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Mechanisms of local regulation of erythropoiesis within bone marrow erythroblastic islands in rats
Abstracts/Experimental Hematology 28 (2000) 31–131
the TfR ASO in these two cell lines by fluorescence and confocal microscopy using a FITC-labelled oligonucleotide. In BV173, the ASO was retained in vesicle scattered throughout the cytoplasm with some diffusion to the cytoplasm and nucleus, while in K562 the oligonucleotide containing vesicles seemed to be sequestered in a region corresponding to the Golgi area with apparently no cytoplasmic or nuclear diffusion. This distribution was partially reverted by coupling the ASO to the transfection reagent lipofectin, increasing the ligonucleotide diffusion to the nucleus and cytoplasm and this correlated with a sharp decrease in TfR protein content in K562 cells. 146
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Molecular Regulation of Hematopoiesis
MECHANISMS OF LOCAL REGULATION OF ERYTHROPOIESIS WITHIN BONE MARROW ERYTHROBLASTIC ISLANDS IN RATS Kalaidjieva V., *Zakharov Yu.M., *Rassokhin A.G. Dept. of Physiology, Fac. of Medicine, Thracian Univ., Stara Zagora, Bulgaria; *Dept. of Physiology, Medical Academy, Chelyabinsk, Russia The purpose of the study was to examine the mechanism of macrophage involvement in erythropoietic process within bone marrow erythroblastic islands (EI). Changes of the tissue oxygen supply following removal of blood cells (2% BW) or transfusion of 2.5mlⲐ100g BW of a 70% suspension of washed isogenous red cellsⲐ2days were modeled in rats. Plasma erythropoietin level (by RIA), the total number of EIⲐfemur and specific indices of erythropoiesis, based on EI of different classes of maturity were estimated during a 10-day period. In bled animals the initial decrease in hematocrit was accompanied by an increased number of absolute EIⲐ femur—628.3⫾102⫻103Ⲑfemur (day 3) vs. 406.7⫾169⫻103Ⲑfemur (P⬍0.01) and proliferating EI appeared elevated on days 3–7. The rate of CFU-E differentiation into erythroblasts in EI and the rate of repeated participation of macrophages in new EI reconstruction were increased during the first days after bleeding (P⬍0.05). Polyglobulia in hypertransfused rats was accompanied by a significant attenuation of EI formation. The number of islands was a 135.6⫾25⫻103Ⲑfemur (day 5) vs. 347.6⫾65⫻103Ⲑfemur (P⬍0.001) in controls. A persistent reduction of young and reconstructive islands, and of the related indices of erythropoiesis was observed. The role of macrophages to organize islands of different classes of maturity was estimated. Our results support the assumption about the specific regulatory role of the centrally-situated macrophages. Macrophage-erythroblast interactions were influenced by the oxygen supply to the tissues after stimulation of reduction of erythropoiesis.
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Monday, July 10, 2000 (12:45–14:15) Session IV-3: Cytokines, Growth Factors and Receptors II
ISOLATION OF PURE MEGAKARYOCYTIC CLONES (32D TPO) FROM THE MURINE MULTIPOTENT IL-3DEPENDENT 32D CELL LINE G. Migliaccio, M. Sanchez, A. de Capoa*, L. Centurione*, R. Rana* and A.R. Migliaccio Cell Biology and Clinical Biochemsitry, ISS, Rome, Genetics and Molecular Biology, Università La Sapienza, Rome and Biomorphology, Università G. D’Annunzio, Chieti, Italy 32D is an IL-3-dependent mast cell line whose cells are often defined multipotent because they generate at low frequency (1 every 105 cells) cells which dependent for growth on factors other than IL-3. using growth factor as selective pressure, differentiation mutant cell lines were isolated from the 32D cells which were dependent on either erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) or granulocyte-monocyte colony stimulating factor (GM-CSF) and had, consistently, either erythroid, granulocytic or monocytic phenotype. The 32D cell line, but none of its subclones, express Mpl (the receptor for thrombopoietin, TPO) by RT-PCR and clone at low frequency in response to TPO under serum-deprived conditions. Using TPO as selective pressure, TPOdependent subclones were isolated and characterized. In contrast with the diploid karyotype of the original 32D cells, the TPOdependent subclones have a nearly tetraploid karyotype (model chromosome number 66). A fraction of the TPO-dependent cells (5–10%) have twice the normal cell size and appear multinucleated although, when carefully analyze,d all the nuclei are interconnected. These giant cells have a chromosome number higher than 120. The 32D TPO cells express high levels of GpIIb–GpIIIa on their surface and release pro-platelets in the supernatant. These cells express high levels of megakaryocytic genes (Mpl, GpIIb and von Willebrand factor) and do not express detectable levels of erythroid (EPO receptor, ␣ and -globin) genes by RT-PCR. Interestingly, the previously isolated erythroid 32D Epo cell lines did not express megakaryocytic gene detectable by RT-PCR. In conclusion, the newly isolated differentiation mutant 32D TPO cells are dependent for growth on TPO and have a pure megakaryocyte morphology. Since 32D TPO and 32D EPO are the only cell lines isolated up to now which are “pure” respectively, for the megakaryocytic and erythroid lineages, they may represent a valuable tool to dissect lineage-specific events in the megakaryocytic and erythroid pathway. 148
Monday, July 10, 2000 (9:45–11:15) Session III-4: Molecular Regulation of Hematopoiesis I
GATA-1LOW MICE RESPOND MORE READILY THAN THEIR NORMAL LITTERMATES TO IN VIVO PERTURBATIONS OF ERYTHROPOIESIS A.M. Vannucchi*, F. Paoletti*, S. Linari*, C. Cellai*, L. Lorenzini*, M. Sanchez, G. Migliaccio and A.R. Migliaccio *Dept. of Hematology, University of Florence, Florence and Cell Biology and Clinical Biochemistry Labs, Istituto Superiore di Sanità, Rome, Italy The observation that megakaryocytes (Mk) are consistently detected in secondary cultures of individual day 5 BFU-E-derived colonies stimulated with thrombopoietin (TPO) (22⫾9 Mk cellsⲐ
the TfR ASO in these two cell lines by fluorescence and confocal microscopy using a FITC-labelled oligonucleotide. In BV173, the ASO was retained in vesicle scattered throughout the cytoplasm with some diffusion to the cytoplasm and nucleus, while in K562 the oligonucleotide containing vesicles seemed to be sequestered in a region corresponding to the Golgi area with apparently no cytoplasmic or nuclear diffusion. This distribution was partially reverted by coupling the ASO to the transfection reagent lipofectin, increasing the ligonucleotide diffusion to the nucleus and cytoplasm and this correlated with a sharp decrease in TfR protein content in K562 cells. 146
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Molecular Regulation of Hematopoiesis
MECHANISMS OF LOCAL REGULATION OF ERYTHROPOIESIS WITHIN BONE MARROW ERYTHROBLASTIC ISLANDS IN RATS Kalaidjieva V., *Zakharov Yu.M., *Rassokhin A.G. Dept. of Physiology, Fac. of Medicine, Thracian Univ., Stara Zagora, Bulgaria; *Dept. of Physiology, Medical Academy, Chelyabinsk, Russia The purpose of the study was to examine the mechanism of macrophage involvement in erythropoietic process within bone marrow erythroblastic islands (EI). Changes of the tissue oxygen supply following removal of blood cells (2% BW) or transfusion of 2.5mlⲐ100g BW of a 70% suspension of washed isogenous red cellsⲐ2days were modeled in rats. Plasma erythropoietin level (by RIA), the total number of EIⲐfemur and specific indices of erythropoiesis, based on EI of different classes of maturity were estimated during a 10-day period. In bled animals the initial decrease in hematocrit was accompanied by an increased number of absolute EIⲐ femur—628.3⫾102⫻103Ⲑfemur (day 3) vs. 406.7⫾169⫻103Ⲑfemur (P⬍0.01) and proliferating EI appeared elevated on days 3–7. The rate of CFU-E differentiation into erythroblasts in EI and the rate of repeated participation of macrophages in new EI reconstruction were increased during the first days after bleeding (P⬍0.05). Polyglobulia in hypertransfused rats was accompanied by a significant attenuation of EI formation. The number of islands was a 135.6⫾25⫻103Ⲑfemur (day 5) vs. 347.6⫾65⫻103Ⲑfemur (P⬍0.001) in controls. A persistent reduction of young and reconstructive islands, and of the related indices of erythropoiesis was observed. The role of macrophages to organize islands of different classes of maturity was estimated. Our results support the assumption about the specific regulatory role of the centrally-situated macrophages. Macrophage-erythroblast interactions were influenced by the oxygen supply to the tissues after stimulation of reduction of erythropoiesis.
147
77
Monday, July 10, 2000 (12:45–14:15) Session IV-3: Cytokines, Growth Factors and Receptors II
ISOLATION OF PURE MEGAKARYOCYTIC CLONES (32D TPO) FROM THE MURINE MULTIPOTENT IL-3DEPENDENT 32D CELL LINE G. Migliaccio, M. Sanchez, A. de Capoa*, L. Centurione*, R. Rana* and A.R. Migliaccio Cell Biology and Clinical Biochemsitry, ISS, Rome, Genetics and Molecular Biology, Università La Sapienza, Rome and Biomorphology, Università G. D’Annunzio, Chieti, Italy 32D is an IL-3-dependent mast cell line whose cells are often defined multipotent because they generate at low frequency (1 every 105 cells) cells which dependent for growth on factors other than IL-3. using growth factor as selective pressure, differentiation mutant cell lines were isolated from the 32D cells which were dependent on either erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) or granulocyte-monocyte colony stimulating factor (GM-CSF) and had, consistently, either erythroid, granulocytic or monocytic phenotype. The 32D cell line, but none of its subclones, express Mpl (the receptor for thrombopoietin, TPO) by RT-PCR and clone at low frequency in response to TPO under serum-deprived conditions. Using TPO as selective pressure, TPOdependent subclones were isolated and characterized. In contrast with the diploid karyotype of the original 32D cells, the TPOdependent subclones have a nearly tetraploid karyotype (model chromosome number 66). A fraction of the TPO-dependent cells (5–10%) have twice the normal cell size and appear multinucleated although, when carefully analyze,d all the nuclei are interconnected. These giant cells have a chromosome number higher than 120. The 32D TPO cells express high levels of GpIIb–GpIIIa on their surface and release pro-platelets in the supernatant. These cells express high levels of megakaryocytic genes (Mpl, GpIIb and von Willebrand factor) and do not express detectable levels of erythroid (EPO receptor, ␣ and -globin) genes by RT-PCR. Interestingly, the previously isolated erythroid 32D Epo cell lines did not express megakaryocytic gene detectable by RT-PCR. In conclusion, the newly isolated differentiation mutant 32D TPO cells are dependent for growth on TPO and have a pure megakaryocyte morphology. Since 32D TPO and 32D EPO are the only cell lines isolated up to now which are “pure” respectively, for the megakaryocytic and erythroid lineages, they may represent a valuable tool to dissect lineage-specific events in the megakaryocytic and erythroid pathway. 148
Monday, July 10, 2000 (9:45–11:15) Session III-4: Molecular Regulation of Hematopoiesis I
GATA-1LOW MICE RESPOND MORE READILY THAN THEIR NORMAL LITTERMATES TO IN VIVO PERTURBATIONS OF ERYTHROPOIESIS A.M. Vannucchi*, F. Paoletti*, S. Linari*, C. Cellai*, L. Lorenzini*, M. Sanchez, G. Migliaccio and A.R. Migliaccio *Dept. of Hematology, University of Florence, Florence and Cell Biology and Clinical Biochemistry Labs, Istituto Superiore di Sanità, Rome, Italy The observation that megakaryocytes (Mk) are consistently detected in secondary cultures of individual day 5 BFU-E-derived colonies stimulated with thrombopoietin (TPO) (22⫾9 Mk cellsⲐ