ABSTRACT
The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.
Subject(s)
Humans , Androstadienes/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases , Cell Differentiation/drug effects , Enzyme Inhibitors/pharmacology , Erythroid Precursor Cells/physiology , Erythroid Precursor Cells/cytology , Erythropoiesis , Flavones/pharmacology , K562 Cells , Leukemia, Myeloid/pathology , Oligonucleotides, Antisense/pharmacology , Quinones/pharmacology , ras Proteins/metabolismABSTRACT
Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.
Subject(s)
Cells, Cultured , Erythroblasts/physiology , Erythroid Precursor Cells/physiology , Flow Cytometry , Fluorescent Antibody Technique , Humans , beta-Thalassemia/bloodABSTRACT
La hipoxia constituye el mejor stress fisiológico para la pertubación del estado estacionario eritropoyético. El present estudio tiende a analizar la respuesta proliferativa eritropoyética esplénica con diferentes dosis de eritropoyentina humana recombinante bajo condiciones hipóxicas a lo largo 18 días mediante el ensayo de síntesis del DNA. Los progenitores esplénicos normóxicos no sufren proliferación eritroide significativa al día 0. Una clara respuesta proliferativa a rh Epo se verificó entre los 2 y 8 días de hipoxia. La proliferación de los progenitores eritroides esplénicos hipóxicos retornó a un patrón basal desde los 10 días hasta el final de la experiencia. La mayor creatividad proliferativa, 25 veces sobre el control (p<0.001), se produjo a los 6 días de condicionamiento desde 62.5 hasta 250mU/ml de rh Epo. estos resultados son concordantes con el concepto que durante la daptación fisiológica a la hipoxia, las células progenitoras eritroides esplénicas modifican transitoriamente su tasa proliferativa observable por variaciones en la relación dosis-respueta a Epo
Subject(s)
Female , Mice , Animals , Spleen/cytology , Erythropoiesis/physiology , Erythropoietin/administration & dosage , Hypoxia/physiopathology , Adaptation, Physiological , Mice, Inbred Strains , Cell Culture Techniques , Erythroid Precursor Cells/physiology , DNA/biosynthesis , Time FactorsABSTRACT
In order to study the effect of running exercise on hematological changes, hematopoietic progenitor cells (CFU-GM) and the fibrinolytic system in humans, a running exercise protocol was developed. The volunteers ran as fast as possible for a distance of 3.8 kilometers. Immediately after exercise, there were significant increases in hematocrit, absolute numbers of total white blood cell count, platelet count and CFU-GM, total protein and plasma fibrinogen, but all values returned to baseline within 15 minutes except hematocrit, platelet and total protein which returned to baseline value in 30, 30 and 60 minutes after exercise, respectively. The fibrinolytic reactivity increased immediately after exercise and returned to baseline level 15 minutes later. The changes in all parameters were transient and the increase in hematopoietic progenitor cells were not useful for stem cell harvesting by hemapheresis.
Subject(s)
Adolescent , Adult , Blood Physiological Phenomena , Erythroid Precursor Cells/physiology , Evaluation Studies as Topic , Female , Fibrinolysis/physiology , Humans , Male , RunningABSTRACT
Sera and leukaemic cell extracts from patients of acute leukaemia were evaluated for their effect on the repopulating ability of the pluripotent stem cells and erythroid differentiation by an in vivo splenic colony count (CFU-S) technique. Normal donor marrow cells of mice were treated with sera and cell extracts from patients of acute leukaemic and healthy controls and injected in the recipient mice. The CFU-S performed on the seventh day to assess repopulating ability of the stem cell showed consistently lower CFU-S counts in the test groups, with leukaemic sera (P less than 0.01) as well as leukaemic cell-extracts (P less than 0.001). The erythroid differentiation assessed by 59Fe uptake by the spleens also showed significantly reduced counts in the two test groups (P less than 0.01 and less than 0.001 respectively). The results indicate that both leukaemic sera and cell-extracts exert a significant suppressive effect on the repopulating ability of the stem cells and on their erythroid differentiation.