ALBERT

Description: Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Description: Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

Description: Bcl-x is a protein in the outer mitochondrial membrane. A member of the Bcl-2 family, Bcl-x protects developing erythroid cells from apoptosis. The exact stage of erythroid development at which Bcl-x exerts its anti-apoptotic effect is not known, but induction of Bcl-x has been proposed to be the mediator of erythropoietin’s (EPO) anti-apoptotic effect in erythroid differentiation. EPO is the principal trophic hormone that controls red blood cell production by regulating apoptosis of erythroid progenitor cells at the CFU-E and early erythroblast stages. Bcl-x has also been reported to be necessary for heme synthesis. In mice, Bcl-x deficiency is embryonically lethal; when Bcl-x deficiency is acquired postnatally by conditional knockout technology, it is associated with splenomegaly, thrombocytopenia, and a profound anemia that is thought to be hemolytic in origin. Objectives: 1)To characterize the defect of erythroid differentiation in conditional Bcl-x −/− mice. 2)To determine whether Bcl-x is the mediator of EPO’s anti-apoptotic action. 3)To determine whether Bcl-x is necessary for heme synthesis. Methods: Phlebotomized or unbled littermate controls and anemic adult Bcl-x −/− mice obtained by cre-lox conditional knockout were bled, sacrificed, and splenectomized. Purified populations of splenic erythroblasts were isolated by sedimentation at unit gravity, cultured with or without EPO, and harvested at 0, 8, 20, 32, and 44 hours for cell counts, cytospin preparations for morphology, flow cytometry analyses for apoptosis (TUNEL) and cell cycle phases, and 59FeCl3 incorporation into heme. Results: Compared to littermate controls, Bcl-x −/− mice were severely anemic (Hgb 2.8 g/dL vs 15.4 g/dL in unbled controls and 7.2 g/dL in bled controls), thrombocytopenic (platelets 23x103/microL vs 905x103/microL in unbled controls and 984x103/microL in bled controls), and reticulocytopenic (82.8x103/microL vs 281x103/microL in unbled controls and 1410x103/microL in bled controls), while WBCs were unaffected. Expanded erythropoiesis led to massive splenomegaly (spleens =4.3gm vs 0.1gm in unbled controls and 0.3gm in bled controls). After 44 hours of culture with EPO, purified erythroblasts from bled controls proliferated 4-fold and differentiated such that the majority enucleated, producing 200–250 reticulocytes per 100 erythroblasts plated, whereas Bcl-x −/− erythroblast numbers doubled during the first 20 hours in culture, but the large majority died by apoptosis between 20 and 44 hours, producing only 9–12 reticulocytes per 100 erythroblasts plated. Bcl-x −/− erythroblast apoptosis occurred after the initiation of heme synthesis and proportionally in all phases of cell cycle. Compared to culture with EPO, Bcl-x −/− erythroblasts cultured without EPO underwent increased apoptosis at earlier times of culture-- at 8 hours (45% vs 29%), 20 hours (71% vs 42%) and 32 hours (83% vs 57%). Conclusions: 1)Bcl-x is required for the survival and differentiation of the late-stage erythroblasts in all phases of cell cycle. Thus, Bcl-x deficiency results in ineffective erythropoiesis rather than hemolytic anemia. 2)Bcl-x is not required for heme synthesis, but has its anti-apoptotic effect during the stage of hemoglobin synthesis. 3)Bcl-x does not mediate EPO’s anti-apoptotic effect in early-stage erythroblasts.

Description: Reticulocytes (Retics) are immature RBCs that are detected by staining of RNA in the internal organelles, mainly the ribosomes, which remain in mammalian erythroid cells following enucleation. Along with other internal organelles, ribosomes are degraded during maturation of Retics, resulting in complete loss of Retic staining in mature erythrocytes. At the same time as internal organelle degradation, plasma membranes of Retics mature with loss of specific proteins including transferrin receptors (TfR) and the alpha-4 component of several integrins (α4). Retic plasma membrane maturation involves an intrinsic mechanism, exocytosis, as well as an extrinsic mechanism that occurs, at least partially, in the spleen. TfR and α4 expressions on Retic plasma membranes and thiazole orange (TO) staining of RNA were examined by flow cytometry during murine Retic maturation under normal and stress conditions. During their 4 days of maturation in vitro, nascent reticulocytes derived from cultured erythroblasts stably expressed TfR in nearly 80% of cells and α4 in 40% of cells on their plasma membranes, while TO staining was completely lost over the 4 days. To compare in vivo and in vitro Retic maturation, mice were bled causing an anemia with reticulocytosis. Retic maturation in vitro was examined in cultures of Retic-rich blood removed from these bled mice, while Retic maturation in vivo was examined after the bled mice were hypertransfused to cease further erythroblast production. Surface TfR and α4 expressions disappeared during Retic maturation in vivo, but not in vitro. As the Retics matured in vivo, a population of erythrocytes with surface TfR expression, but without TO staining, accumulated to a maximum of 6% of the RBCs. These TfR+/TO− RBCs then disappeared gradually in vivo over several days indicating that they were developmentally between Retics and mature erythrocytes. These TfR+/TO− RBCs were not found in unbled, control mice (

Description: Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

Description: Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Description: Interferon γ (IFNγ) has been shown to inhibit proliferation and differentiation of erythroid progenitor cells and to produce apoptosis of erythroid cells, whereas stem cell factor (SCF ), erythropoietin (EP), and insulin-like growth factor-I (IGF-I) have distinct roles in enhancing erythroid cell production and preventing apoptosis. The mechanism by which IFNγ exerts an inhibitory effect on the positive roles of these growth factors is unknown. Although some inhibitory cytokines including IFNγ have been shown to downregulate growth factor receptors, the effect of IFNγ on SCF, EP, and IGF-I receptors of human erythroid progenitor cells has not been defined. We obtained highly purified day-5 or day-6 erythroid colony-forming cells (ECFCs) from human blood in sufficient quantity and purity for radiolabeled cytokine binding studies and analysis of mRNA. When day-5 ECFCs were incubated with increasing concentrations of recombinant human (rh) IFNγ for 24 hours at 37°C, specific binding of 125I-rhSCF to SCF receptors was significantly decreased by 25% to 40% in a dose-dependent fashion, with the maximum effect at 2,500 to 5,000 U/mL of IFNγ. The decrease was apparent by 12 hours of incubation and was only slightly lower by 24 hours. The numbers of SCF and EP receptors, but not of IGF-I receptors, per ECFC, calculated by Scatchard analysis, were significantly decreased by 30% and 23% to 25%, respectively, after incubation with 2,500 U/mL rhIFNγ for 24 hours at 37°C, whereas the binding affinities were not affected. This decrease in SCF receptors was confirmed by flow cytometry using an anti–c-kit mouse monoclonal antibody. Northern blot analysis showed that the mRNAs for the SCF and EP receptors, but not for the IGF-I receptors, were decreased by 50% to 60% after 3 hours of incubation at 37°C with 2,500 U/mL of rhIFNγ. This persisted for 24 hours without alteration of the stability of the SCF and EP receptor mRNAs. These observations suggest that one means by which IFNγ inhibits erythroid cell proliferation and differentiation and produces apoptosis may be through the reduction of the number of target receptors for SCF and EP and that this occurs through transcriptional inhibition of the corresponding mRNAs.

Description: Interferon γ (IFNγ) inhibits the growth and differentiation of highly purified human erythroid colony-forming cells (ECFCs) and induces erythroblast apoptosis. These effects are dose- and time-dependent. Because the cell surface receptor known as Fas (APO-1; CD95) triggers programmed cell death after activation by its ligand and because incubation of human ECFCs with IFNγ produces apoptosis, we have investigated the expression and function of Fas and Fas ligand (FasL) in highly purified human ECFCs before and after incubation with IFNγ in vitro. Only a small percentage of normal human ECFCs express Fas and this is present at a low level as detected by Northern blotting for the Fas mRNA and flow cytometric analysis of Fas protein using a specific mouse monoclonal antibody. The addition of IFNγ markedly increased the percentage of cells expressing Fas on the surface of the ECFCs as well as the intensity of Fas expression. Fas mRNA was increased by 6 hours, whereas Fas antigen on the cell surface increased by 24 hours, with a plateau at 72 hours. This increase correlated with the inhibitory effect of IFNγ on ECFC proliferation. CH-11 anti-Fas antibody, which mimics the action of the natural FasL, greatly enhanced IFNγ-mediated suppression of cell growth and production of apoptosis, indicating that Fas is functional. Expression of FasL was also demonstrated in normal ECFCs by reverse transcriptase-polymerase chain reaction and flow cytometric analysis with specific monoclonal antibody. FasL was constitutively expressed among erythroid progenitors as they matured from day 5 to day 8 and IFNγ treatment did not change this expression. Apoptosis induced by IFNγ was greatly reduced by the NOK-2 antihuman FasL antibody and an engineered soluble FasL receptor, Fas-Fc, suggesting that Fas-FasL interactions among the ECFCs produce the erythroid inhibitory effects and apoptosis initiated by IFNγ.