losartan-potassium and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Topics: Colony-Stimulating Factors; Erythropoietin; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interferon Type I; Interferon-gamma; Interferons; Interleukin-3; Leukemia; Leukemia, Hairy Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Myelodysplastic Syndromes; Recombinant Proteins; Thrombocythemia, Essential

We report on six cases of unrelated UCB transplant in adult patients with hematological malignancies: three chronic myelocytic leukemias and three acute leukemias. Their median age and body weight were respectively: 28 years (range 15.5-40) and 55.5 kg (range 46-90). The cord blood units were from the New York Blood Center. The median number of nuclear cells provided, evaluated before thawing, was 2.1 x 10(7)/kg (range 1 x 10(7)/kg-4.7 x 10(7)/kg). The degree of HLA disparity was 1/6: two patients, 2/6: three patients, 3/6: one patient. The patients received a pretransplant regimen including total body irradiation. They were given graft-versus-host disease prophylaxis which consisted of cyclosporin A and corticosteroids. They were all given a combination of G-CSF and erythropoietin. The median time of white blood cell and platelet reconstitution were respectively 24 days (range 12-43) and 60 days (range 23-90). All the patients had a full chimerism. A grade I acute GVHD was observed in four patients and two patients do not have any GVHD. No chronic GVHD has been observed yet. Three patients died from toxicity. Three patients are alive and well in complete remission at 2 years, 1 year and 11 months post-graft.

Topics: Adolescent; Adrenal Cortex Hormones; Adult; Burkitt Lymphoma; Cyclosporine; Erythropoietin; Female; Fetal Blood; Graft vs Host Disease; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Histocompatibility Testing; Humans; Immunosuppressive Agents; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male

Topics: Adult; Aged; Aged, 80 and over; Anemia; Erythropoietin; Female; Humans; Injections, Subcutaneous; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Recombinant Proteins

Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA-identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.

Topics: Acute Disease; Adult; Antigens, CD34; Bone Marrow; Cell Division; Cyclosporine; Erythropoietin; Female; Filgrastim; Graft vs Host Disease; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Immunosuppressive Agents; Leukapheresis; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Male; Methotrexate; Middle Aged; Platelet Transfusion; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Recombinant Proteins; T-Lymphocytes; Transplantation, Homologous

We have used recombinant human erythropoietin (rHuEPO) in a phase I/II clinical trial to evaluate its ability to reverse refractory anemia in hematologic disorders. rHuEPO was administered subcutaneously 5 days per week at escalating doses (50 to 150 U/kg per day). The aim of treatment was a hemoglobin (Hb) level greater than or equal to 10 g/dL without blood transfusion. Of 25 patients treated, 17 were evaluable, most of them with a regular need for transfusion. Eight of these had lymphoproliferative disorders (three cases of malignant lymphoma and five of monoclonal gammopathy) and were exposed to cytotoxic therapy. The other nine patients had hematopoietic stem cell disorders (four cases of myelodysplastic syndrome, three of idiopathic myelofibrosis, and two of chronic myelogenous leukemia). All patients with lymphoproliferative disorder had serum EPO levels inappropriately low for the degree of anemia, while patients with stem cell disorder showed variable values. Erythroid marrow activity was inadequate in all cases. Seven of eight patients with lymphoproliferative disorder responded to treatment maintaining Hb above 10 g/dL without transfusion. The median dose of rHuEPO required for correction of anemia was 75 U/kg. In four cases response was maintained with 50 U/kg, three times per week. There was no complete response among patients with hematopoietic stem cell disorder, although transfusion requirement was eliminated or reduced in four cases. Four patients developed functional iron deficiency during rHuEPO treatment and required iron supplementation to obtain response. Aggravation of splenomegaly was observed in two cases of myeloproliferative disorder. We conclude that: (1) subcutaneous administration of rHuEPO can be effective and safe in patients with lymphoproliferative disorder exposed to chemotherapy and showing inappropriate EPO response to anemia; (2) this is less likely in hematopoietic stem cell disorders, although favorable responses may be observed in occasional patients; and (3) functional iron deficiency as a cause of nonresponse to rHuEPO is frequent also in nonrenal anemia.

Topics: Adult; Aged; Anemia, Refractory; Bone Marrow; Drug Evaluation; Erythroid Precursor Cells; Erythropoietin; Female; Ferritins; Hematologic Diseases; Humans; Iron; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoproliferative Disorders; Male; Middle Aged; Myelodysplastic Syndromes; Primary Myelofibrosis; Receptors, Transferrin; Recombinant Proteins

In elderly patients with chronic myeloid leukemia (CML) responsive to imatinib, the incidence of clinically significant (CS) late chronic anemia is still unknown.. To highlight this issue, we revised retrospectively 81 CML patients aged >60 years treated at our Institution with front-line imatinib for at least 24 months in durable complete cytogenetic response (CCyR). CS late chronic anemia was defined as the presence of persistent (>6 months) and otherwise unexplained Hb levels ≤10 g/dL, which occurred >6 months from imatinib start.. A condition of CS late chronic anemia occurred in 22 out of 81 patients (27.2%) at different intervals from imatinib start. Seven out of 22 patients (31.8%) needed packed red cell transfusions during the follow-up. At diagnosis, patients who developed CS late chronic anemia were significantly older and had a lower Hb median level. Six out of 22 patients with CS late chronic anemia received subcutaneous recombinant alpha-erythropoietin (EPO) at the standard dosage of 40,000 IU weekly: all 6 patients achieved an erythroid response. A significantly worse event-free survival (EFS) in patients with untreated CS late chronic anemia was observed (p = 0.012).. CS late chronic anemia during long-term treatment with imatinib is a common complication in responsive elderly patients, with worse EFS if untreated. Results with EPO are encouraging, but larger studies are warranted to define its role.

Topics: Age Factors; Aged; Aged, 80 and over; Anemia; Erythrocyte Transfusion; Erythropoietin; Female; Humans; Imatinib Mesylate; Incidence; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Recombinant Proteins; Retrospective Studies; Treatment Outcome

Polycythemia Vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms respectively characterized by erythrocytosis and thrombocytosis; other disease features include leukocytosis, splenomegaly, thrombosis, bleeding, microcirculatory symptoms, pruritus, and risk of leukemic or fibrotic transformation.. PV is defined by a JAK2 mutation, whose absence, combined with normal or increased serum erythropoietin level, makes the diagnosis unlikely. JAK2, CALR, and MPL mutations are the mutually exclusive "driver" mutations in ET with respective incidences of 55%, 25%, and 3%; approximately 17% are triple-negative. However, the same molecular markers might also be present in prefibrotic myelofibrosis, whose morphological distinction from ET is prognostically relevant.. Median survivals are approximately 14 years for PV and 20 years for ET; the corresponding values for younger patients (age <60 years) are 24 and 33 years. Life-expectancy in ET is inferior to the control population. Driver mutational status has not been shown to affect survival in ET whereas the presence of JAK2/MPL mutations has been associated with higher risk of arterial thrombosis and that of MPL with higher risk of fibrotic progression. Risk factors for overall survival in both ET and PV include advanced age, leukocytosis and thrombosis. Leukemic transformation rates at 20 years are estimated at <10% for PV and 5% for ET; fibrotic transformation rates are slightly higher. Most recently, ASXL1, SRSF2, and IDH2 mutations have been associated with inferior overall, leukemia-free or fibrosis-free survival in PV; similarly adverse mutations in ET included SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2.. Current risk stratification in PV and ET is designed to estimate the likelihood of recurrent thrombosis. Accordingly, PV includes two risk categories: high-risk (age >60 years or thrombosis history) and low-risk (absence of both risk factors). In ET, risk stratification includes four categories: very low risk (age ≤60 years, no thrombosis history, JAK2/MPL un-mutated), low risk (age ≤60 years, no thrombosis history, JAK2/MPL mutated), intermediate risk (age >60 years, no thrombosis history, JAK2/MPL un-mutated), and high risk (thrombosis history or age >60 years with JAK2/MPL mutation). In addition, presence of extreme thrombocytosis (platelets >1000 × 10(9)/L) might be associated with acquired von Willebrand syndrome (AvWS) and, therefore, risk of bleeding.. The main goal of therapy in PV and ET is to prevent thrombohemorrhagic complications. All patients with PV require phlebotomy to keep hematocrit below 45% and once-daily aspirin (81 mg). In addition, high-risk patients with PV require cytoreductive therapy. Very low risk ET patients might not require any form of therapy while low-risk patients require at least once-daily aspirin therapy. Cytoreductive therapy is also recommended for high-risk ET patients but it is not mandatory for intermediate-risk patients. First-line drug of choice for cytoreductive therapy, in both ET and PV, is hydroxyurea and second-line drugs of choice are interferon-α and busulfan. We currently do not recommend treatment with ruxolutinib or other JAK2 inhibitors in PV or ET, unless in the presence of severe and protracted pruritus or marked splenomegaly that is not responding to the aforementioned drugs. Screening for AvWS is recommended before administrating aspirin, in the presence of extreme thrombocytosis. Am. J. Hematol. 92:95-108, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Aspirin; Calreticulin; Diagnosis, Differential; Erythropoietin; Humans; Hydroxyurea; Interferon-alpha; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Polycythemia Vera; Primary Myelofibrosis; Receptors, Thrombopoietin; Risk Assessment; Thrombocythemia, Essential; Thrombocytosis

This 12th biannual report of the Cochrane Haematological Malignancies Group highlights recently published randomized controlled trials in the field of hemato-oncology, covering the publication period from September 1, 2009, through June 30, 2010. Implication for clinical practice and methodological aspects are the main principles used to select trials for this report. Studies on tyrosine kinase inhibitors for patients with chronic myeloid leukemia were identified through electronic search of MEDLINE with a broad search filter that covered all topics in hemato-oncology combined with a highly sensitive search filter for randomized studies as described in the Cochrane Handbook for Systematic Reviews of Interventions.

Topics: Adrenal Cortex Hormones; Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cyclophosphamide; Dasatinib; Diphosphonates; Disease-Free Survival; Epoetin Alfa; Erythropoietin; Evidence-Based Medicine; Gemtuzumab; Graft vs Host Disease; Hematologic Neoplasms; Hodgkin Disease; Humans; Imatinib Mesylate; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Methotrexate; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrimidines; Randomized Controlled Trials as Topic; Recombinant Proteins; Rituximab; Stem Cell Transplantation; Thiazoles; Treatment Outcome; Vidarabine

Nilotinib (Tasigna) is a second-generation BCR-ABL kinase inhibitor, recently introduced and used for the treatment of chronic or accelerated phase CML patients, intolerant or resistant to imatinib. This treatment represents and important step forward for the disease control of such patients but can lead to side effects, sometimes serious, which can limit its optimal use. We propose here some guidelines that might be of help in daily practice, in order to manage properly these side effects.

Topics: Antineoplastic Agents; Benzamides; Drug Eruptions; Drug Interactions; Drug Resistance, Neoplasm; Erythropoietin; Fertility; Food-Drug Interactions; France; Granulocyte Colony-Stimulating Factor; Heart Diseases; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metabolic Diseases; Neutropenia; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrimidines; Recombinant Proteins

Myelodysplastic syndrome (MDS) may be induced by certain mutagenic environmental or chemotherapeutic toxins; however, the role of susceptibility genes remains unclear. The G/G genotype of the single-nucleotide polymorphism (SNP) rs1617640 in the erythropoietin (EPO) promoter has been shown to be associated with decreased EPO expression. We examined the association of rs1617640 genotype with MDS.. We genotyped the EPO rS1617640 SNP in 189 patients with MDS, 257 with acute myeloid leukemia (AML), 106 with acute lymphoblastic leukemia, 97 with chronic lymphocytic leukemia, 353 with chronic myeloid leukemia, and 95 healthy controls.. The G/G genotype was significantly more common in MDS patients (47/187; 25.1%) than in controls (6/95; 6.3%) or in patients with other leukemias (101/813; 12.4%) (all P < 0.001). Individuals with the G/G genotype were more likely than those with other genotypes to have MDS (odd ratio = 4.98; 95% CI = 2.04-12.13). Clinical and follow up data were available for 112 MDS patients and 186 AML patients. There was no correlation between EPO promoter genotype and response to therapy or overall survival in MDS or AML. In the MDS group, the GG genotype was significantly associated with shorter complete remission duration, as compared with the TT genotype (P = 0.03). Time to neutrophils recovery after therapy was significantly longer in MDS patients with the G/G genotype (P = 0.02).. These findings suggest a strong association between the rs1617640 G/G genotype and MDS. Further studies are warranted to investigate the utility of screening for this marker in individuals exposed to environmental toxins or chemotherapy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Erythropoietin; Genetic Association Studies; Genotype; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Middle Aged; Myelodysplastic Syndromes; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Treatment Outcome; Young Adult

Topics: Adult; Blood Preservation; Blood Transfusion, Autologous; Erythropoietin; Female; Hematopoietic Stem Cell Transplantation; Humans; Jehovah's Witnesses; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Recombinant Proteins; Transplantation Conditioning; Transplantation, Homologous

Myeloproliferative disorders (MPDs) are heterogeneous diseases that occur at the level of a multipotent hematopoietic stem cell. They are characterized by increased blood cell production related to cytokine hypersensitivity and virtually normal cell maturation. The molecular pathogenesis of the MPDs has been poorly understood, except for chronic myeloid leukemia (CML), where the Bcr-Abl fusion protein exhibits constitutive kinase activity. Since some rare MPDs are also related to a dysregulated kinase activity, a similar mechanism was thought to be likely responsible for the more frequent MPDs. We investigated the mechanisms of endogenous erythroid colony formation (EEC) by polycythemia vera (PV) erythroid progenitor cells and found that EEC formation was abolished by a pharmacological inhibitor of JAK2 as well as an siRNA against JAK2. JAK2 sequencing revealed a unique mutation in the JH2 domain leading to a V617F substitution in more than 80% of the PV samples. This mutation in the pseudokinase autoinhibitory domain results in constitutive kinase activity and induces cytokine hypersensitivity or independence of factor-dependent cell lines. Retroviral transduction of the mutant JAK2 into murine HSC leads to the development of an MPD with polycythemia. The same mutation was found in about 50% of patients with idiopathic myelofibrosis (IMF) and 30% of patients with essential thrombocythemia (ET). Using different approaches, four other teams have obtained similar results. The identification of the JAK2 mutation represents a major advance in our understanding of the molecular pathogenesis of MPDs that will likely permit a new classification of these diseases and the development of novel therapeutic approaches.

Topics: Amino Acid Substitution; Cell Division; Cytokines; Erythrocytes; Erythropoietin; Humans; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Myeloproliferative Disorders; Polycythemia Vera; Receptors, Erythropoietin; Signal Transduction

The tyrosine kinase activity of p210BCR-ABL is essential to its leukemogenic potential, but the role of other functional domains in primary human hematopoietic cells has not been previously investigated. Here we show that infection of normal human CD34+ cord blood (CB) cells with a retroviral vector encoding p210BCR-ABL rapidly activates a factor-independent phenotype and autocrine interleukin-3/granulocyte colony-stimulating factor/erythropoietin production in the transduced cells. These changes are characteristic of primitive chronic myeloid leukemic (CML) cells and are important to the leukemogenicity of BCR-ABL-transduced murine hematopoietic stem cells. When BCR-ABL-transduced human CB cells were incubated with imatinib mesylate, an inhibitor of the p210BCR-ABL kinase, or when human CB cells were transduced with a BCR-ABL cDNA lacking the SH2 domain (p210DeltaSH2), factor independence was significantly reduced. In contrast, deletion of the SH2 domain had little impact on the p210BCR-ABL kinase-dependent promotion of erythropoietic differentiation also seen immediately following the BCR-ABL transduction of primitive human CB cells, but not in naturally occurring CML. Thus, p210BCR-ABL has distinct biological effects in primary human hematopoietic cells, which variably mimic features of human CML, and activation of these changes can show different dependencies on the integrity of the SH1 and SH2 domains of p210BCR-ABL.

Topics: Antigens, CD34; Cell Division; Cell Lineage; Erythropoietin; Fetal Blood; Fusion Proteins, bcr-abl; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; src Homology Domains; Transduction, Genetic

Myelosuppression occurs in up to 50% of patients with chronic myeloid leukemia (CML) who are treated with imatinib and > or = Grade 3 myelosuppression is reported in approximately 10% of patients.. The authors investigated the prognostic significance of anemia occurring during therapy with imatinib in patients with CML in chronic phase.. Of 338 patients treated with imatinib (150 patients after interferon failure and 188 patients with newly diagnosed CML), 230 (68%) developed anemia. In a multivariate analysis, factors associated with an increased probability of developing anemia were a starting hemoglobin level < 12 g/dL, age > or = 60 years, female gender, higher imatinib dose, and intermediate or high Sokal risk group. Of these 230 patients, 102 patients received treatment with 40,000 U of recombinant human erythropoietin administered subcutaneously once weekly. An increase in the hemoglobin level of > or = 2 g/dL was achieved in 69 patients (68%) and 22 patients (22%) had an increase of 1-1.9 g/dL. Patients who developed anemia had a trend toward a lower probability of complete cytogenetic remission compared with patients without anemia (68% vs. 77%; P = 0.14), as well as a trend for inferior survival. Patients with anemia and other manifestations of myelosuppression were found to have a significantly worse outcome than those with isolated anemia.. The authors concluded that erythropoietin is safe and effective in patients in chronic-phase CML who develop anemia with imatinib therapy.

Topics: Anemia; Antineoplastic Agents; Benzamides; Erythropoietin; Female; Humans; Imatinib Mesylate; Injections, Subcutaneous; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Chronic-Phase; Middle Aged; Piperazines; Prognosis; Protein-Tyrosine Kinases; Pyrimidines; Recombinant Proteins; Survival Rate; Treatment Outcome

Chronic myeloid leukemia (CML) is a hematological neoplasia that results from the transformation of a hematopoietic stem cell. It is characterized by the expansion of the myeloid lineage, which results in the accumulation of mature and immature granulocytes in peripheral blood and bone marrow. However, when CML marrow cells are cultured in Dexter-type long-term cultures (LTMC) hematopoiesis is defective and can be sustained for only a few weeks. One possible explanation for the deficient growth of hematopoietic cells in CML LTMC is that some factors that act as key regulators of hematopoiesis are absent in this experimental system. Thus, we tested this hypothesis by adding recombinant cytokines to these cultures. As a first approach, we added recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), rhGranulocyte-CSF (rhG-CSF) and rhErythropoietin (rhEPO); each factor was added individually once a week. Addition of rhGM-CSF and rhG-CSF resulted in a significant increase in the levels of nucleated cells and myeloid progenitors; the highest effects were seen in the presence of rhGM-CSF. Interestingly, such a cytokine also induced a significant decrease in the levels of erythroid progenitors. Recombinant hEPO had no significant effects on nucleated cells or myeloid progenitors, however, it induced a significant, although transient, increase in the levels of erythroid cells. The above results indicate that the hematopoietic regulators used here (rhGM-CSF, rhG-CSF and rhEPO) are capable of stimulating the growth of hematopoietic cells in LTMC from CML patients. Thus, this study demonstrates that it is, indeed, possible to manipulate CML LTMC by the addition of recombinant cytokines; this observation may be of particular relevance, since this in vitro experimental system has already been used as a method for purging of leukemic cells in autologous transplant settings. By using specific recombinant hematopoietic modulators it might be possible to make LTMC a more efficient system for such a clinical purpose.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow Purging; Cell Lineage; Erythropoietin; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Humans; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Neoplastic Stem Cells; Recombinant Proteins; Tumor Cells, Cultured; Tumor Stem Cell Assay

Activin A, one member of the transforming growth factor (TGF)-beta superfamily, is known to be a commitment factor for cell death and differentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or differentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A-induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid differentiation. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined effect of activin A and EPO on the parental KU812 cells, activin A induced differentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/mcl cells, as well as parental cells, were only differentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular differentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to differentiation signals to the erythroid lineage in CML cells.

Topics: Activins; Apoptosis; Caspase Inhibitors; Cell Differentiation; Cell Division; Drug Interactions; Erythropoietin; Inhibins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2

We describe a complete cytogenetic response to interferon-alpha in a patient with chronic myelogenous leukemia undergoing chronic hemodialysis. Although IFN-alpha therapy has been applied to patients with chronic hepatitis C receiving hemodialysis, the pharmacokinetics of IFN-alpha in patients with poor renal function still remain unclear. In the present patient, the serum IFN-alpha concentration remained high even 48 hours after injection (42.9 IU/ml), and IFN-alpha was almost completely removed by hemodialysis (< 6 UI/ml). The patient was treated with IFN-alpha (3 x 10(6) IU, three times a week), and cytogenetic disappearance (0%) of the Ph-positive clone was confirmed 31 months after the start of therapy. Recombinant human erythropoietin (Epo) was used to treat anemia due to renal failure and IFN-alpha therapy. The anemia was controllable with Epo, and no adverse effect was observed.

Topics: Anemia; Erythropoietin; Hepatitis C; Humans; Interferon-alpha; Kidney Failure, Chronic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Philadelphia Chromosome; Recombinant Proteins; Renal Dialysis; Treatment Outcome

We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disorders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV, 76 patients) or essential thrombocythemia (ET, 27 patients) were grown in collagen-based, serum-free, cytokine-free semisolid medium. Colony analysis at day 8 or 10 showed that this collagen assay is specific, as endogenous growth of erythroid colonies was never observed in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (CML) patients. Endogenous erythroid colony formation was observed in 53.3% of patients suspected of PV, with only 15.4% of positive cultures for patients with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for patients with > or =2 minor or 1 major PV criterion. Similarly, endogenous growth of erythroid colonies was found in 44.4% of patients suspected of ET, with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with > or =2 ET criteria. In addition, we found that in collagen gels, tests of erythropoietin (EPO) hypersensitivity in the presence of 0.01 or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be used to detect PV or ET, as these tests were positive for, respectively, 21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retrospective analysis suggests that collagen assays are more sensitive than methylcellulose assays to assess endogenous growth of erythroid colonies. In summary, serum-free collagen-based colony assays are simple and reliable assays of endogenous growth of erythroid colonies in myeloproliferative diseases. They also appear to be more sensitive than methylcellulose-based assays.

Topics: Bone Marrow; Cells, Cultured; Collagen; Colony-Forming Units Assay; Culture Media, Serum-Free; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Female; Gels; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Megakaryocytes; Methylcellulose; Polycythemia Vera; Thrombocytosis

Deregulating the expression of Bcl-xL, an inhibitor of apoptosis, in an erythropoietin-dependent erythroblast cell line averts apoptosis induced by the withdrawal of erythropoietin. Since in polycythemia vera an abnormal clone of erythroid progenitors is independent of erythropoietin, we investigated whether the endogenous expression of Bcl-xL was deregulated in these cells.. Erythroid colonies from patients with polycythemia vera and normal subjects were cultured in the presence and absence of erythropoietin and assessed by immunocytochemical and flow-cytometric analysis with anti-Bcl-x antibodies that recognize the two species of Bcl-x (Bcl-xL and Bcl-xS). Reverse-transcriptase-polymerase-chain-reaction analysis was used to determine which one of the two species was responsible for anti-Bcl-x staining. Bone marrow mononuclear cells from 8 healthy bone marrow donors, 14 patients with polycythemia vera, 19 patients with other myeloproliferative syndromes, and 12 patients with secondary erythrocytosis were analyzed by flow cytometry with antibodies against Bcl-x and glycophorin A, an erythroid marker.. Erythroid cells from patients with polycythemia vera survived in vitro without erythropoietin, and this finding correlated with the expression of Bcl-x protein (Bcl-xL messenger RNA was the main species of Bcl-x found), even in mature erythroblasts that normally do not express Bcl-x. The mean (+/-SD) percentage of cells positive for both glycophorin A and Bcl-x in the 14 patients with polycythemia vera (21.8+/-3.6 percent) was significantly higher than that in 8 normal donors (6.62+/-1.58 percent), 12 patients with secondary erythrocytosis (6.87+/-1.95 percent), 9 patients with essential thrombocythemia (3.81+/-0.97 percent), and 10 patients with chronic myeloid leukemia (2.7+/-0.41 percent).. Deregulated expression of Bcl-x may contribute to the erythropoietin-independent survival of erythroid-lineage cells in polycythemia vera and thereby contribute to the pathogenesis of this disease.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; bcl-X Protein; Case-Control Studies; Cell Survival; Child; Erythroid Precursor Cells; Erythropoietin; Female; Flow Cytometry; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Polycythemia Vera; Proto-Oncogene Proteins c-bcl-2; Reference Values; Thrombocytosis

Topics: Cells, Cultured; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Recombinant Proteins; Stem Cell Factor

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

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

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.

Topics: Apoptosis; Calcium; Cell Differentiation; Cytokines; Erythropoietin; Hemoglobins; Humans; Interleukin-3; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nuclear Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Recombinant Proteins; RNA, Antisense; Tumor Suppressor Protein p53

Galectin 1 (GAL1) is a beta-galactoside-binding lectin involved in cell cycle progression. GAL1 overexpression is associated with neoplastic transformation and loss of differentiation. The gene encoding for human GAL1 resides on chromosome 22(q12; q13), and its expression is developmentally regulated. Although devoid of signal peptide GAL1 can be externalized from cells by a mechanism independent of the normal secretory process. We report here on a study of the effects of erythroid differentiation of the human leukemia cell line K562 on GAL1 protein expression. In undifferentiated K562 cells, GAL1 was expressed into the cytosol. However, the amount of GAL1 was surprisingly weaker in K562 cells than in other leukemia cell lines such as TF-1 or KG1a. Treatment of K562 cells with erythropoietin (EPO) or with aphidicolin (APH), an inhibitor for DNA polymerase alpha, induced an erythroid phenotype and led to the externalization of cytosolic GAL1 which was then bound to ligands on cell surface in a galactoside-inhibitable fashion. Our results demonstrate that acquisition of an erythroid phenotype is associated with an externalization of GAL1. The autocrine binding of GAL1 to cell surface ligands of non adherent cells such as K562 suggest that GAL1 is implicated rather in signal transduction than in cell-cell or cell-matrix interaction. Moreover, the reciprocal translocation involving chromosomes 9 and 22 t(9;22) present in K562 cells might explain the weak expression of GAL1 in K562 leukemia cells.

Topics: Aphidicolin; Blotting, Western; Cell Differentiation; Cell Membrane; Cytosol; DNA Polymerase I; Enzyme Inhibitors; Erythroid Precursor Cells; Erythropoietin; Galectin 1; Glycoconjugates; Hemagglutinins; Humans; Immunoenzyme Techniques; Immunophenotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Tumor Cells, Cultured

Topics: Adult; Antineoplastic Agents, Alkylating; Busulfan; Dose-Response Relationship, Drug; Erythrocyte Transfusion; Erythropoiesis; Erythropoietin; Female; Hematopoiesis; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Platelet Count; Platelet Transfusion; Recombinant Proteins; Time Factors

In August, 1992, we established a leukemic cell line (NS-Meg) from a patient in megakaryoblastic transformation of Philadelphia chromosome-positive chronic myeloid leukemia. The NS-Meg cells were positive for alpha-naphthyl acetate esterase and periodic acid-Schiff (PAS) staining and for surface CD4, CD7, CD13, CD34, CD41a, and glycophorin A antigens. Ultrastructurally, the cells had alpha-granules, demarcation membranes, and platelet peroxidase activity. The NS-Meg cells spontaneously produced platelet-like particles which contained alpha-granules, mitochondria and dense bodies, strongly suggesting platelet production. Erythropoietin (Epo), granulocyte/macrophage colony stimulating factor(GM-CSF), and interleukin 3 (IL-3) promoted the growth of NS-Meg cells. Phorbol-12-myristate-13-acetate increased the expression of both CD41a and CD61 antigens. Ten-day exposure to Epo induced mature erythroblasts and red cells. These benzidine-positive cells were positive for hemoglobin F staining. Untreated NS-Meg cells expressed mRNA for the Epo receptor (EpoR), for GATA-1, and for alpha 1, alpha 2 and gamma globin genes. These results indicate that NS-Meg cells undergo terminal differentiation of both megakaryocytic and erythroid lineages. This cell line should be a very useful tool for the investigation of both megakaryocytic and erythroid maturation.

Topics: Adult; Base Sequence; Blood Platelets; Cell Differentiation; Cell Division; Cell Line; Cytokines; DNA Primers; DNA-Binding Proteins; Erythroid Precursor Cells; Erythroid-Specific DNA-Binding Factors; Erythropoiesis; Erythropoietin; Female; GATA1 Transcription Factor; Gene Expression; Globins; Hematopoiesis; Humans; Immunophenotyping; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Megakaryocytes; Microscopy, Electron; Molecular Sequence Data; Receptors, Erythropoietin; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured

The mechanisms of the chronic myeloid leukemia (CML) clones proliferative advantage over normal clones are currently unknown. They may involve an insensitivity to a negative regulation of a growth factor-independent proliferation. Clonogenic progenitors from CML patient blood or marrow in chronic phase were grown either in the presence or absence of recombinant growth factors. No erythroid colonies were observed in the absence of any cytokine. In contrast, erythroid colonies composed of fully mature hemoglobinized erythroblasts (day 12 burst-forming units-erythroid) were obtained in the presence of Steel factor (SF) alone. Addition of erythropoietin (Epo) to SF either had no effect on the cloning efficiency or increased up to 50% the number of erythroid colonies. No erythroid growth was observed when cultures were stimulated by interleukin-3 or granulocyte-macrophage colony-stimulating factor alone. Similar erythroid growth in the presence of SF but without Epo was obtained in "serum-free" cultures when purified blood CML CD34+ cells were grown. This growth of erythroid colonies in the absence of Epo was not accounted for by an autocrine stimulation loop by Epo, because neutralizing antibodies against Epo did not inhibit it. This abnormal response to growth factor was specifically observed in the CML clone, as shown by the presence of the BCR-ABL transcript in all of these erythroid colonies. The direct implication of BCR-ABL was further documented (1) by studies of alpha-interferon-treated patients with a chimerism in which the abnormal growth correlates with the presence of the malignant clone and (2) by the use of antisense oligonucleotide against BCR-ABL transcript, which abrogated this abnormal growth. Finally, erythroid growth in the SF presence was greatly diminished by herbimycin A, whereas, at the same concentration, this tyrosine kinase inhibitor had no marked effect on erythroid colony formation in the presence of SF plus Epo on CML or normal marrow cells. This result suggests that the BCR-ABL kinase activity leads directly to this Epo-independent terminal differentiation requiring, however, the presence of SF.

Topics: Antigens, CD; Antigens, CD34; Base Sequence; Bone Marrow; Cell Division; Cells, Cultured; DNA Primers; Drug Interactions; Erythroblasts; Erythropoietin; Exons; Fusion Proteins, bcr-abl; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Proteins; Stem Cell Factor; Transcription, Genetic

In order to elucidate the role of c-myb gene in erythroid differentiation of K562 cell induced by hemin (Hm) and erythropoietin (Epo), we constructed recombinant plasmid that could produce antisense myb RNA after induction with dexamethasone. During treatment with Hm, K562 cells constitutively expressed c-myb mRNA, and 50% of them began to synthesize hemoglobin (Hb). Expression of antisense myb RNA reduced the amount of c-myb mRNA, and the percentage of Hb-synthesizing cells was decreased to 20%. In the presence of Epo, c-myb mRNA declined and 20% of K562 cells synthesized Hb regardless of antisense myb RNA expression. It is suggested that constitutive expression of c-myb mRNA is necessary for Hm-induced differentiation, and that a decrease in the amount of c-myb mRNA induced by antisense myb RNA expression suppresses Hm-induced differentiation. The amount of c-myb mRNA in K562 cells was reduced during the differentiation induced by Epo. Expression of GATA-1 mRNA was almost constant during Hm-induced differentiation, but increased during Epo treatment. It is supposed that the mechanism of Hm-induced differentiation is distinguished from that of Epo-induced differentiation in K562 cells.

Topics: Analysis of Variance; Cell Differentiation; Dexamethasone; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Erythroid Precursor Cells; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; Gene Expression Regulation, Neoplastic; Globins; Glycophorins; Hemin; Hemoglobins; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; RNA, Antisense; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured

Previous studies have suggested that erythroid progenitors derived from patients with chronic myelogenous leukemia (CML) in chronic phase may have reduced proliferative capacity. Considering recent evidence that mast cell growth factor (MGF) enhances the proliferative capacity of normal erythroid burst-forming units (BFU-E), we examined whether MGF could increase the proliferative potential of CML erythroid progenitors to normal capacity. To evaluate the total proliferative capacity achieved, the BFU-E were divided into four subpopulations (XL = extra large, L = large, M = medium, S = small) and colonies were aspirated to determine the cellularity of BFU-E from each subpopulation. MGF alone or in combination with MoT cell line conditioned medium (MoCM) or granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-3 (IL-3) significantly increased the proliferative capacity of erythropoietin (EPO) dependent CML and normal BFU-E. Although the total number of BFU-E generated were similar, the number of BFU-E with high proliferative potential were considerably less in CML BFU-E populations. BFU-E designated XL (129,000-431,000 cells) were only found in MGF cultures and only normal BFU-E had this proliferative capacity. BFU-E designated L were increased in both normal and CML BFU-E populations but less CML BFU-E had this proliferative capacity (mean number 25% of normal) and CML L BFU-E from 2/3 CML patients comprised fewer cells than normal L BFU-E. Normal BFU-E populations comprised 16-24% high proliferative BFU-E (XL + L) in contrast to 4-5% high proliferative BFU-E (L only) comprising CML BFU-E populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cells, Cultured; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Hematopoietic Cell Growth Factors; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Philadelphia Chromosome; Stem Cell Factor; Tumor Cells, Cultured

Interleukin-4 (IL-4) is a cytokine with pleiotropic activities. In normal bone marrow cultures grown in the presence of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), IL-4 suppresses granulocyte-macrophage colony-forming unit (CFU-GM) proliferation but it enhances the colony-stimulatory effect of granulocyte colony-stimulating factor (G-CSF). We studied the effect of IL-4 on chronic myelogenous leukemia (CML) bone marrow or peripheral blood cells from 30 patients using the CFU-granulocyte-erythrocyte-monocyte-megakaryocyte colony culture assay. In several repetitive experiments, IL-4 inhibited CFU-GM colony replication by 24 to 65% in a dose-dependent fashion at concentrations ranging from 0.01 to 10 micrograms/ml when patients' cells were cultured in the presence of erythropoietin alone or with phytohemagglutinin-conditioned medium, GM-CSF, or IL-3. The addition of 100 U/ml of IL-1 beta to the CML cultures partially reversed the inhibitory effect of IL-4. Incubation of CML low-density peripheral blood cells with IL-4 resulted in down-regulation of IL-1 beta and IL-6 production in three of four samples, suggesting that the suppressive effect of IL-4 is mediated by inhibition of IL-1 and by other mechanisms including inhibition of IL-6 production. In contrast to the stimulatory effect exerted by IL-4 on G-CSF-dependent CFU-GM progenitor proliferation in normal marrow, the addition of IL-4 to CML cultures grown in the presence of G-CSF resulted in a divergent effect: suppression of CML CFU-GM in two, stimulation in three, and no significant effect in two CML patients' samples. It is therefore possible that IL-4 may have an in vivo antiproliferative effect in a subpopulation of CML patients.

Topics: Adult; Aged; Colony-Forming Units Assay; Culture Media, Conditioned; Erythropoietin; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Interleukin-1; Interleukin-4; Interleukin-6; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Phytohemagglutinins

Juvenile chronic myelogenous leukemia (JCML) is a good model for the study of myeloproliferation because JCML hematopoietic progenitor cells grow in vitro at very low cell densities without the addition of exogenous stimulus. Previous studies have demonstrated that this proliferation is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF), and that removal of monocytes from the cell population before culture eliminates this "spontaneous" myeloproliferation, suggesting a paracrine role of monocyte stimulation. However, subsequent studies have shown that increased GM-CSF production from the JCML monocytes is not a consistent finding and therefore not a plausible sole mechanism. In examining hematopoietic growth factor dose-response curves, both JCML GM and erythroid nonadherent progenitor cell populations displayed a marked and selective hypersensitivity to GM-CSF. Responses to interleukin-3 and G-CSF were identical to control dose-response curves. This is the first demonstration of a myeloid leukemia in which hypersensitivity to a specific growth factor appears to be involved in the pathogenesis of the disease.

Topics: Cell Division; Child, Preschool; Dose-Response Relationship, Drug; Erythropoietin; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive

Juvenile chronic myelocytic leukemia (JCML) is a rare hematopoietic neoplasia of early childhood with distinct hematologic and biochemical features. We studied the biologic properties and the globin synthetic profiles of JCML erythroid cells both in vivo and in vitro from a total of 24 patients. In these cases we observed the exuberant colony-forming unit-macrophage (CFU-M) colony growth, as reported previously. Furthermore, in contrast to previous reports, we found significant erythroid colony growth in most of our cases (average: 1,182 burst-forming unit-erythroid [BFUe] per 10(5) plated cells, range: 40 to 6,927). This growth was by and large erythropoietin-dependent and was not greatly influenced by other added cytokines. By several criteria all erythroid colony growth detected in vitro was derived from JCML progenitors. The globin synthetic profile of JCML erythroid cells showed high levels of fetal hemoglobin both in vivo and in vitro (gamma/gamma + beta: 53% to 94% in reticulocytes, 62% to 98% in BFUe-derived cells). In addition (in seven cases studied) we detected embryonic globins (epsilon and zeta) at the protein and messenger RNA level, a novel finding for primary leukemic cells. We speculate that the transformed erythroid cells in JCML harbor a trans environment supporting expression of developmentally earlier genes (fetal, embryonic). However, in contrast to other acute or subacute leukemias, JCML erythroid cells also have the ability to reach full maturation to the red cell level, thus allowing detection of this primitive program in vivo.

Topics: Bone Marrow; Cells, Cultured; Child, Preschool; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Globins; Humans; Infant; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Reticulocytes; RNA, Messenger

A novel erythroid cell line, RM10, was established from a long-term bone marrow culture of a patient with chronic myelogenous leukemia (CML). RM10 cells were positive for periodic acid Schiff (PAS), but negative for peroxidase and dual esterase. RM10 cells had la, pre B (CD10), myeloid (CD13, CD14, CD33) and erythroid (glycophorin A) markers, but had no other lymphoid, megakaryocytic, or mesenchymal cell markers. RM10 cells spontaneously synthesized hemoglobin, which was markedly enhanced with hemin. Isoelectric focusing of the cell lysates and northern blot analysis of the total cellular RNA revealed hemoglobin synthesis in the cells. Using 125I-labeled recombinant human erythropoietin (Epo), two classes of Epo receptors were demonstrated in the RM10 cells. However, Epo did affect neither growth nor erythroid differentiation of the cells. RM10 cells rapidly differentiated to monocytic cells in the presence of 12-0-tetradecanoylphorbol-13-acetate, and simultaneously expressed glycoprotein IIb/IIIa. RM10 cells had Philadelphia chromosome (Ph), and expressed p210bcr-abl using immunoprecipitation with anti-c-abl and anti-phosphotyrosine antibodies. These results indicate that the RM10 cells have the characteristics of multipotential hemopoietic cells originating from Ph-positive CML and that high affinity Epo receptor class is not a sufficient condition for Epo responsiveness.

Topics: Adult; Antigens, CD; Antigens, Differentiation; Antigens, Neoplasm; Cell Differentiation; Erythropoietin; Female; Fusion Proteins, bcr-abl; Hemoglobins; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neprilysin; Receptors, Cell Surface; Receptors, Erythropoietin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The authors established a new hemopoietic cell line (JK-1) from a patient with chronic myelogenous leukemia in erythroid crisis. This JK-1 line predominantly consists of immature cells, but a small number of mature erythroblasts and red cells can be consistently seen without any specific differentiation inducer. The JK-1 cells grow in suspension culture supplemented with human plasma and carry double Philadelphia chromosomes. Hemoglobin staining with benzidine was positive for about 20% of cells and the type of the hemoglobin was for the most part HbF. Surface-marker analysis revealed JK-1 cells positive for glycophorin A, EP-1, and HAE9. The proportion of mature cells was elevated by the addition of delta-aminolevulinic acid. Erythropoietin (EPO) enhanced the growth of JK-1 cells either in the suspension or in methylcellulose semisolid culture. The total number of EPO receptors was 940 per cell, of which 220 sites had an affinity higher than the other 720 sites. This is the first report of an established human erythroid cell line which spontaneously undergoes terminal differentiation.

Topics: Antigens, Surface; Cell Differentiation; Cell Division; Erythroid Precursor Cells; Erythropoietin; Growth Substances; Humans; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Receptors, Cell Surface; Receptors, Erythropoietin; Tumor Cells, Cultured

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

Topics: Antigens, Differentiation; Biomarkers; Blood Group Antigens; Cell Differentiation; Cell Line; Colony-Stimulating Factors; Erythroblasts; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Hematopoietic Stem Cells; Hemin; Humans; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Receptors, Cell Surface; Receptors, Erythropoietin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The ability of erythroid cultures to distinguish among myeloproliferative disorders was examined. We studied 14 patients with polycythemia vera (PV), 11 with chronic myelogenous leukemia (CML), four with non-PV erythrocytosis, two with agnogenic myeloid metaplasia, as well as three normal fetuses and greater than 25 normal adults. Endogenous, i.e. grew without added erythropoietin, bone marrow CFU-E-derived colonies were observed in all but one PV patient. However, endogenous blood BFU-E-derived bursts were observed in only eight of 14 PV patients. Endogenous erythroid colonies were not seen in cultures from any normal adults or fetuses, or patients with CML, erythrocytosis, or myeloid metaplasia. In PV, relative HbF synthesis was always greater in cultures without erythropoietin, while in cultures from all other patients relative HbF synthesis was similar to that observed in cultures from normal individuals. We conclude that PV and CML are distinguishable in culture since CML patients do not have endogenous growth. Most important, endogenous bone marrow CFU-E-derived colonies are the only consistently unique observation in patients with PV, and endogenous CFU-E- and BFU-E-derived colonies and bursts are not uniformly observed in PV blood cultures. In-vitro studies of erythropoiesis to confirm the diagnosis of PV, therefore, require marrow when endogenous colonies and bursts are absent from blood cultures.

Topics: Adolescent; Adult; Aged; Bone Marrow; Child; Colony-Forming Units Assay; Erythroblasts; Erythropoietin; Globins; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; Polycythemia; Polycythemia Vera