losartan-potassium and Melanoma

The utilisation of nonviral gene delivery methods has been increasing steadily, however, a drawback has been the relative low efficiency of gene transfer with naked DNA compared with viral delivery methods. In vivo electroporation, which has previously been used clinically to deliver chemotherapeutic agents, also enhances the delivery of plasmid DNA and has been used to deliver plasmids to several tissue types, particularly muscle and tumour. Recently, a large number of preclinical studies for a variety of therapeutic modalities have demonstrated the potential of electrically mediated gene transfer. Although clinical trials using gene transfer with in vivo electroporation have not as yet been realised, the tremendous growth of this technology suggests that the first trials will soon be initiated.

Topics: Animals; Carcinoma, Squamous Cell; Electroporation; Erythropoietin; Forecasting; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Growth Substances; Hematologic Diseases; Humans; Injections, Intramuscular; Interleukin-12; Melanoma; Plasmids; Protein Deficiency; Toxins, Biological; Vaccines, DNA

Epoetin alfa (EPO, PROCRIT) pharmacokinetics and pharmacodynamics were evaluated in children with malignant solid tumors receiving chemotherapy.. Children initially received IV EPO 600 IU/kg (max dose 40,000 IU) or placebo once weekly for 16 weeks. Dose was increased to 900 IU/kg (max dose 60,000 IU) for patients not achieving a 1 g/dl increase in hemoglobin by study week 3 or 4. Serial PK samples were collected for 24 hr after the first study dose, and after the 10th or 11th dose. Serum EPO concentrations were analyzed using an ELISA assay, and pharmacokinetics were evaluated using compartmental methods.. Twelve children participated; six (median age 15.2 years; range 9.3-18.6 years) were randomized to receive EPO. All children required dosage increases to 900 IU/kg due to no response. The median (range) apparent EPO AUC0-24 and clearance (CL) were 67.1 IU/ml.hr (13.8-102.6) and 0.26 L/hr/m2 (0.19-1.08), respectively. After the 10th or 11th EPO dose in four of these six EPO patients, the median (range) apparent AUC0-24 and CL of EPO was 126.5 IU/ml.hr (107.3-161.1) and 0.21 L/hr/m2 (0.15-0.25), respectively. No significant correlations were observed between pharmacokinetic parameters and pharmacodynamic effects.. EPO disposition in our patients was similar to other pediatric patient populations or adults receiving IV EPO. Interesting but insignificant trends were noted in pharmacodynamic effects.

Topics: Adolescent; Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Child; Chondrosarcoma; Dose-Response Relationship, Drug; Double-Blind Method; Epoetin Alfa; Erythropoietin; Female; Hematinics; Humans; Injections, Intravenous; Male; Melanoma; Osteosarcoma; Placebos; Recombinant Proteins; Skin Neoplasms; Time Factors; Treatment Outcome

In order to study the relation to the metastatic profile and survival, we evaluated the association between pretreatment serum levels of IL-6, TNF-alpha, and erythropoietin (EPO) and metastatic distribution and survival in 16 patients with metastatic malignant melanoma. IL-6 and TNF-alpha immunoassay (R&D Systems, Inc. Minneapolis, USA) employs the quantitative sandwich enzyme immunoassay technique. The Quantikine IVD EPO ELISA (R&D Systems, Inc.) is based on the double-antibody sandwich method. The baseline serum IL-6 and EPO levels were significantly higher in patients with metastatic malignant melanoma than in the control group (p = 0.009 and p = 0.033, respectively). Serum IL-6 levels were higher in patients with weight loss (p = 0.02), who were anemic (p = 0.026), with elevated serum LDH levels (p = 0.028), and who were chemotherapy nonresponding (p = 0.016). The relationship of serum IL-6 concentration to the tumor burden was not determined. Only serum EPO level was influenced by hemoglobin status (p = 0.001). No difference was shown among these three parameters. Elevated serum IL-6 concentration (p = 0.002) was found to be an adverse prognostic factor in patients with poor performance status, weight loss, low serum hemoglobin, elevated serum LDH, and unresponsive to chemotherapy. On the other hand, both serum TNF-alpha and EPO levels were of no prognostic value. Serum levels of IL-6 were found to be prognostic factors as valuable as serum LDH levels in patients with metastatic malignant melanoma. Their prognostic value should be further evaluated in a larger patient population.

Topics: Adult; Aged; Biomarkers, Tumor; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Follow-Up Studies; Humans; Interleukin-6; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Predictive Value of Tests; Prognosis; Skin Neoplasms; Tumor Necrosis Factor-alpha

Endogenous cytokines are thought to mediate numerous biologic processes and may account for some adverse effects experienced following the administration of recombinant proteins. This study describes the pattern of endogenous cytokine exposure following high-dose chemotherapy. Blood concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage colony-stimulating factor (M-CSF), and erythropoietin (EPO) were measured by enzyme-linked immunosorbent assay (ELISA) methods in 68 patients receiving the same ablative chemotherapy regimen (cyclophosphamide, cisplatin, carmustine). Patients were grouped according to cellular support (autologous bone marrow [BM] CSF-primed peripheral blood progenitor cells [PBPCs]) and prescribed growth factor (recombinant human granulocyte or granulocyte-macrophage colony-stimulating factor [rHuG-CSF or rHuGM-CSF]). Leukocyte reconstitution was most accelerated in the groups treated with PBPCs and rHuG-CSF. IL-6, M-CSF, and TNF-alpha concentrations were higher in the groups treated with rHuGM-CSF and without PBPCs. Maximal endogenous cytokine concentrations occurred approximately 12 days after BM reinfusion. High concentrations of EPO occurred in patients experiencing significant hypotension despite routine transfusions for hematocrit < 42%. High M-CSF and IL-6 levels were associated with increased platelet transfusion requirements. Concentrations of all four cytokines were significantly higher in patients experiencing renal or hepatic toxicity, with elevations occurring in a predictable sequence and M-CSF elevations occurring first. This report shows that endogenous cytokine concentrations may be influenced by either cellular or CSF support and are associated with differences in platelet reconstitution and organ toxicity.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Breast Neoplasms; Carmustine; Cisplatin; Combined Modality Therapy; Cyclophosphamide; Cytokines; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Kinetics; Macrophage Colony-Stimulating Factor; Melanoma; Neoplasm Staging; Recombinant Proteins; Transplantation, Autologous; Tumor Necrosis Factor-alpha

Recombinant human erythropoietin (EPO) is standard treatment for anemia in cancer patients. Recent clinical trials suggest that EPO may accelerate tumor progression and increase mortality. However, the evidence supporting a growth-promoting effect of EPO has remained controversial. Employing an in vivo model of B16 murine melanoma, we observed that administration of EPO to tumor bearing C57BL/6 mice resulted in pronounced acceleration of melanoma growth. Our in vitro studies demonstrate that B16 murine melanoma cells express EPOR, both at the protein and mRNA levels. Interestingly, expression of EPOR was retained in the established tumors. EPO stimulation of B16 cells enhanced proliferation and protein synthesis rates, and correlated with activation of the receptor associated Janus kinase 2 (Jak2) as well as phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and Akt kinases. Treatment with EPO and Jak-2 antagonists significantly inhibited EPO-mediated B16 cell proliferation. Moreover, EPO dose-dependently induced the phosphorylation and activation of the translation initiation factor eIF4E as well as the phosphorylation of its repressor, the eIF4E binding protein 4E-BP1. Finally, using eIF4E small interfering RNA (siRNA), we observed that EPO-mediated stimulation of B16 cell proliferation is eIF4E-dependent. Our results indicate that EPO exerts a powerful stimulatory effect on cell proliferation and de novo protein synthesis in melanoma cells through activation of the initiation factor eIF4E.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Erythropoietin; Gene Expression; Humans; Melanoma; Melanoma, Experimental; Mice; Protein Binding; Protein Biosynthesis; Receptors, Erythropoietin; Recombinant Proteins; RNA, Small Interfering; Signal Transduction; Transcription Factors

Erythropoietin (Epo) is widely used clinically to treat anemia associated with various clinical conditions including cancer. Data from several clinical trials suggest significant adverse effect of Epo treatment on cancer patient survival. However, controversy exists whether Epo receptor (EpoR) is functional in cancer cells. In this study, we demonstrated that EpoR mRNA expression was detectable in 90.1% of 65 melanoma cell lines, and increased copy number of the Epo and EpoR loci occurred in 30 and 24.6% of 130 primary melanomas, respectively. EpoR knockdown in melanoma cells resulted in diminished ERK phosphorylation in response to Epo stimulation, decreased cell proliferation and increased response to the inhibitory effect of hypoxia and cisplatin in vitro. EpoR knockdown significantly decreased melanoma xenograft size and tumor invasion in vivo. On the contrary, constitutive activation of EpoR activated cell proliferation pathways in melanoma cells and resulted in increased cell proliferation and resistance to hypoxia and cisplatin treatment in vitro. EpoR activation resulted in significantly larger xenografts with increased tumor invasion of surrounding tissue in vivo. Daily administration of recombinant Epo fails to stimulate melanoma growth in vivo, but the treatment increased vascular size in the xenografts. Increased local recurrence after excision of the primary tumors was observed after Epo treatment. Epo induced angiogenesis in Matrigel plug assays, and neutralization of Epo secreted by melanoma cells results in decreased angiogenesis. These data support that EpoR is functional in melanoma and EpoR activation may promote melanoma progression, and suggest that Epo may stimulate angiogenesis and increase survival of melanoma cells under hypoxic condition in vivo.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Progression; DNA Copy Number Variations; Epoetin Alfa; Erythropoietin; Gene Knockdown Techniques; Humans; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; Skin Neoplasms; Transcriptional Activation

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Beclin-1; Blotting, Western; Cell Movement; Cell Proliferation; Endothelial Cells; Erythropoietin; Heterozygote; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lung; Melanoma; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Protein Stability; RNA, Small Interfering; Stress, Physiological

Cancer is known to be an important cause of anaemia due to several factors including iron deficiency and inflammation. Hepcidin, a key regulator of iron metabolism, is up-regulated by iron and inflammatory stimuli such as interleukin 6, and decreased by iron deficiency, enhanced erythropoiesis and hypoxia. It is supposed to play a crucial role in changes of iron metabolism in anaemia of chronic disease, which is characterized by sequestering iron in macrophages and decreasing its availability for red blood cell production. To study the effect of tumour growth on hepcidin expression, we implanted human melanoma cells into mice and studied the changes of the amount of liver hepcidin mRNA by real-time PCR. We observed development of anaemia, which correlated with the size of the tumour. Hepcidin expression significantly decreased with the anaemia development, but in late stages we observed an increase of its expression together with an increase of mRNA for interleukin 6. However, the increase of hepcidin expression could be inhibited by exogenous erythropoietin administration. In our model of tumour growth, hepcidin expression reflected anaemia development and iron deficiency, erythropoietin administration and inflammation, and we suppose that it could therefore serve as a useful marker of these clinical situations common in cancer patients and play a role in the pathogenesis of cancer-associated anaemia.

Topics: Anemia; Animals; Antimicrobial Cationic Peptides; Cell Proliferation; Erythropoietin; Gene Expression Regulation, Neoplastic; Hepcidins; Humans; Inflammation; Injections, Subcutaneous; Interleukin-6; Iron Deficiencies; Liver; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Proteins; RNA, Messenger; Xenograft Model Antitumor Assays

Recombinant human erythropoietin (Epo) is used to prevent and treat tumor-related anemia and improve quality of life in cancer patients. Recent evidence suggested that Epo may adversely affect the survival of selected cancer patients by promoting tumor growth, inhibition of apoptosis, and induction of migration. Epo unfolds its effect on the Epo receptor (EpoR). We show--to the best of our knowledge for the first time--significantly increased EpoR expression in clinical melanoma metastases and primary melanomas in comparison with different sets of nevi by quantitative real-time reverse transcriptase-PCR, immunohistochemistry, and western blot analysis. When assessing the functionality of the EpoR-signaling pathway, recombinant human Epo led to the phosphorylation of JAK-2, signal transducers and activators of transcription 3 (STAT3), and ERK1/2 in several of the melanoma cell lines that were analyzed. Besides, Epo counteracted cisplatin-induced cell death in BLM and MV3 cells. Finally, Epo promoted cell migration of MV3 cells, whereas inhibition of the JAK/STAT and ERK1/2 pathways reduced Epo-mediated migration. In summary, we show the overexpression of functional EpoR expression in about half of the analyzed clinical melanoma metastasis specimens and show anti-apoptotic as well as pro-migratory effects of Epo, which is of importance for the treatment of anemia in advanced melanoma.

Topics: Anemia; Apoptosis; Biopsy; Cell Movement; Cells, Cultured; Erythropoietin; Humans; Janus Kinase 2; Melanocytes; Melanoma; Mitogen-Activated Protein Kinase 3; Phosphorylation; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor

In this study, the extent of angiogenesis, evaluated as microvascular volume density, immunoreactivity of tumour cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human primary melanoma specimens. Results showed that Epo/EpoR expression correlate with angiogenesis and tumour thickness. These findings suggest that Epo is secreted by tumour cells and it affects vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner, playing an important role in melanoma angiogenesis.

Topics: Adult; Aged; Aged, 80 and over; Erythropoietin; Female; Humans; Immunohistochemistry; Male; Melanoma; Middle Aged; Neovascularization, Pathologic; Receptors, Erythropoietin; Skin; Skin Neoplasms

Erythropoietin (Epo) is used commonly to treat cancer and/or therapy-related anemia. Until recently, Epo was considered to be a specific stimulator of erythropoiesis, acting via its receptor, EpoR. It becomes clear, however, that EpoR is expressed in a variety of cell types other than hematopoietic cells, and that Epo is a potent cytoprotective cytokine increasing cell survival under hypoxic conditions. Epo and EpoR are also expressed in various malignant tumors, and EpoR expression shows association with tumor invasion and progression. Recently, a functional Epo autocrine signaling mechanism was also detected in human melanoma cells. In this study, we examined the hypothesis that Epo activates the Akt signaling pathway in human melanoma cells and thus promotes the survival of tumor cells. The Akt signaling pathway in response to Epo was examined in melanoma. Similar to Epo, the expression of EpoR was up-regulated in response to hypoxia and Epo stimulation in melanoma cells. Melanoma cells constitutively expressed Akt with variable expression of mammalian target of rapamycin, and Epo dose-dependently induced their activity. Epo increased Akt kinase activity, which was abrogated by co-treatment with LY294002, a specific blocker of phosphoinositide 3-kinase. LY294002 also inhibited the cytoprotective effects of Epo in melanoma cells under both normoxic and hypoxic conditions. Our results suggest that Epo promotes melanoma cell survival by activating an Akt-dependent signaling pathway.

Topics: Cell Proliferation; Cells, Cultured; Enzyme Activation; Erythropoietin; Glycogen Synthase Kinase 3; Humans; Hypoxia; Melanocytes; Melanoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; Skin Neoplasms

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

Consorcial projects focused on 5 cancer types, breast-, colorectal-, head and neck- and pediatric cancers, and malignant melanoma. Breast cancer studies revealed unique splicing mechanisms concerning BRCA1. In sporadic breast cancers the involvement of DNA-repair genes was proved to be dependent on the histological type. Bone-metastatic tumors have been characterized by decreased NM23 and increased c-met and p53 expressions. C-erbB2 genotype of the primary tumor was not maintained frequently in bone metastases. Application of DNA-microarray and quantitative PCR technologies improved the prediction of therapeutic sensitivity of breast cancers. Colorectal cancer studies revealed regional inhomogenities (clusters) in various geographical regions of Hungary, which were distinct in the case of colonic and rectal cancers. To increase the sensitivity of fecal blood test of colorectal cancer screening, a new double-antibody test was developed and tested in a large cohort of patients. Genetic analysis revealed that hypermethylation is a significant factor in microsatellite instability which, and plays a role in silencing of APC and E-cadherin genes as well. The Hungarian pattern of TS polymorphism was also determined and was correlated not only with the efficacy of 5-FU treatment but with the progression of the disease as well. Population-based studies have been carried out in head and neck cancer patients (HNC) and smokers as well to reveal the genetic background of increasing tumor incidence. These studies revealed polymorphism in XRCC1/3 methylation enzyme gene which has preventive role. Other studies found frequent local immunosuppression in HNC patients. Studies indicated that the success of irradiation in this cancer type is dependent on the anti-vascular effects. Pediatric cancer studies determined the parameters of neuroblastoma screening based on VMA measurements. New splice variants of the WT1 gene involved in the monitoring of MRD of ALL patients was also described this year. We also obtained positive experimental data for the retinoic acid therapy of ALL. Melanoma studies extensively used DNA-microarray technology which identified 4 melanoma-specific and 2 melanoma progression-specific genes. In experimental human melanoma xenograft models we have identified 3 anti-metastatic agents: low molecular weight heparin, 2-methoxyestradiol and erythropoietin-alpha, where the later was characterized by specific effects on tumor vasculature.

Topics: 2-Methoxyestradiol; Adult; Animals; Antineoplastic Agents; Biomarkers, Tumor; Biomedical Research; Bone Neoplasms; Breast Neoplasms; Child; Colorectal Neoplasms; Disease Models, Animal; Disease Progression; DNA Methylation; Epoetin Alfa; Erythropoietin; Estradiol; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Markers; Head and Neck Neoplasms; Heparin, Low-Molecular-Weight; Humans; Hungary; Incidence; Male; Melanoma; Microsatellite Repeats; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Polymorphism, Genetic; Predictive Value of Tests; Recombinant Proteins; Transplantation, Heterologous

Tumor necrosis factor-alpha (TNF-alpha) has been intensively studied because of the specific toxicity of this cytokine toward cells that undergo malignant transformation. However, its proinflammatory and immunoregulatory properties always represented a drawback to the TNF-alpha administration in cancer therapy. In this study, we describe an adenovirus-based strategy in which the tumoricidal activity of TNF-alpha can be selectively triggered to eradicate tumors without administering TNF-alpha. This strategy might allow us to prevent TNF-alpha effects on normal tissues and, therefore, to bypass its systemic toxic effects. We inserted the constitutively active version of the Mr 55,000 TNF receptor, which was shown previously (F. Bazzoni et al., Proc. Natl. Acad. Sci. USA, 92: 5376-5380, 1995) to be capable of killing cells upon expression in the absence of its ligand, into a replication-deficient adenovirus, and under the control of a melanoma-specific promoter/enhancer element. We show that, upon infection, the recombinant gene reaches high level of expression in melanoma cell lines and triggers apoptosis by activating the caspase cascade. Expression and function of this receptor is restricted to melanoma cell lines, because morphology, viability, and proliferation of other cell types exposed to the recombinant adenovirus infection are not affected. We show for the first time that a TNF-like apoptotic response can be triggered in the absence of TNF-alpha and can be selectively confined to specific cellular targets. Killing activity and tissue specificity of the recombinant TNF receptor adenovirus, together with the advantage of triggering a TNF-like antitumor activity in the absence of TNF-alpha itself, are ideal features for a vector that might be the choice for gene therapy aimed to eradicate malignant cells.

Topics: Adenoviridae; Animals; Antigens, CD; Apoptosis; Cell Division; Cell Line; Cell Survival; Enhancer Elements, Genetic; Erythropoietin; Humans; Melanoma; Mice; Monophenol Monooxygenase; Promoter Regions, Genetic; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Recombinant Fusion Proteins; Signal Transduction; Transduction, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Virus Replication

Treatment of metastatic melanoma with biochemotherapy results in the rapid onset of anemia, requiring blood transfusion in 9 of 13 (69%) patients. Prophylactic use of weekly subcutaneous recombinant epoetin alfa eliminated the need for transfusion in all but 1 of 21 (5%) patients.

Topics: Anemia; Blood Transfusion; Cost-Benefit Analysis; Drug Therapy; Drug-Related Side Effects and Adverse Reactions; Epoetin Alfa; Erythropoietin; Hematinics; Humans; Melanoma; Neoplasm Metastasis; Quality of Life; Recombinant Proteins

Topics: Adult; Anemia, Iron-Deficiency; Erythropoietin; Humans; Intestinal Neoplasms; Leg; Lymphatic Metastasis; Male; Melanoma

Patients with malignant disease frequently develop anaemia. To investigate the role of erythropoietin (EPO) in this anaemia, serum levels were determined in patients with solid tumours. The study population consisted of 84 patients (44 males, 40 females) with solid tumours and 99 healthy control subjects, and 13/84 patients were anaemic. Serum EPO was clearly elevated in the anaemic tumour patients, but this increase was less than in patients suffering from iron deficiency anaemia. As in iron deficiency anaemia, the correlation between EPO levels and haemoglobin values was inverse. When compared to healthy control subjects, the levels of EPO in the tumour patients without anaemia were decreased. We conclude that there may be an inhibition in the expression or secretion of EPO in patients with solid tumours which, as yet, has not been further defined. Based on this, the treatment of anaemia in cancer patients with erythropoietin appears promising.

Topics: Adult; Aged; Aged, 80 and over; Anemia; Anemia, Iron-Deficiency; Erythropoietin; Esophageal Neoplasms; Female; Gastrointestinal Neoplasms; Hemoglobins; Humans; Iron; Male; Melanoma; Middle Aged; Sex Factors; Transferrin

Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells results in significant tumor regression in patients with advanced cancer. We have investigated the kinetics of circulating erythroid (BFU-E) and granulocytic-macrophage (CFU-GM) progenitors after IL-2 therapy in 11 cancer patients, mainly affected by metastatic melanoma and renal cell carcinoma. Administration of IL-2 from day 1 through day 5 constantly induced a dramatic decrease of the number of circulating BFU-E and CFU-GM, which then showed a striking rebound (up to values fourfold and sevenfold higher, respectively, than the pretherapy levels) on discontinuation of IL-2, ie, from day 5 through day 10. A similar kinetic pattern was observed during and after the second cycle of IL-2 administration. 3[H]-thymidine killing experiments showed that the cycling activity of the progenitors was virtually unmodified in the rebound phases. To explore the mechanism(s) underlying this kinetic pattern, we have analyzed the plasma concentration of several hematopoietic growth factors, including IL-1 beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and erythropoietin (Ep). No modifications in the levels of IL-3, GM-CSF, or IL-1 beta were observed, whereas a pronounced increase of IL-6 and G-CSF concentration was monitored, starting at day 3 and peaking at day 5 of treatment (a parallel, but modest, increase of Ep level was also observed). The elevation of IL-6 and G-CSF concentration is directly correlated with and may, at least in part, underlie the subsequent rebound of circulating hematopoietic progenitors. Furthermore, the increase in IL-4 level observed at day 10 of therapy may mediate the eosinophilia gradually starting at this stage of treatment.

Topics: Adult; Aged; Carcinoma, Renal Cell; Cell Count; Erythroid Precursor Cells; Erythropoietin; Female; Granulocyte Colony-Stimulating Factor; Granulocytes; Growth Substances; Hematopoietic Stem Cells; Humans; Immunotherapy, Adoptive; Interleukin-2; Interleukin-6; Kidney Neoplasms; Kinetics; Macrophages; Male; Melanoma; Middle Aged; Neoplasms

Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells. The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator. Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension. When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed. When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated. This was shown to be because of the drop in pH, possibly due to CO2 accumulation, rather than due to oxygen depletion. It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator. The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator. This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000-1 of culture fluid. Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable. Studies showed that the Epo was produced in serum-free systems containing peptones.

Topics: DNA, Recombinant; Erythropoietin; Gene Expression; Humans; Hydrogen-Ion Concentration; Melanoma; Recombinant Proteins; Tissue Plasminogen Activator; Tumor Cells, Cultured