TF-1 is a cell line of immature erythroid origin that requires GM-CSF, IL-3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors.

Topics: Adult; Antigens, Surface; Bone Marrow; Cell Line; Cell Transformation, Neoplastic; Colony-Stimulating Factors; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Male; Mitosis; Stem Cells; Tumor Cells, Cultured

In vitro infection of murine fetal liver cells with a retrovirus containing v-raf and v-myc oncogenes has produced continuous lines of immature erythroid cells that are leukemogenic. These cells synthesized a factor that stimulated their growth in vitro before autonomous variants emerged. Approximately 1000 high-affinity erythropoietin receptors could be detected per cell, and the hormone induced terminal differentiation in these cells. The lines were generated at an extremely low frequency (approximately 1 in 10(7) cells), suggesting that the combination of raf and myc is insufficient to develop erythroid cell lines and that additional events are necessary for transformation.

Topics: Abelson murine leukemia virus; Animals; Antigens, Surface; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Erythropoietin; Hematopoietic Stem Cells; Leukemia Virus, Murine; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Moloney murine sarcoma virus; Nucleic Acid Hybridization; Oncogenes; Receptors, Cell Surface; Receptors, Erythropoietin; Sarcoma Viruses, Murine

The transforming capacity of the normal and mutant human EGF receptor (EGFR) was investigated in primary chicken cells. In fibroblasts, both N- and C-terminal truncations resulted in a weak, additive oncogenic activity. However, not even double truncations caused a v-erbB-like phenotype. Upon EGF-binding, on the other hand, both normal and C-terminally truncated EGFRs resembled v-erbB in their fibroblast transforming potential. In erythroblasts, N-terminal truncation was sufficient to induce constitutive self-renewal, which was enhanced by deletion of 32 C-terminal amino acids but abolished by a larger truncation of 202 amino acids. In contrast to the normal EGFR, the receptor lacking 32 C-terminal amino acids resembled v-erbB in conferring erythropoietin independence for spontaneous differentiation to the transformed erythroblasts. Our results indicate that the C-terminal domain of the EGFR is non-essential in fibroblast transformation, but seems to be crucial for both self renewal induction and specificity of receptor function in erythroblasts.

Topics: Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; DNA Mutational Analysis; Epidermal Growth Factor; ErbB Receptors; Erythroblasts; Erythropoietin; Fibroblasts; Gene Expression Regulation; Humans; Leukemia, Experimental; Mitosis; Oncogene Proteins, Viral; Protein Binding; Structure-Activity Relationship

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.

Topics: Ammonium Chloride; Animals; Cell Line; Cell Transformation, Neoplastic; Chloroquine; Endocytosis; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred Strains; Receptors, Cell Surface; Receptors, Erythropoietin

Erythropoietin (Epo) is a glycoprotein hormone that regulates erythroid development and interacts with surface receptors on developing erythroid cells. In this laboratory, a cell system with a relatively pure population of erythroid cells that respond to Epo has been developed. Immature erythroid cells are obtained from the spleens of mice infected with the anemia strain of Friend virus. The binding of 125I-labeled Epo (125I-Epo) to plasma membranes from these cells was studied in this investigation. 125I-Epo binding reached equilibrium within 20 min at 37 degrees C. Twenty percent of the receptors bound 125I-Epo with a Kd of 0.08 X 10(-9) M, while the remaining receptors bound the hormone with a Kd of 0.6 X 10(-9) M. In this study, a receptor for Epo was identified by cross-linking 125I-Epo to the receptor in intact cells and plasma membrane preparations using disuccinimidyl suberate. Polyacrylamide gel electrophoresis revealed two labeled bands of 100 and 85 kDa. The 85-kDa band was more heavily labeled (65%) than the 100-kDa band. Both bands were equally decreased when increasing amounts of unlabeled Epo were included in the binding mixture, indicating a specific interaction of 125I-Epo with the receptor.

Topics: Animals; Cell Membrane; Cell Transformation, Neoplastic; Erythrocytes; Erythropoietin; Friend murine leukemia virus; Humans; Kinetics; Mice; Mice, Inbred Strains; Molecular Weight; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Spleen

The erythroleukemia cell line IW32, derived by transformation with the Friend murine leukemia virus, has been shown previously to produce erythropoietin (EPO) constitutively. Here we demonstrate that, in addition to the normal mouse EPO locus, this cell line has another EPO locus which has undergone rearrangement and amplification. Both loci were cloned, and the rearrangement breakpoint of the second EPO locus was located within a 1.1-kilobase region upstream of an otherwise apparently normal EPO gene. There are no viral sequences present in the immediate vicinity of the rearranged EPO gene. DNase I digestion studies suggest that the rearranged gene is in a region where the chromatin is more sensitive to DNase hydrolysis than is the site of the normal gene. We conclude, tentatively, that the rearranged EPO locus is probably the transcriptionally active one and that either proviral sequences are acting at a distance to activate the EPO gene or the rearrangement itself has served to activate the gene.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; Erythropoietin; Friend murine leukemia virus; Genes; Genes, Viral; Mice; RNA, Viral; Transcription, Genetic

Avian leukosis viruses induce erythroblastosis in chicks by integrating into the c-erbB gene and thus activating c-erbB transcription. We characterized Rous-associated virus 1-induced leukemic erythroblasts in vitro and showed that they mostly resemble erythropoietin-independent but otherwise normal erythroid progenitors. Some leukemic cells, however, were able to both differentiate and proliferate extensively in vitro. All 14 leukemias studied expressed high levels of erbB-related proteins that were 5 to 10 kilodaltons larger but otherwise very similar to the gp74erbB protein of avian erythroblastosis virus ES4 with respect to biosynthesis, glycosylation, and cell surface expression. Two leukemias contained and released retroviruses that transduced erbB. Chicken embryo fibroblasts fully infected with these viruses expressed high levels of erbB RNA and protein but retained a normal phenotype. Our results suggest that certain forms of c-erbB, activated by long terminal repeat insertion or viral transduction, are capable of inducing erythroleukemia but unable to transform fibroblasts.

Topics: Animals; Avian Leukosis Virus; Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chickens; ErbB Receptors; Erythroblasts; Erythropoietin; Fibroblasts; Glycoproteins; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Oncogene Proteins, Viral; Oncogenes; Phenotype; Receptors, Cell Surface; Retroviridae; RNA, Viral; Transcription, Genetic; Transduction, Genetic

A human renal carcinoma from a patient with erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures. After reaching confluency, the cultured cells formed multicellular hemicysts (domes), which became more abundant as the cultures approached saturation density. Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase after the cultures reached confluency, in parallel with dome formation. Dibutyryl adenosine 3',5'-cyclic monophosphate significantly (P less than .01) stimulated Ep production and dome formation in the semiconfluent and confluent cultures of the renal carcinoma.

Topics: Animals; Bucladesine; Carcinoma; Cell Transformation, Neoplastic; Cells, Cultured; Erythropoietin; Humans; Kidney Neoplasms; Mice; Mice, Nude

Tritiated erythropoietin with full biological activity has been prepared, and a relatively homogeneous population of enriched progenitor cells that respond to the hormone has been generated by infection of mice with the Friend virus that produces anemia. These cells, obtained from the spleens of infected mice, develop into mature erythroblasts and erythrocytes in the presence of erythropoietin. We have measured the binding of erythropoietin to these target cells; 62% of the binding was inhibited by excess unlabeled erythropoietin, but no inhibition occurred with albumin, serum, or a variety of growth factors and glycoproteins. Apparent equilibrium was reached by 2 hr at 37 degrees C and by 3.5-4 hr at 10 degrees C. The extent of specific binding increased linearly with cell concentration. In binding experiments at 10 degrees C, apparent saturation of specific binding occurred at approximately equal to 8.7 nM. Scatchard analysis showed a single class of binding sites. The dissociation constant is 5.2 nM with an average of 660 binding sites per cell. At 0.06 nM, where most of the cells are induced to terminally differentiate in vitro, an average of only 8 erythropoietin molecules bound per cell. These studies indicate that erythropoietin attaches to specific binding sites, which are most likely receptors since they manifest high affinity and specificity, and that the biologic effect of the hormone may be produced by attachment of a very small number of erythropoietin molecules.

Topics: Anemia; Animals; Cell Transformation, Neoplastic; Erythropoietin; Friend murine leukemia virus; Humans; Kinetics; Mice; Mice, Inbred BALB C; Receptors, Cell Surface; Receptors, Erythropoietin; Spleen; Tritium

The present studies report erythropoietin (Ep) production in primary cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted to BALB/c nude mice. The levels of erythropoietin in the culture media were estimated using the exhypoxic polycythemic mouse assay (EHPCMA), fetal mouse liver erythroid colony-forming technique (FMLC), and a radioimmunoassay (RIA). The spent culture media of the exponentially growing cells contained less than 10 mU/ml of Ep measured by RIA. However, after the cells became confluent, Ep levels (RIA) in the spent media showed a marked increase to approximately 300 mU/ml. Ep levels estimated using the FMLC and EHPCMA were approximately 2/3 and 1/10, respectively, of those measured by RIA. Rabbit antiserum to highly purified human urinary Ep (70,400 U/mg protein) was utilized for immunocytochemical (peroxidase-antiperoxidase method) localization of Ep in the cultured cells. Very few of the cells in exponential growth exhibited Ep-like immunoreactivity, whereas intense Ep-like immunoreactivity was observed in the cytoplasm of the cells maintained in culture for a prolonged period after reaching confluency. The most intense staining was observed in some of the cells forming domes. The domes developed after the cells reached confluency, and their numbers increased with increasing time in confluent culture, in parallel with the increase in Ep levels in the spent media. This primary cell culture system of a renal cell carcinoma maintained in nude mice, which produces immunologically and biologically active Ep, may provide a useful model for studies of the mechanism of Ep production.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Erythropoietin; Female; Histocytochemistry; Humans; Immunoenzyme Techniques; Kidney Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Polycythemia

Studies were carried out on the role of endogenous prostaglandin E2 (PGE2) in erythropoietin (Ep) production and dome formation in primary monolayer cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted into BALB/c athymic nude mice. The metabolism of [14C]arachidonic acid (14C-AA) by cultured renal carcinoma cells, which were plated in 25-cm2 flasks at a density of 2 X 10(4) cells/cm2 and grown for 6, 12 (confluence, 13 X 10(4) cells/cm2), 16, 24, and 30 d in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum, was examined by using radiometric thin-layer chromatography (TLC). TLC revealed PGE2 to be the major metabolite of 14C-AA produced by the cultured cells throughout the 30 d of cultivation. In addition, the cultured cells at each time period were incubated for 24 h in 5 ml of serum-free Eagle's MEM and the levels of PGE2 and Ep in the incubated media were measured via radioimmunoassay. PGE2 levels in the serum-free media incubated with the cultured cells grown for 6 d were significantly (P less than 0.001) elevated (174 +/- 2.5 pg/ml, n = 5), compared with the unincubated control media (1.5 +/- 0.19 pg/ml, n = 5) and gradually decreased at each time period to 97.6 +/- 4.4 pg/ml (n = 5) at 30 d. On the other hand, the levels of Ep in the incubated media of the cells grown for 6 d were 11.5 +/- 0.52 mU/ml (n = 5) compared with 7.6 +/- 0.62 mU/ml (n = 5) in the control media. However, after the cultured cells became confluent, the levels of Ep in the incubated media showed a marked increase to 222.9 +/- 5.26 mU/ml (n = 5) at 30 d of cultivation. Multicellular hemicysts (domes) developed after the cultured cells reached confluence and their numbers increased with increasing time in confluence in parallel with the increase in Ep. Meclofenamate (MF) (3 X 10(-6)-3 X 10(-5) M), a prostaglandin synthesis inhibitor, produced a significant dose-related decrease in PGE2, Ep, and dome formation without producing a significant effect on cell viability in the 30-d cells. This inhibitory effect of MF on Ep production and dome formation was completely abolished by the addition of 10(-8) M PGE2 to the incubation medium. In conclusion, endogenous PGE2 plays an important role in supporting and/or stimulating Ep production and dome formation in cultured renal carcinoma cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Carcinoma; Cell Transformation, Neoplastic; Cells, Cultured; Dinoprostone; Erythropoietin; Humans; Indomethacin; Kidney Neoplasms; Meclofenamic Acid; Mice; Mice, Nude; Prostaglandin Antagonists; Prostaglandins E; Rabbits

It has been shown previously in this laboratory that in vitro infection of mouse bone marrow cells with the anemia strain of Friend leukemia virus leads to growth of large bursts of erythroid cells which are arrested in development prior to hemoglobin synthesis but can respond to erythropoietin (EP) to complete the late stage of erythroblast differentiation. In this study, the effect of EP on the metabolism of 45Ca2+ in these cells was examined. At 4 degrees C, an increased rate of 45Ca2+ uptake and efflux as well as an increase in the steady state level of 45Ca2+ in treated cells was observed. Exchange of 45Ca2+ from preloaded cells at 4 degrees C indicated that treatment with EP increased the size of a rapidly exchanging pool of 45Ca2+ from 5 to 12% of total 45Ca2+ in the cell. The effect of treatment with EP can be seen as increased exchange of extracellular 45Ca2+ with cellular Ca2+; however, an effect of EP on the net level of Ca2+ in these cells cannot be excluded. This investigation demonstrates one of the earliest effects of EP on erythroid cells and suggests that alterations in Ca2+ metabolism may contribute to the progression of erythroid cells to their final development.

Topics: Animals; Biological Transport, Active; Bone Marrow; Calcium Chloride; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Erythroblasts; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Mice

Two strains of Friend virus differ in their in vivo actions in that one strain induces anemia (FVA), while the other induces polycythemia (FVP). This study characterizes differences in the in vitro effects of these viruses on hematopoietic cells of (BALB/c x DBA/2)F1 mice. Both variants induced erythroid bursts that proliferated and differentiated without added erythropoietin (EPO). However, while the bursts induced by FVP were well "hemoglobinized" (i.e., most cells contained hemoglobin), the cells of FVA-induced bursts contained little or no hemoglobin. The nonhemoglobinized bursts, induced by FVA, were established to be erythroid by cytochemistry, electron microscopy, and hormone sensitivity. FVA-induced cells appeared to be hypersensitive to EPO, since small concentrations of the hormone produced marked increases in hemoglobin production--even when the hormone was added to the culture 3 days post infection. Time-lapse photography documented that EPO stimulated hemoglobin synthesis in virally transformed cells rather than uninfected erythroid precursors. This observation of FVA-induced hypersensitivity prompted the reexamination of the hormone requirements of FVP-induced bursts--previously considered to be EPO-independent. Reduction of the serum in the cultures allowed the demonstration that FVP-induced erythroid cells also were hypersensitive to EPO. Thus FVA and FVP can be readily distinguished in vitro by the relative EPO sensitivity of virus-induced bursts. From these findings, a hypothesis is drawn: i.e., oncogenic transformation may result from increased sensitivity of progenitor cells for natural, physiologic regulators, and transformation is not necessarily accompanied by a block in differentiation. In addition, since hypersensitive virus-induced bursts could be recognized and picked from the cultures, these studies provide a method for obtaining highly purified erythroid precursors for the study of the regulation of terminal differentiation by EPO and other regulatory factors.

Topics: Animals; Bone Marrow; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Kinetics; Mice; Mice, Inbred Strains; Microscopy, Electron

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dimethyl Sulfoxide; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Helper Viruses; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred BALB C; Spleen

Recent data concerning in vitro studies of erythroid progenitors in polycythemia vera (PV) are reviewed. As shown by different laboratories, at least two populations of CFUE seem to coexist in the bone marrow of such patients: one exhibits normal in vitro behavior; the other, probably of clonal origin, is abnormally sensitive to erythropoietin. This abnormal population of CFUE is highly sensitive to trace amounts of erythropoietin and is probably responsible for the in vivo development of polycythemia despite a depressed level of erythropoietin. At the BFUE level, two populations also seem to coexist. However, the abnormal behavior of erythroid progenitors is probably not expressed at this early level of differentiation but only at the late CFUE level. This is in agreement with some of the data, which suggest that in the differentiation of erythroid progenitors into erythroblasts, erythropoietin is only needed at a level very close to CFUE.

Topics: Animals; Cell Transformation, Neoplastic; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera

A patient with Ph1-positive chronic myelogenous leukemia (CML) who entered an erythroblastic transformation prior to the development of a typical myeloblastic crisis is described. In vitro methylcellulose cultures obtained at the time of erythroblastic transformation revealed that the erythroid progenitors were responsive to erythropoietin. Thus, similar to the findings in polycythemia vera, the erythroid progenitors in this case of erythroblastic transformation of CML retained responsiveness to erythropoietin in vitro.

Topics: Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Erythroblasts; Erythrocytes; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Methylcellulose; Middle Aged

Topics: Animals; Bone Marrow; Cell Transformation, Neoplastic; Cells, Cultured; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Leukemia, Experimental; Male; Mice; Mice, Inbred BALB C; Spleen; Tumor Virus Infections

Techniques that allow the selective stimulation of the erythropoietin-responsive cell population in mice with suppressed multipotential hemopoietic stem cells were used to identify (1) the in vivo target cell transformed by Friend virus (FV) into a tumor colony-forming unit and (2) a target cell for FV replication in vivo. Plethoric mice with busulphan-induced reductions in stem cell populations (characterized as colony-forming units) and stimulated erythropoietin-responsive cell compartments were given FV; control groups, not receiving erythropoietin, also received FV. A comparison of the number of target cells transformed in each group provided evidence identifying the ERC as the in vivo compartment in which the target cell detected by tumor colony formation resides. Differences in plasma virus titers revealed that the erythropoietin-responsive cell is also predominantly responsible for the production of FV as measured by focus-forming activity.

Topics: Animals; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Leukemia, Experimental; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neuraminidase; Tumor Virus Infections; Virus Replication

B.M. cells of RLV-infected BALB/c mice can proliferate in methylcellulose in the absence of E.P., while normal B.M. cells cannot (12). Not only the more primitive BFU-E shows hormone-independency (18). This phenomenon is in favour of the view that the Rauscher virus induced erythroblastosis is a true neoplasia although transplantation experiments failed so far. The experiments in which transformation in vitro of B.M. cells by RLV is established (19) show that the CFU-E can serve as a target for the virus. Treatment of normal mice with CFA leads to a rapid increase in CFU-E in the bone marrow (18). Splenomegaly of RLV-infected mice is enhanced by CFA-treatment probably due to an increase in targets. Transfection with proviral DNA also can transform the CFU-E of BALB-c mice. This approach allows in vitro studies on the resistence of mouse strains to RLV in vitro. The studies are of interest for the human disease in two aspects. In vitro transformation assays are needed to study the oncogenic potential of putative human leukemia viruses. Furthermore the studies have yielded some new insight in the pathogenesis of virally induced erythroblastosis. This might serve as a model for e.g. acute myeloid leukemia in man.

Topics: Anemia; Avian Sarcoma Viruses; Bone Marrow; Bone Marrow Cells; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Freund's Adjuvant; Phenylhydrazines; Rauscher Virus; Transformation, Genetic

Busulphan (BU) treatment of DBA/2 mice with hypertransfusion (HT)-induced polycythemia resulted in an ablation of detectable hematopoietic stem cells (CFUS) in pooled marrow from the long bones. Daily injections of erythropoietin (EP) stimulated an EP-responsive population of cells in the absence of detectable CFUS. Mice treated with BU and EP and having HT-induced polycythemia were inoculated with the polycythemia-inducing strain of Friend virus (FVP) and determinations were made for the presence of tumor colony-forming units (tCFU). No change in CFUS/10(6) bone marrow cells was detected as a result of EP treatment. However, tCFU were increased more than 100-fold in HT-BU-EP-treated mice compared with saline-treated controls. The demonstration of tCFU in mice in which CFUS were not detectable indicated that this leukemogenic effect of FVP could occur in the absence of the pluripotent stem cell. Furthermore, the increased numbers of this FVP target cell in the EP-stimulated, BU-treated mice with HT-induced polycythemia supported the model licating the target for this effect in the EP-responsive cell population.

Topics: Animals; Busulfan; Cell Transformation, Neoplastic; Erythrocytes; Erythropoietin; Female; Friend murine leukemia virus; Hematopoietic Stem Cells; Mice; Mice, Inbred DBA; Polycythemia

We have investigated the production of erythroid colonies in plasma culture by bone-marrow and spleen cells taken form C3Hf/Bi mice previously infected with a polycythemic strain of Friend virus (FV). Inclusion of erythropoietin (Epo) in the medium was found unnecessary for erythroid colony formation in vitro by these cells, although it was essential for the production of erythroid colonies by hemopoietic cells from normal animals. Development of erythroid colonies also proceeded umimpeded when cells from FV-infected animals were cultivated in medium pretreated with rabbit anti-serum that was shown to inactivate Epo. Thus, the hemopoietic tissues of FV-infected mice contained erythroid colony-forming units (CFU-Es) which appeared to be Epo-independent. When spleen cells from FV-infected mice were exposed to antiserum directed against syngeneic FV-infected spleen cells and complement, and then cultured with or without Epo, the number of erythroid colonies that developed was drastically reduced, indicating that the CFU-Es in these animals carried FV-induced antigen(s), and must themselves have been infected with virus. Electron microscopy of erythroid colonies produced by cells from FV-infected mice revealed the presence of budding and abundant free type-C virus particles. The efficiency of erythroid colony formation in vitro either with or without Epo by hemopoietic cells from FV-infected mice was substantially increased over that of cells from normal mice. The increase in the number of CFU-Es in these animals was due mainly to an increase in the number of Epo-independent CFU-Es. Epo-independent CFU-Es were first detected in bone marrow and spleen as early as 3 days after FV infection; thereafter their numbers progressively increased for at least 9 days. Hypertransfusion with red blood cells prior to FV infection reduced, while bleeding greatly increased, the efficiency of erythoid colony formation without Epo by cells from the spleens of the infected mice. The phenomenon of erythroid colony formation in plasma cultures lacking Epo provides a sensitive and reliable means of detecting Epo-independent CFU-Es, which appear to play a fundamental part in pathogenesis of the disease resulting from infection with the polycythemic strain of FV.

Topics: Animals; Bone Marrow Cells; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Complement System Proteins; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Immune Sera; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Spleen; Virus Replication

Bone marrow cells from BALB/c mice infected with Rauscher erythroblastosis virus produced five to twenty-five times more erythroid colonies in vitro in the absence of erythropoietin (EP) as compared to normal cells. A good correlation existed between the state of the disease and the number of hormone-independent erythroid colony-forming cells (CFU-E). A significant number of hormone-independent CFU-E was found as early as 3 days after infection. A linear relationship existed between the number of cells plated and the number of erythroid colonies formed in vitro. Addition of EP did not enhance colony formation, even at low cell concentrations. Feeder layer experiments demonstrated that EP-independent colony formation was not due to the production of endogenous EP. Repeated injections of phenylhydrazine into normal mice did not lead to the loss of EP responsiveness in vitro; this indicated that the hormone independency induced by the virus was not due to continuous erythropoietic stimulation in vivo. Besides hormone independency, the CFU-E from infected mice required less serum in the culture medium. Normal erythroid colonies regressed after 4 days of culture, but EP-independent colonies from infected mice persisted for more than 2 weeks. These three phenomena may be regarded as indicative for a physiologic transformation.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Dose-Response Relationship, Drug; Erythroblasts; Erythrocytes; Erythropoietin; Female; Hematopoietic Stem Cells; Mice; Mice, Inbred BALB C; Organ Size; Phenylhydrazines; Rauscher Virus; Spleen; Stimulation, Chemical; Time Factors

Topics: Agammaglobulinemia; Antibiotics, Antineoplastic; Antibodies, Heterophile; Blood Protein Disorders; Cell Differentiation; Cell Transformation, Neoplastic; Clone Cells; Erythropoietin; Fluorescent Antibody Technique; Humans; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Multiple Myeloma; Myeloma Proteins; Plasma Cells; Waldenstrom Macroglobulinemia

Topics: Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Depression, Chemical; Dimethyl Sulfoxide; DNA, Neoplasm; Erythropoietin; Friend murine leukemia virus; Heme; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Neoplasm Proteins; RNA, Neoplasm; Stimulation, Chemical

Topics: Animals; Cell Transformation, Neoplastic; Erythrocytes; Erythropoiesis; Erythropoietin; Inclusion Bodies, Viral; Iron Isotopes; L-Lactate Dehydrogenase; Male; Mice; Mice, Inbred BALB C; Rauscher Virus; Retroviridae; RNA Viruses; Spleen; Splenomegaly

Topics: Alpharetrovirus; Animals; Biological Assay; Cell Transformation, Neoplastic; Erythropoietin; Friend murine leukemia virus; Helper Viruses; Humans; Leukemia; Leukemia Virus, Murine; Leukemia, Experimental; Leukemia, Lymphoid; Mice; Polycythemia; Splenic Neoplasms

Topics: Animals; Blood Protein Electrophoresis; Cell Count; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Erythropoietin; Friend murine leukemia virus; Heme; Hemoglobins; Iron Isotopes; Leukemia, Experimental; Mice; Polycythemia

Topics: Animals; Blood Platelets; Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Dactinomycin; Erythrocytes, Abnormal; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Heme; Hemoglobins; Iron Isotopes; Mice; Mice, Inbred Strains; Polycythemia; Reticulocytes; Spleen; Virus Replication