Role of NF-kappa B in regulation of apoptosis of erythroid progenitor cells

Erythropoietin (EPO) and interferon-gamma (IFN-gamma) added to human erythroid progenitor cells purified from peripheral blood (erythroid colony-forming cells; ECFC) significantly reduces apoptosis as assessed by flow cytometry (FCM) using annexin V. To clarify the role of NF-kappaB in the regulation of the apoptosis of erythroid progenitor cells, cyclosporin A (CsA), which blocks dissociation of the NF-kappaB complex, was added to serum-free cultures of ECFC. CsA induced the apoptosis of ECFCs in the presence of EPO or IFN-gamma, but at different magnitudes. In the presence of a relatively low concentration of CsA (10 microm), apoptosis was induced only in cultures with EPO. The direct involvement of NF-kappaB was then assessed by Western blotting and confocal microscopy. In the presence of EPO, NF-kappaB was abundant both in the cytoplasm and in the nucleus, and nuclear expression was diminished after adding CsA. In contrast, NF-kappaB was undetectable in the nucleus in the presence of IFN-gamma. The effect of CsA on mitochondrial function was investigated by determining the DeltaPsim and reactive oxygen species production. CsA disturbed the transmembrane potential in the presence of either EPO or IFN-gamma, although the viability of the cells was maintained in the presence of IFN-gamma plus CsA. These results indicate that IFN-gamma reduced the apoptosis of erythroid progenitor cells through a unique signaling pathway that is independent of NF-kappaB translocation, and which is not mediated by modulating mitochondrial function, whereas EPO reduced apoptosis through NF-kappaB translocation to the nucleus.     

Activation of the human delta-globin gene promoter in primary adult erythroid cells

Restoration of the CCAAT box or insertion of an erythroid Krüppel-like factor (EKLF) binding site in the delta promoter activates its expression in several erythroid cell lines. We extended these studies using a novel primary human adult erythroid cell ( h AEC) system to investigate these effects at the late erythroblast stage. Restoration of the CCAAT box at −70 bp, or insertion of an EKLF binding site at −85 bp or −95 bp in the promoter significantly increased delta globin gene expression in h AEC. Our results demonstrate that the altered CCAAT box (CCAAC) and the lack of an EKLF binding site in δ-globin contribute to its low level of expression in the h AEC model as well.     

Stem cell factor and erythropoietin inhibit apoptosis of human erythroid progenitor cells through different signalling pathways

Erythropoietin (EPO) and stem cell factor (SCF) are two important factors in human erythropoiesis. We have recently demonstrated that SCF and EPO synergistically activate mitogen-activated protein (MAP) kinase, thereby promoting growth of human erythroid colony-forming cells (ECFCs). In the present study, we have examined the intracellular mechanisms by which SCF and EPO maintain survival of these cells. In the absence of SCF and EPO, human ECFCs underwent rapid apoptosis. The process was significantly inhibited by addition of a single factor and was totally prevented in the presence of both factors. Treatment of ECFCs with wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI3K), inhibited the antiapoptotic effect of SCF but had no effect on that of EPO, indicating that SCF but not EPO inhibits apoptosis through the PI3K pathway. In contrast, treatment of ECFCs with PD98059, a specific inhibitor of MAP kinase/ERK kinase (MEK), inhibited cell growth but had no effect on the antiapoptotic activity of either SCF or EPO, suggesting that SCF and EPO prevent apoptosis of human ECFCs independent of the extracellular signal-regulated kinase (ERK) pathway. Interestingly, both EPO and SCF induced activation of PI3K. However, through PI3K, SCF caused activation of protein kinase B (PKB), an anti-apoptosis signal, whereas EPO led to activation of ERKs. Furthermore, the SCF- and EPO-maintained expression of antiapoptotic protein Bcl-XL was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl-XL may not have a major role in preventing apoptosis of human ECFCs. Phosphorylated BAD was not affected by SCF, EPO or wortmannin. Taken together with our previous results, the present study indicates that SCF and EPO support survival and growth of human ECFCs through different signalling pathways and that they transduce distinctly different signals through activation of PI3K.     

Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum-free cell-culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum-free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL-3 was TPO able to stimulate a small increase (∼25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34⁺ cells were cultured for 7 d in serum-free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre-cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit-erythroid (BFU-E) colonies as cells which were pre-cultured in the absence of TPO. Further, when primitive CD34⁺, Kit⁺ MNC were cultured for 3–7 d under serum-free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.     

红系造血分化中分化调节因子的表达及调控

目的探讨红系分化调节因子（EDRF）在红白血病及正常造血组织中的表达差异及可能的调控机制。方法应用Northern blot方法检测红白血病细胞HB22.2、CB3中EDRF基因的表达,并以正常小鼠胚胎肝脏组织作为对照;通过改变培养条件,诱导红白血病细胞系HB60-5成熟分化,探讨红系分化中EDRF表达的变化;采用Southern blot方法对EDRF基因进行DNA水平检测;通过转基因的方法探讨红系造血调控因子GATA-1、NF-E2、Fli-1对EDRF表达的影响。结果在胚胎肝组织中EDRF表达水平较高,在NF-E2缺陷的红白血病细胞株HB22.2及CB3中,EDRF表达降低,同时α-珠蛋白表达缺失;Southern blot检测未发现红白血病细胞与正常胚胎肝脏组织EDRF基因的差别;HB22.2及CB3转染NF-E2基因后,血红蛋白及EDRF表达水平均明显上调;诱导HB60-5分化成熟后,NF-E2、GATA-1、α-珠蛋白及EDRF的表达逐渐增高,具有一定的相关性;HB60-5细胞转染GATA-1基因后可明显上调EDRF表达,转染Fli-1基因后则EDRF表达水平下降。结论 EDRF是红系造血相关基因,在正常红系细胞中具有高水平表达;红白血病细胞系中EDRF的表达受抑,这种表达减低不是DNA突变所致;红系造血因子GATA-1、NF-E2可上调EDRF的表达,Fli-1可下调EDRF表达。     

Binding and uptake of exogenous isoferritins by cultured human erythroid precursor cells

Summary. The interaction of extracellular human isoferritins with normal erythroid precursors developing in a two-phase liquid culture was studied. Cells at the stage of polychromatic normoblasts exhibited substantial specific binding of radio-iodinated placental isoferritins. Considerably more acidic isoferritin was bound than basic isoferritin. The binding of ferritin was significantly higher at 37°C than at 4°C. All of the ¹²⁵I-acidic isoferritin bound at 4°C, but only part of that bound at 37°C, could be dislodged by the addition of a 500-fold excess of non-labelled acidic isoferritin. Acidic isoferritin displaced radio-iodinated acidic isoferritin from the erythroid cells more efficiently than intermediate or basic isoferritins. Kinetic analysis suggests a dissociation constant ( K _d) of 3·9 × 10^-8 m for acidic ferritin and 3·7 × 10^-7 m for basic isoferritin. The average number of binding sites for acidic isoferritin was 1·3 × 10⁵ per cell. The results point to specific binding and receptor-mediated internalization for predominantly acidic isoferritin by developing human erythroid cells.     

Comparative ultrastructure of late rabbit-embryo erythroid cells in liver and peripheral blood

A morphological study of hemoglobin biosynthesis activity in rabbit-embryo liver and peripheral blood was comparatively developed. Orthochromatic erythroblasts and reticulocytes of the same maturing degree were analysed through thin sections, as to their organellar constitution and behaviour regarding iron incorporation. It was found that peripheral blood erythroid cells contain mainly hemosomes, organelles taken as sites of final hemoglobin molecule biosynthesis. The ratio between the mean number of hemosomes in blood erythroid cell sections and that in liver erythroid cell sections reaches a little more than 6:1. Inversely, late liver erythroid cells are predominantly constituted by mitochondria that directly or indirectly participate in hemosome formation. The ratio between the mean number of mitochondria in blood erythroid cell sections and that in liver erythroid cell sections reaches 1:11. Besides this, the iron incorporation activity is higher in peripheral blood erythroid cells than in liver erythroblasts and reticulocytes. It is apparent that erythroid cells in the liver accumulate heme, given the ratio of the means of iron incorporation activity per cell to hemosome per cell (3.67), while blood erythroid cells, with a 1.08 ratio, synthesize heme, which is immediately afterwards integrated into the globin chains. The sudden increase in the formation of hemosomes when liver orthochromatic erythroblasts and reticulocytes enter the peripheral blood, reflecting an enhancement of hemoglobin synthesis, agrees with biochemical findings of other authors.     

Effects of mitochondrial ferritin overexpression in normal and sideroblastic erythroid progenitors

In myelodysplastic syndromes with ring sideroblasts (MDS‐RS), the iron deposited in the mitochondria of RS is present in the form of mitochondrial ferritin (FTMT), but it is unknown whether FTMT overexpression is the cause or the result of mitochondrial iron deposition. Lentivirus FTMT‐transduced CD34⁺ bone marrow cells from seven healthy donors and CD34⁺ cells from 24 patients with MDS‐RS were cultured according to a procedure that allowed the expansion of high numbers of erythroid progenitors. These cells were used to investigate the possible influence of experimentally‐induced FTMT overexpression on normal erythropoiesis and the functional effects of FTMT in sideroblastic erythropoiesis. In MDS‐RS progenitors, FTMT overexpression was associated with reduced cytosolic ferritin levels, increased surface transferrin receptor expression and reduced cell proliferation; FTMT effects were independent of SF3B1 mutation status. Similarly, FTMT overexpressing normal erythroid progenitors were characterized by reduced cytosolic ferritin content and increased CD71 expression, and also by higher apoptotic rate in comparison with the FTMT‐ controls. Significantly lower levels of STAT5 phosphorylation following erythropoietin stimulation were found in both sideroblastic and normal FTMT⁺ erythroid cells compared to the FTMT‐ cells. In conclusion, experimental overexpression of FTMT may modify mitochondrial iron availability and lead to ineffective erythropoiesis.     

Differential in vitro sensitivity of marrow erythroid and granulocytic colony forming cells to chloramphenicol

The effects of chloramphenicol (CAP) and thiamphenicol (TAP) on mouse and human in vitro erythroid (CFU-E) and granulocytic (CFU-C) colony forming units have been studied. Both drugs inhibited CFU-E growth in a concentration-dependent, stereospecific, manner. Complete inhibition of human CFU-E growth was observed at a CAP concentration of 10 microgram/ml while a concentration ofer 50 microgram/ml was required to inhibit CFU-C growth. Furthermore, whereas the inhibition of CFU-C growth could be blocked in vitro by high colony stimulating factor concentrations, inhibition of CFU-E growth was not affected by increased erythropoietin (ESF) levels. The lack of protection by ESF may account for the apparent vulnerability of erythroid cells to CAP in vivo.     

T cells and erythroid burst forming units in chronic lymphocytic leukemia

Substantial evidence exists indicating T cell abnormalities in chronic lymphocytic leukemia (CLL). There is also evidence that the T cell is an important source of burst promoting activity (BPA) for the peripheral blood (PB) erythroid burst forming unit (BFU-e). We studied the BPA of T cells and response of BFU-e in normals and untreated early stage B cell CLL patients in a methylcellulose colony assay. Normal null cell cultures grew significantly more BFU-e than CLL null cell cultures. Addition of autologous T cells to normal or CLL null cells significantly increased BFU-e only in normals. Allogeneic coculture of T cells from CLL patients with null cells from normals yielded normal responses of BFU-e in five of six cases. In contrast, allogeneic coculture of normal T cells with CLL null cells yielded a normal response in only one of six studies. Furthermore, adding increasing quantities of autologous or normal allogeneic T cells to CLL null cells did not augment the BFU-e response. Accounting for the expanded lymphocyte pool in CLL, BFU-e are decreased in concentration but the absolute number is normal or increased. The decrease in concentration could be secondary to expansion of the null cell fraction in CLL by pre-B cells. CLL T cells appeared to augment normal allogeneic PB BFU-e in a normal fashion, whereas, in several cases, CLL BFU-e were hyporesponsive to autologous or normal allogeneic T cells. It is therefore apparent that in untreated early stage B cell CLL, erythroid progenitor cells are present in the peripheral blood but are diluted in an expanded null cell compartment and may, in some cases, be hyporesponsive to T cell BPA. T cell BPA of CLL T cells in this early stage of disease is preserved.     

Fetal to adult hemoglobin switch in cultures of early erythroid precursors from human fetuses and neonates.

Erythroid burst colonies derived from the cord blood of nine neonates and from the blood and liver of three fetuses aborted after 20 weeks of gestation were grown in plasma clot culture. Their quantitative study revealed a higher proportion of burst-forming units (BFU-Es) in cord blood than in cord blood of normal adults. In addition, colony-forming units (CFU-Es) were present in cord blood but absent from adult blood. Study of haemoglobin synthesis in 14-day cultures of cord blood BFU-Es showed a significantly higher degree of Hb A synthesis than was found in reticulocytes from fresh cord blood; this proportion was, however, similar to that expected in vivo about three weeks after birth. These data suggest that the hemoglobin switch is already programmed in most of the early erythroid precursors present in cord blood or full-term neonates and indicate that the differentiation time is probably of the same order of magnitude in vivo and in vitro. The proportion of Hb A and F synthesis in erythroid bursts was not influenced by the concentration of erythropoietin in the range studied--ie, from 0.5 to 12 international units. Low but identical proportions of Hb A synthesis were found both in erythroid cells from liver after two hours of incubation with [3H]-leucine, and in 14-day liver bursts from fetuses aborted at 20 weeks of gestation.     

Developing human erythroid cells grown in transferrin-free medium utilize iron originating from extracellular ferritin

In addition to transferrin, ferritin can also function as a source of iron for heme synthesis (Gelvin D, et al. Blood 1996;88:3200-3207; Meyron-Holtz EG, et al. Blood 1999;94:3205-3211). In the present study we investigate the effect of external ferritin on the proliferation and hemoglobinization of human erythroid precursors grown in transferrin-free cultures.     

Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells

The present study aimed to determine whether rapamycin could increase the expression of gamma-globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti-tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors and patients with beta-thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, gamma-globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four beta-thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human gamma-globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including beta-thalassaemia and sickle cell anaemia.     

microRNA expression in erythropoiesis and erythroid disorders

MicroRNAs are a recently discovered class of small (c. 22 nt) naturally occurring non‐coding RNA molecules that regulate gene expression through binding to the un‐translated region of target mRNA. MicroRNAs play key roles in many cellular pathways including haematopoiesis and aberrant expression is a common feature of haematological malignancies. Whilst other areas of haematopoiesis have been extensively reviewed the involvement of microRNAs in red cell production (erythropoiesis) and disorders of this pathway is lacking. In this review the rapidly accumulating evidence that points to the major role microRNAs play in both erythropoiesis and erythroid disorders is discussed.