Mamoru Sakaguchi

The expression of functional erythropoietin receptors on an interleukin-3 dependent cell line

We report the expression of the erythropoietin receptors on an interleukin-3 dependent cell line. By the transfer into medium supplemented with erythropoietin, DA-1 cells were converted to an erythropoietin dependent growth state. [125I] erythropoietin was used to detect receptors specific for this hormone on the cell surface. Binding studies revealed that erythropoietin bound to 131 +/- 23 receptors/cell with a Kd of 0.54 +/- 0.2 nM. When the cells were incubated at 37 degrees C, trichloroacetic acid soluble radioactivity appeared in the medium after [125I] erythropoietin binding began to decrease, suggesting that the decline represents the degradation of cell associated [125I] erythropoietin- receptor complexes.

Two regulation points for differentiation in the cell cycle of human megakaryocytes

To examine the relationship between cell cycle progression and differentiation in megakaryocytes, variances in the cell cycle during PMA (phorbol 12‐myristate‐13‐acetate)‐induced differentiation were analysed in synchronized growing CMK cells. The cells stimulated with PMA in early S phase showed cell cycle arrest in G2 phase. In addition, the cells stimulated with PMA in early G1 phase showed cell cycle arrest in late G1. The expression of gpIIb/IIIa, megakaryocytic differentiation marker, was markedly induced in G2 phase when the cells were stimulated in early S phase, and was also induced in late G1 phase when cells were stimulated in early G1 phase. Therefore the induction of gpIIb/IIIa expression seems to be associated with cell cycle blockage in both G2 and late G1. Expression of the transcription factor GATA‐1 in cells stimulated in early S phase was much higher than that in control cells at G2 phase. Furthermore, GATA‐1 expression in cells stimulated in early G1 tended to be higher than that in controls at late G1. These periods corresponded to the time that the cell cycle arrest and gpIIb/IIIa expression were induced by PMA. These results suggest that the cell cycle in human megakaryocytic lineage cells may have two regulation points for differentiation.

A new system that analyzes erythropoietin‐mediated early signal transduction: Transfection of the c‐fos enhancer·promoter‐luciferase gene into a murine erythroid cell line

Abstract:  Erythropoietin (Epo) exerts its effects by binding specific receptors on the surface of reactive cells. However, the signal transduction system after binding has not been well described. To develop a system to analyze the steps of signal transduction, we transfected the human c‐fos‐enhancer/promoter linked with the Photinus pyralis luciferase gene (pfosluc2) into a murine erythroleukemia cell line ELM‐I‐1, in which we previously showed that c‐fos mRNA is rapidly induced upon Epo‐stimulation. A stable transfectant was obtained. The cells transfected with pfosluc2 were stimulated with Epo and luciferase activity in the cells was measured as light intensity. The light intensity integrated for 2 min (LI2.0) was 3202 ± 80 unit/1.5 × 105 cells before stimulation. This increased up to 5869 ± 321 unit/1.5 × 105 cells by incubating the cells with 5 U/ml Epo for 2 h. After Epo stimulation, light intensity began to increase at 30 min, reached a peak (about 1.8 times the basal level) at 120 min, and then gradually dropped. The effect of Epo was dose‐dependent; significant action occurred at as low as 0.5 U/ml, with a maximum at 5 U/ml. A similar response was observed when the cells were stimulated with interleukin‐3 (IL‐3) although the response was apparently lower than that with Epo. It was also found that IL‐3 had an additive action with Epo on c‐fos activity in this system. Thus, the above method was proven to be simple, rapid and sensitive enough to use to determine the early phase of signal transduction of Epo.

Human urinary megakaryocyte colony-stimulating factor in thrombopoietic disorders

Summary The level of urinary Meg‐CSF activity in patients with various thrombopoietic disorders was studied. Five out of eight patients with idiopathic thrombocytopenic purpura had megakaryocytic hyperplasia in the marrow and increased Meg‐CSF activity in the urine. Urinary Meg‐CSF activity in patients with polycythaemia vera and essential thrombocythaemia was normal. There was a significant inverse correlation between urinary Meg‐CSF activity and peripheral blood platelet count but not bone marrow megakaryocyte mass. There was a significant increase of urinary Meg‐CSF activity during the period of thrombocytopenia after chemotherapy in patients with acute leukaemia who were in complete remission. The timing of maximal Meg‐CSF levels corresponded to the nadirs of platelet counts. These results support the concept that Meg‐CSF may play a significant role in the regulation of megakaryopoiesis and/or thrombopoiesis in vivo.

Analysis of Antibody to Neutrophils Associated with Autoimmune Neutropenia: Possible Recognition of Fc-Receptor-Related Molecules

The serum of a 50-year-old male with neutropenia and a bout of bacterial infection was studied. Anti-neutrophil IgG antibody was detected by indirect immunofluorescence using a laser flow cytometry system. Purified IgG from our patients serum did not inhibit chemotaxis of neutrophils, but inhibited rosette formation of neutrophils with ox red blood cells (ORBC) coated with anti-ORBC rabbit IgG dose-dependently. Surface iodination of neutrophils followed by their immunoprecipitation by purified IgG and sodium dodecylsulfate polyacrylamide gel electrophoresis showed a single band that corresponded to 45 kilodaltons. Possibly the IgG antibody in our patients serum recognizes molecules related to Fc receptors.

Human urinary erythropoietin: preparation with high potency.

Human urinary erythropoietin has been purified to homogeneity. The seven-step procedure yielded a preparation with a potency of 225,000 U/mg protein. SDS-polyacrylamide gel electrophoretic analysis of the purified hormone revealed a single protein band with a molecular weight of about 35,000 that migrated with the biological activity. As to its stability, the purified hormone retained its activity in the presence of 0.001% Tween 20.