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Analysis of constitutively activated stat signaling molecules in primary acute myeloid leukemia cells
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Abstracts/Experimental Hematology 28 (2000) 31–131
AML cells. Although a high percentage of these primary AML cells showed constitutively activated p38 MAPK, the cells from only five of 22 primary AML cells survived in culture for more than 2–3 weeks. Among them, two samples had the constitutively activated p38 MAPK. Thus, our data indicated that (1) there is a differential pattern of the activation of p38, P44/p42 and NF-B molecules in primary AML cells; (2) there is no correlation of activation among these signaling molecules, and p38 MAPK activation is not depended on the activation of either p44/p42 MAPK or NF-B; (3) the mechanisms resulting in p38 MAPK activation in AML cells is unclear; and (4) constitutive activation of p38, p44/ p42 MAPK, and NFB had no obvious role, but themselves, in promoting growth of AML cells in culture. 260
Sunday, July 9, 2000 (10:30–12:30) Session I-1: Acute Leukemia: Basic Research
ANALYSIS OF CONSTITUTIVELY ACTIVATED STAT SIGNALING MOLECULES IN PRIMARY ACUTE MYELOID LEUKEMIA CELLS R. Y. Liu, C. Fan, G. Q. Liu and K. S. Zuckerman Division of Medical Oncology and Hematology, and Department of Internal Medicine, University of South Florida, and H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida We reported previously that constitutive activation of the JAK2/STAT5 signaling pathway was detected in several leukemic cell lines including HEL and Meg-01 cells, and the constitutively activated STAT5 correlated with growth factor independence for these cell lines (Blood, 93:2369, 1999). To begin to determine the role of constitutively activated STAT molecules in survival of leukemia cells, we analyzed the activation of STAT signaling molecules in primary leukemia cells from bone marrow or peripheral blood of patients with acute myeloid leukemia (AML). When nuclear translocation of STAT5 was analyzed by Western blotting, significant amounts of STAT5 were detected in 15 or 22 (68%) nuclear extracts from AML patients. Similar results were obtained with two other assays of STAT5 activation—electrophoretic gel mobility shift assays and Western blotting of cellular extracts with anti-phospho-STAT5 antibody. The results of analysis of phosphorylation status of STAT1 and STAT3 showed that constitutively phosphorylated STAT1 was detected in 3 of 22 (13.6%) primary AML samples and phosphorylated STAT3 in 4 of 22 (18%) of patients. Of them, leukemic cells from two patients had constitutively phosphorylated and activated STAT1, STAT3 and STAT5, and cells from one patient had a small amount of phosphorylated STAT1 and STAT3, but only barely detectable phosphorylated STAT5. The majority of these primary leukemia cells died in culture within 2–3 weeks, but cells from one patient continued to grow in culture for two months and cells from one patient continue to grow in culture more than 4 months. The leukemia cells from all 5 patients which continued to proliferate in culture for more than 2 weeks had constitutively activated STAT5. Thus, these data indicate a predominance of STAT5 activation in these leukemia cells. The constitutively activation STAT molecules, especially STAT5, apparently played a role in the survival of these primary leukemia cells in culture.
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Monday, July 10, 2000 (12:45–14:15) Session IV-6: Stem Cell Biology IV
REVERSIBLE DIFFERENTIATION IN LIN⫺RHOLOHOLO CULTURED STEM CELLS C. Miller*, J. Reilly*, P. J. Quesenberry Cancer Center, UMASS Medical School We have shown that Lin⫺RholoHolo cells stimulated with IL-3, -6, -11, and SCF enter S phase by 18–20 hr. and complete mitosis by 30–36 hr. In this study, we chose to determine if single cell stem cells could alter their differentiation over time. Marrow was collected from Balb/C mice and single Lin⫺RholoHolo cells were sorted by Mo-Flo analysis to microtiter wells containing four factors or EPO with IL-3. Cloning efficiencies (%HPP colonies/cells plated) were as follows for 0, 18, 20, 24, 44, 48 hr: 17, 58, 37, 38, 75, 45%, respectively. Two week cultured colonies were, cytofuged, WrightGiemsa stained, and characterized based upon individual hematopoietic cell type. Cells cultured in four factors showed 35, 4, and 24% colonies with monocytes at 0, 18, and 20 hr., respectively while megakaryocyte containing colonies were 70, 14 and 35% at 18, 20, and 24 hr. respectively. Cells cultured in EPO showed changes in the percentage of neutrophil containing colonies of 89, 43, and 70% at 20, 24, and 32 hr., respectively while macrophage containing colonies changed over this same period: 44, 0 and 70%. We next determined colony type. Colonies grown in four factors showed a decrease in granulocyte colonies from 28 to 0% of the total colonies over 44 hr. while the mixed colonies increased from 71 to 100% in this same time period. EPO cultured colonies, at 20 and 24 hr., resulted in 90–100% small mononuclear cell colonies while at 32 hr, 100% of the colonies were mixed. These results indicate that total colony number and differentiation pathways fluctuate during cell cycle progression and alteration of phenotypes are reversible. 262
Sunday, July 9, 2000 (10:30–12:30) Session I-5: Stem Cell Biology I
A POPULATION OF SATELLITE CELLS EXPRESSING CD34 REPRESENT A TYPE OF MUSCLE STEM CELL IN MICE Zhuqing Qu Petersen, Ryan Pruchnic, Baohong Cao, Ron Jankowski, Jim Cummins, Charley Gates and Johnny Huard Department of Orthopaedic Surgery and Molecular Genetics & Biochemistry, Musculoskeletal Research Center, Children’s Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, PA* Satellite cells were proposed as muscle stem cells based on their proliferation and differentiation behaviors. However, recent studies have shown that at least two populations of satellite cells exist in skeletal muscles that behave differently both in vitro and in vivo. In the present study, we report that two populations of satellite cells were isolated from mouse skeletal muscle according to their adhesion characteristics and proliferation behaviors. A main population (MP) of myoblasts (rich M-Cadherin⫹ cells) adhered to culture dishes earlier than the other population, and rapidly fused into myotubes with distinct phenotypes, expressing committed precursor muscle cell characteristics. A sub-population (SP) of myoblasts (rich CD34⫹Sca-1⫹ cells) exhibited a strong ability of self-renewal and multi-potential differentiation in vitro, indicating a stem cell characteristic. We further investigated whether the CD34⫹Sca-1⫹ population (SP) originated from muscle fibers or from blood. We
Abstracts/Experimental Hematology 28 (2000) 31–131
AML cells. Although a high percentage of these primary AML cells showed constitutively activated p38 MAPK, the cells from only five of 22 primary AML cells survived in culture for more than 2–3 weeks. Among them, two samples had the constitutively activated p38 MAPK. Thus, our data indicated that (1) there is a differential pattern of the activation of p38, P44/p42 and NF-B molecules in primary AML cells; (2) there is no correlation of activation among these signaling molecules, and p38 MAPK activation is not depended on the activation of either p44/p42 MAPK or NF-B; (3) the mechanisms resulting in p38 MAPK activation in AML cells is unclear; and (4) constitutive activation of p38, p44/ p42 MAPK, and NFB had no obvious role, but themselves, in promoting growth of AML cells in culture. 260
Sunday, July 9, 2000 (10:30–12:30) Session I-1: Acute Leukemia: Basic Research
ANALYSIS OF CONSTITUTIVELY ACTIVATED STAT SIGNALING MOLECULES IN PRIMARY ACUTE MYELOID LEUKEMIA CELLS R. Y. Liu, C. Fan, G. Q. Liu and K. S. Zuckerman Division of Medical Oncology and Hematology, and Department of Internal Medicine, University of South Florida, and H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida We reported previously that constitutive activation of the JAK2/STAT5 signaling pathway was detected in several leukemic cell lines including HEL and Meg-01 cells, and the constitutively activated STAT5 correlated with growth factor independence for these cell lines (Blood, 93:2369, 1999). To begin to determine the role of constitutively activated STAT molecules in survival of leukemia cells, we analyzed the activation of STAT signaling molecules in primary leukemia cells from bone marrow or peripheral blood of patients with acute myeloid leukemia (AML). When nuclear translocation of STAT5 was analyzed by Western blotting, significant amounts of STAT5 were detected in 15 or 22 (68%) nuclear extracts from AML patients. Similar results were obtained with two other assays of STAT5 activation—electrophoretic gel mobility shift assays and Western blotting of cellular extracts with anti-phospho-STAT5 antibody. The results of analysis of phosphorylation status of STAT1 and STAT3 showed that constitutively phosphorylated STAT1 was detected in 3 of 22 (13.6%) primary AML samples and phosphorylated STAT3 in 4 of 22 (18%) of patients. Of them, leukemic cells from two patients had constitutively phosphorylated and activated STAT1, STAT3 and STAT5, and cells from one patient had a small amount of phosphorylated STAT1 and STAT3, but only barely detectable phosphorylated STAT5. The majority of these primary leukemia cells died in culture within 2–3 weeks, but cells from one patient continued to grow in culture for two months and cells from one patient continue to grow in culture more than 4 months. The leukemia cells from all 5 patients which continued to proliferate in culture for more than 2 weeks had constitutively activated STAT5. Thus, these data indicate a predominance of STAT5 activation in these leukemia cells. The constitutively activation STAT molecules, especially STAT5, apparently played a role in the survival of these primary leukemia cells in culture.
261
Monday, July 10, 2000 (12:45–14:15) Session IV-6: Stem Cell Biology IV
REVERSIBLE DIFFERENTIATION IN LIN⫺RHOLOHOLO CULTURED STEM CELLS C. Miller*, J. Reilly*, P. J. Quesenberry Cancer Center, UMASS Medical School We have shown that Lin⫺RholoHolo cells stimulated with IL-3, -6, -11, and SCF enter S phase by 18–20 hr. and complete mitosis by 30–36 hr. In this study, we chose to determine if single cell stem cells could alter their differentiation over time. Marrow was collected from Balb/C mice and single Lin⫺RholoHolo cells were sorted by Mo-Flo analysis to microtiter wells containing four factors or EPO with IL-3. Cloning efficiencies (%HPP colonies/cells plated) were as follows for 0, 18, 20, 24, 44, 48 hr: 17, 58, 37, 38, 75, 45%, respectively. Two week cultured colonies were, cytofuged, WrightGiemsa stained, and characterized based upon individual hematopoietic cell type. Cells cultured in four factors showed 35, 4, and 24% colonies with monocytes at 0, 18, and 20 hr., respectively while megakaryocyte containing colonies were 70, 14 and 35% at 18, 20, and 24 hr. respectively. Cells cultured in EPO showed changes in the percentage of neutrophil containing colonies of 89, 43, and 70% at 20, 24, and 32 hr., respectively while macrophage containing colonies changed over this same period: 44, 0 and 70%. We next determined colony type. Colonies grown in four factors showed a decrease in granulocyte colonies from 28 to 0% of the total colonies over 44 hr. while the mixed colonies increased from 71 to 100% in this same time period. EPO cultured colonies, at 20 and 24 hr., resulted in 90–100% small mononuclear cell colonies while at 32 hr, 100% of the colonies were mixed. These results indicate that total colony number and differentiation pathways fluctuate during cell cycle progression and alteration of phenotypes are reversible. 262
Sunday, July 9, 2000 (10:30–12:30) Session I-5: Stem Cell Biology I
A POPULATION OF SATELLITE CELLS EXPRESSING CD34 REPRESENT A TYPE OF MUSCLE STEM CELL IN MICE Zhuqing Qu Petersen, Ryan Pruchnic, Baohong Cao, Ron Jankowski, Jim Cummins, Charley Gates and Johnny Huard Department of Orthopaedic Surgery and Molecular Genetics & Biochemistry, Musculoskeletal Research Center, Children’s Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, PA* Satellite cells were proposed as muscle stem cells based on their proliferation and differentiation behaviors. However, recent studies have shown that at least two populations of satellite cells exist in skeletal muscles that behave differently both in vitro and in vivo. In the present study, we report that two populations of satellite cells were isolated from mouse skeletal muscle according to their adhesion characteristics and proliferation behaviors. A main population (MP) of myoblasts (rich M-Cadherin⫹ cells) adhered to culture dishes earlier than the other population, and rapidly fused into myotubes with distinct phenotypes, expressing committed precursor muscle cell characteristics. A sub-population (SP) of myoblasts (rich CD34⫹Sca-1⫹ cells) exhibited a strong ability of self-renewal and multi-potential differentiation in vitro, indicating a stem cell characteristic. We further investigated whether the CD34⫹Sca-1⫹ population (SP) originated from muscle fibers or from blood. We