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Functional characterization of the two alternative promoters of human p45 NF-E2 Gene
Abstracts/Experimental Hematology 28 (2000) 31–131
examining the effects of series of different, targeted culture conditions. CD34⫺/Lin⫺ cells isolated from human adult bone marrow were used in cultures specific for hematopoietic stem cell (HSC), mesenchymal stem cell (MSC) and neural stem cell (NSC) differentiation. After 3 to 4 weeks, the cultures were harvested and evaluated by flow cytometry. MSC cultures, which consisted of MSC growth medium and growth factors, resulted in the development CD45⫺Cytokeratin⫹ (epithelial) and CD45⫺Vimentin⫹ (mesenchymal) cells. When vascular endothelial growth factor was added to the culture medium, cells expressing von Willebrands Factor were detected. Non-adherent cells expressing hematopoietic phenotype CD45⫹CD34⫹CD38⫹ were isolated from hematopoietic growth culture medium supplemented with hematopoietic growth factors. Cells cultured in neuronal growth medium supplemented with neuronal growth factors were observed to express neurofilament. The morphology of the cells in these three growth specific culture media was markedly different. To extend this work and assess the in vivo attributes of in vitro-derived cells, bone marrow CD34⫺Lin⫺ cells cultured for 3 weeks under conditions for neuronal cell differentiation were injected via the tail vein into irradiated NOD/SCID mice. After 18 days, mice were sacrificed and cells from the brain, muscle, thymus, spleen, and bone marrow were collected and stained with antibodies specific to human CD45 and MHC class-1. Human cells (CD45⫹ or MHC class 1⫹) were only identified in the brain of transplanted mice. To confirm the FACS observation, PCR specific for human globin was performed and has demonstrated the presence of human cells only in the brain of the transplanted mice. These data indicate that HSC, MSC and NSC may originate from a common precursor or pool of precursors in the CD34⫺/Lin⫺ fraction of human bone marrow. These precursor cells may have strong requirements for defined growth environments to enable their differentiation into particular lineages. The data also suggest that the homing of the injected cells may be organ specific. Immunohistochemistry should further reveal the nature of these cells. 52
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
ACCELERATION OF ERYTHROPOIESIS MEDIATED BY ERYTHROID ACCELERATING FACTOR (EAF) IN VERY EARLY STAGE OF DRUG INDUCED HEMOLYSIS IN RATS M. Yokoyama, H. Maruyama, C. Tohno, J. Kitazawa, T. Toki, K. Terui and E. Itoh Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan Erythroid accelerating factor (EAF) is a peculiar factor because the activity is definitely exhibited in an early stage of hemolytic stress in rats. A presumption can be made that EAF contributes to the acceleration of erythroid differentiation during initial period of sudden loss of erythrocytes. To further assess the relationship between serum EAF and a rapid rise of erythropoiesis in response to acute reduction of red blood cells, We determined the sequential changes of serum EAF and Epo in concomitant with blood parameters, bone marrow (BM) CFU-E and the erythroblastic islets in spleens at short intervals from 0time through 24h after acetylphenyhydrazine (APH) injection to induce hemolysis in rats. Serum EAF was significantly elevated as early as 6h following APH injection, reached a peak at 24h. On the other hand, serum
47
Epo levels were gradually increased from 12h, with peaking on day 3 of the experiment. The number of colonies derived from CFU-E and erythroblastic ratios in BM showed peak increases at 8h after injection of APH. Histologic analysis of spleens revealed that the erythroblastic islets increased from (⫹) to (3⫹) at 6h. These results suggest that EAF mediates an early enhance of erythropoietic process during initial period of urgent anemia as acute hemolysis. 53
Tuesday, July 11, 2000 (10:15–12:15) Session V-2: Gene Expression and Transcription Factors
FUNCTIONAL CHARACTERIZATION OF THE TWO ALTERNATIVE PROMOTERS OF HUMAN p45 NF-E2 GENE E. Ito*, T. Toki*, K. Arai*, K. Terui*, M. Yokoyama Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. Expression of the p45 subunit is restricted to selected blood lineages, including erythroid and megakaryocytic lineage cells. Here we report results of studies aimed to understand the regulatory mechanisms controlling p45 gene expression in erythroid cells. Human p45 mRNAs have the two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both NF-E2 isomers by RT-PCR analysis. To examine the expression of both NF-E2 isomers in primary erythroid and megakaryocytic cells, CD34⫹ cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells, whereas aNF-E2 was predominantly expressed in megakaryocytic cells. Although both isomers were abundantly expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12myristate 13-acetate (PMA) resulted in exclusive down regulation of fNF-E2. A functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. These results suggest that NF-E2 resides downstream of the GATA proteins in the transcription factor hierarchy that governs the differentiation of erythroid lineage cells. 54
Tuesday, July 11, 2000 (8:15–9:45) Plenary Session VI: Presidential Symposium
IN VITRO AND IN VIVO EVIDENCES FOR THE LONGTERM MULTILINEAGE (MYELOID, B, NK & T) RECONSTITUTION CAPACITY OF EX VIVO EXPANDED HUMAN CD34ⴙ CORD BLOOD CELLS L. DOUAY, L. KOBARI*, F. PFLUMIO*, M.C. GIARRATANA*, X. LI*, M. TITEUX*, B. IZAC*, F. LETEUTRE*, L. COULOMBEL St Antoine and St louis hospitals, Paris, Institut Gustave Rousy, Villejuif, France We have identified clinically relevant culture conditions that support the expansion of HSC. After 14 days of stroma-free, serum-free
examining the effects of series of different, targeted culture conditions. CD34⫺/Lin⫺ cells isolated from human adult bone marrow were used in cultures specific for hematopoietic stem cell (HSC), mesenchymal stem cell (MSC) and neural stem cell (NSC) differentiation. After 3 to 4 weeks, the cultures were harvested and evaluated by flow cytometry. MSC cultures, which consisted of MSC growth medium and growth factors, resulted in the development CD45⫺Cytokeratin⫹ (epithelial) and CD45⫺Vimentin⫹ (mesenchymal) cells. When vascular endothelial growth factor was added to the culture medium, cells expressing von Willebrands Factor were detected. Non-adherent cells expressing hematopoietic phenotype CD45⫹CD34⫹CD38⫹ were isolated from hematopoietic growth culture medium supplemented with hematopoietic growth factors. Cells cultured in neuronal growth medium supplemented with neuronal growth factors were observed to express neurofilament. The morphology of the cells in these three growth specific culture media was markedly different. To extend this work and assess the in vivo attributes of in vitro-derived cells, bone marrow CD34⫺Lin⫺ cells cultured for 3 weeks under conditions for neuronal cell differentiation were injected via the tail vein into irradiated NOD/SCID mice. After 18 days, mice were sacrificed and cells from the brain, muscle, thymus, spleen, and bone marrow were collected and stained with antibodies specific to human CD45 and MHC class-1. Human cells (CD45⫹ or MHC class 1⫹) were only identified in the brain of transplanted mice. To confirm the FACS observation, PCR specific for human globin was performed and has demonstrated the presence of human cells only in the brain of the transplanted mice. These data indicate that HSC, MSC and NSC may originate from a common precursor or pool of precursors in the CD34⫺/Lin⫺ fraction of human bone marrow. These precursor cells may have strong requirements for defined growth environments to enable their differentiation into particular lineages. The data also suggest that the homing of the injected cells may be organ specific. Immunohistochemistry should further reveal the nature of these cells. 52
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
ACCELERATION OF ERYTHROPOIESIS MEDIATED BY ERYTHROID ACCELERATING FACTOR (EAF) IN VERY EARLY STAGE OF DRUG INDUCED HEMOLYSIS IN RATS M. Yokoyama, H. Maruyama, C. Tohno, J. Kitazawa, T. Toki, K. Terui and E. Itoh Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan Erythroid accelerating factor (EAF) is a peculiar factor because the activity is definitely exhibited in an early stage of hemolytic stress in rats. A presumption can be made that EAF contributes to the acceleration of erythroid differentiation during initial period of sudden loss of erythrocytes. To further assess the relationship between serum EAF and a rapid rise of erythropoiesis in response to acute reduction of red blood cells, We determined the sequential changes of serum EAF and Epo in concomitant with blood parameters, bone marrow (BM) CFU-E and the erythroblastic islets in spleens at short intervals from 0time through 24h after acetylphenyhydrazine (APH) injection to induce hemolysis in rats. Serum EAF was significantly elevated as early as 6h following APH injection, reached a peak at 24h. On the other hand, serum
47
Epo levels were gradually increased from 12h, with peaking on day 3 of the experiment. The number of colonies derived from CFU-E and erythroblastic ratios in BM showed peak increases at 8h after injection of APH. Histologic analysis of spleens revealed that the erythroblastic islets increased from (⫹) to (3⫹) at 6h. These results suggest that EAF mediates an early enhance of erythropoietic process during initial period of urgent anemia as acute hemolysis. 53
Tuesday, July 11, 2000 (10:15–12:15) Session V-2: Gene Expression and Transcription Factors
FUNCTIONAL CHARACTERIZATION OF THE TWO ALTERNATIVE PROMOTERS OF HUMAN p45 NF-E2 GENE E. Ito*, T. Toki*, K. Arai*, K. Terui*, M. Yokoyama Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. Expression of the p45 subunit is restricted to selected blood lineages, including erythroid and megakaryocytic lineage cells. Here we report results of studies aimed to understand the regulatory mechanisms controlling p45 gene expression in erythroid cells. Human p45 mRNAs have the two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both NF-E2 isomers by RT-PCR analysis. To examine the expression of both NF-E2 isomers in primary erythroid and megakaryocytic cells, CD34⫹ cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells, whereas aNF-E2 was predominantly expressed in megakaryocytic cells. Although both isomers were abundantly expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12myristate 13-acetate (PMA) resulted in exclusive down regulation of fNF-E2. A functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. These results suggest that NF-E2 resides downstream of the GATA proteins in the transcription factor hierarchy that governs the differentiation of erythroid lineage cells. 54
Tuesday, July 11, 2000 (8:15–9:45) Plenary Session VI: Presidential Symposium
IN VITRO AND IN VIVO EVIDENCES FOR THE LONGTERM MULTILINEAGE (MYELOID, B, NK & T) RECONSTITUTION CAPACITY OF EX VIVO EXPANDED HUMAN CD34ⴙ CORD BLOOD CELLS L. DOUAY, L. KOBARI*, F. PFLUMIO*, M.C. GIARRATANA*, X. LI*, M. TITEUX*, B. IZAC*, F. LETEUTRE*, L. COULOMBEL St Antoine and St louis hospitals, Paris, Institut Gustave Rousy, Villejuif, France We have identified clinically relevant culture conditions that support the expansion of HSC. After 14 days of stroma-free, serum-free