Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.

Fyn kinase is a novel modulator of erythropoietin signaling and stress erythropoiesis.

The signaling cascade induced by the interaction of erythropoietin (EPO) with its receptor (EPO-R) is a key event of erythropoiesis. We present here data indicating that Fyn, a Src-family-kinase, participates in the EPO signaling-pathway, since Fyn-/- mice exhibit reduced Tyr-phosphorylation of EPO-R and decreased STAT5-activity. The importance of Fyn in erythropoiesis is also supported by the blunted responsiveness of Fyn-/- mice to stress erythropoiesis. Fyn-/- mouse erythroblasts adapt to reactive oxygen species (ROS) by activating the redox-related-transcription-factor Nrf2. However, since Fyn is a physiologic repressor of Nrf2, absence of Fyn resulted in persistent-activation of Nrf2 and accumulation of nonfunctional proteins. ROS-induced over-activation of Jak2-Akt-mTOR-pathway and repression of autophagy with perturbation of lysosomal-clearance were also noted. Treatment with Rapamycin, a mTOR-inhibitor and autophagy activator, ameliorates Fyn-/- mouse baseline erythropoiesis and erythropoietic response to oxidative-stress. These findings identify a novel multimodal action of Fyn in the regulation of normal and stress erythropoiesis.

Erythropoietin regulates the expression of dimeric form of acetylcholinesterase during differentiation of erythroblast.

Acetylcholinesterase (AChE; EC 3.1.1.7) is known to hydrolyze acetylcholine at cholinergic synapses. In mammalian erythrocyte, AChE exists as a dimer (G2 ) and is proposed to play role in erythropoiesis. To reveal the regulation of AChE during differentiation of erythroblast, erythroblast-like cells (TF-1) were induced to differentiate by application of erythropoietin (EPO). The expression of AChE was increased in parallel to the stages of differentiation. Application of EPO in cultured TF-1 cells induced transcriptional activity of ACHE gene, as well as its protein product. This EPO-induced event was in parallel with erythrocytic proteins, for example, α- and ß-globins. The EPO-induced AChE expression was mediated by phosphorylations of Akt and GATA-1; because the application of Akt kinase inhibitor blocked the gene activation. Erythroid transcription factor also known as GATA-1, a downstream transcription factor of EPO signaling, was proposed here to account for regulation of AChE in TF-1 cell. A binding sequence of GATA-1 was identified in ACHE gene promoter, which was further confirmed by chromatin immunoprecipitation (ChIP) assay. Over-expression of GATA-1 in TF-1 cultures induced AChE expression, as well as activity of ACHE promoter tagged with luciferase gene (pAChE-Luc). The deletion of GATA-1 sequence on the ACHE promoter, pAChEΔGATA-1 -Luc, reduced the promoter activity during erythroblastic differentiation. On the contrary, the knock-down of AChE in TF-1 cultures could lead to a reduction in EPO-induced expression of erythrocytic proteins. These findings indicated specific regulation of AChE during maturation of erythroblast, which provided an insight into elucidating possible mechanisms in regulating erythropoiesis.

Non-apoptotic functions of caspases in myeloid cell differentiation.

Subtle caspase activation is associated with the differentiation of several myeloid lineages. A tightly orchestrated dance between caspase-3 activation and the chaperone HSP70 that migrates to the nucleus to protect the master regulator GATA-1 from cleavage transiently occurs in basophilic erythroblasts and may prepare nucleus and organelle expel that occurs at the terminal phase of erythroid differentiation. A spatially restricted activation of caspase-3 occurs in maturing megakaryocytes to promote proplatelet maturation and platelet shedding in the bloodstream. In a situation of acute platelet need, caspase-3 could be activated in response to IL-1α and promote megakaryocyte rupture. In peripheral blood monocytes, colony-stimulating factor-1 provokes the formation of a molecular platform in which caspase-8 is activated, which downregulates nuclear factor-kappa B (NF-κB) activity and activates downstream caspases whose target fragments such as those generated by nucleophosmin (NPM1) cleavage contribute to the generation of resting macrophages. Human monocytes secrete mature IL-1ß in response to lipopolysaccharide through an alternative inflammasome activation that involves caspase-8, a pathway that does not lead to cell death. Finally, active caspase-3 is part of the proteases contained in secretory granules of mast cells. Many questions remain on how these proteases are activated in myeloid cell lineages, which target proteins are cleaved, whereas other are protected from proteolysis, the precise role of cleaved proteins in cell differentiation and functions, and the link between these non-apoptotic functions of caspases and the death of these diverse cell types. Better understanding of these functions may generate therapeutic strategies to control cytopenias or modulate myeloid cell functions in various pathological situations.

Identification of NuRSERY, a new functional HDAC complex composed by HDAC5, GATA1, EKLF and pERK present in human erythroid cells.

To clarify the role of HDACs in erythropoiesis, expression, activity and function of class I (HDAC1, HDAC2, HDAC3) and class IIa (HDAC4, HDAC5) HDACs during in vitro maturation of human erythroblasts were compared. During erythroid maturation, expression of HDAC1, HDAC2 and HDAC3 remained constant and activity and GATA1 association (its partner of the NuRD complex), of HDAC1 increased. By contrast, HDAC4 content drastically decreased and HDAC5 remained constant in content but decreased in activity. In erythroid cells, pull down experiments identified the presence of a novel complex formed by HDAC5, GATA1, EKLF and pERK which was instead undetectable in cells of the megakaryocytic lineage. With erythroid maturation, association among HDAC5, GATA1 and EKLF persisted but levels of pERK sharply decreased. Treatment of erythroleukemic cells with inhibitors of ERK phosphorylation reduced by >90% the total and nuclear content of HDAC5, GATA1 and EKLF, suggesting that ERK phosphorylation is required for the formation of this complex. Based on the function of class IIa HDACs as chaperones of other proteins to the nucleus and the erythroid-specificity of HDAC5 localization, this novel HDAC complex was named nuclear remodeling shuttle erythroid (NuRSERY). Exposure of erythroid cells to the class II-selective HDAC inhibitor (HDACi) APHA9 increased γ/(γ+ß) globin expression ratios (Mai et al., 2007), suggesting that NuRSERY may regulate globin gene expression. In agreement with this hypothesis, exposure of erythroid cells to APHA9 greatly reduced the association among HDAC5, GATA1 and EKLF. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic therapies of hemoglobinopaties and other disorders.

Differential sensitivity to JAK inhibitory drugs by isogenic human erythroblasts and hematopoietic progenitors generated from patient-specific induced pluripotent stem cells.

Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD34+ hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.

p-ERK1/2 is a predictive factor of response to erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes.

Serum erythropoietin level less than 100U/L and a transfusion requirement of less than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes. To investigate the factors influencing the response to erythropoiesis-stimulating agents, we enrolled 127 low/int-1 myelodysplastic syndrome patients at diagnosis in a biological study of erythropoiesis. The 54 non-responders had a significantly lower number of burst-forming unit-erythroid and colony-forming unit-erythroid than responders. Erythropoietin-dependent proliferation and survival, and phospho (p)-ERK1/2 expression in steady state and after erythropoietin stimulation were defective in cultured erythroblasts. By flow cytometry, p-ERK1/2 was significantly lower in bone marrow CD45(-)/CD71(+)/GPA(-)cells from non-responders compared to responders or controls. Receiver Operator Characteristic curve analysis showed that this flow cytometry test was a sensitive biomarker for predicting the response to erythropoiesis-stimulating agents.

[Contents of monovalent cations and ATP in erythrocytes of marine fishes in experimental hypoxia].

Coupling of membrane and metabolic functions in nuclear erythrocytes was investigated under experimental hypoxia conditions in fishes (Liza aurata, Scorpaena porcus) with different tolerance to oxygen deficiency. It was shown, that resistant to hypoxia Scorpaena porcus keeps in erythrocytes transmembrane gradients of K+ and Na+ and cellular concentration ATP under 15% of oxygen saturation of sea water. It was connected with the decrease in Na+, K+ -ATPase and hexokinase activity. The reaction to oxygen deficiency was opposite in sensitive to hypoxia Liza aurata erythrocytes. A decrease in ionic gradients and concentration of ATP in red blood cells was observed while the activity of Na+, K+ -ATPase and hexokinase was high. The reasons of the differences obtained are discussed.

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen.

BACKGROUND: The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated. DESIGN AND METHODS: Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry. RESULTS: Erythropoiesis in Rac1(-/-);Rac2(-/-) mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen. Conclusions These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.

Erythropoietin down-regulates stem cell factor receptor (Kit) expression in the leukemic proerythroblast: role of Lyn kinase.

Overexpression of the transcription factor Spi-1/PU.1 by transgenesis in mice induces a maturation arrest at the proerythroblastic stage of differentiation. We have previously isolated a panel of spi-1 transgenic erythroleukemic cell lines that proliferated in the presence of either erythropoietin (Epo) or stem cell factor (SCF). Using these cell lines, we observed that EpoR stimulation by Epo down-regulated expression of the SCF receptor Kit and induced expression of the Src kinase Lyn. Furthermore, enforced expression of Lyn in the cell lines increased cell proliferation in response to Epo, but reduced cell growth in response to SCF in accordance with Lyn ability to down-regulate Kit expression. Together, the data suggest that Epo-R/Lyn signaling pathway is essential for extinction of SCF signaling leading the proerythroblast to strict Epo dependency. These results highlight a new role for Lyn as an effector of EpoR in controlling Kit expression. They suggest that Lyn may play a central role in during erythroid differentiation at the switch between proliferation and maturation.

Biological and clinical relevance of matrix metalloproteinases 2 and 9 in acute myeloid leukaemias and myelodysplastic syndromes.

We analysed by immunocytochemistry metalloproteinase (MMP)-2 and MMP-9 expression in bone marrow cells from 54 acute myeloid leukaemia (AML) patients, 153 myelodysplastic syndrome (MDS) patients, and 52 non-haemopathic subjects, in order to evaluate whether MMP expression abnormalities were associated with relevant laboratory or clinical findings. In normal samples MMP-2 was detected in rare myeloid cells, MMP-9 in most maturing myeloid cells. In MDS MMP-2 myeloid levels were higher than in controls (P < 0.0001); MMP-2 and MMP-9 were often co-expressed. Also many erythroblasts expressed MMP-2. There was a positive correlation between MMP-2 erythroblast expression and erythroid dysplasia (P = 0.002) and an inverse correlation between MMP-2 or MMP-9 myeloid expression and blast cell percentage (P = 0.05 and P = 0.04 respectively). High MMP levels in myeloid cells were associated with longer overall survival (P = 0.03) and evolution-free survival (P = 0.04). In AML MMP-2 levels were lower than in MDS (P < 0.0001) and MMP-9 levels lower than in MDS and controls (P < 0.0001). MMP levels did not predict response to therapy. The release of active MMPs was detected by colorimetric analysis in cell cultures from representative MDS and AML cases. In conclusion, we have demonstrated an abnormal MMP expression in AML as well as in MDS. The production and release of these enzymes may influence haematopoietic cell behaviour. In MDS, the detection of MMP deregulated expression may be important also from the clinical point of view: it may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.

Histone deacetylase inhibitor FK228 is a potent inducer of human fetal hemoglobin.

We investigated the induction of the human fetal globin gene using five potent histone deacetylase (HDAC) inhibitors: FK-228, HC-Toxin, Trichostatin, MS-275, and Apicidin, using in vitro assays and cultures of primary human erythroblasts. The results showed that FK228 is the most potent inducer of fetal hemoglobin and exhibits its effects in picomolar concentrations. FK228 should be considered as a potential therapeutic for induction of fetal hemoglobin in patients with beta chain hemoglobinopathies.

A novel role for STAT1 in regulating murine erythropoiesis: deletion of STAT1 results in overall reduction of erythroid progenitors and alters their distribution.

Erythropoietin (EPO) activates many distinct signal transduction cascades on engagement of its receptor. Deletion of the EPO, EPO receptor (EPO-R), or JAK2 genes in mice results in embryonic lethality due to a fatal anemia. EPO activates signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5a/b transcription factors in erythroid cell lines. Studies have focused on STAT5 as the primary target of EPO-dependent JAK2 activation. However, STAT5a/b(-/-) mice are viable, displaying a nonfatal anemia during embryogenesis, and delayed differentiation in adult erythropoiesis. Importantly, EPO-R cytoplasmic tyrosines are dispensable for viability in vivo. Interestingly, no cytoplasmic tyrosines are required for phosphorylation of STAT1. This led us to examine whether STAT1-deficient mice have altered erythropoiesis. A shift in erythropoiesis was observed in STAT1(-/-) mice, with reduced bone marrow-derived erythroid colony-forming units (CFU-Es) and a compensatory increase in splenic burst-forming units (BFU-Es) and CFU-Es. Both types of splenic-derived cells displayed EPO hyperresponsiveness. A 1.6-fold reduction in total CFU-Es was observed in STAT1-deficient mice, whereas total BFU-Es were comparable. Flow cytometry of STAT1-deficient erythroid cells revealed a less differentiated phenotype, associated with increased apoptosis of early erythroblasts. STAT1-deficient erythroblasts from phenylhydrazine-primed mice displayed enhanced phosphorylation of STAT5a/b, Erk1/2, and protein kinase B (PKB)/Akt. These results illustrate that STAT1 plays an important role in the regulation of erythropoiesis.

[Erythropoiesis: a paradigm for the role of caspases in cell death and differentiation]. / L'erythropoïèse : un paradigme pour l'etude du rôle des caspases dans la mort et la différenciation cellulaire.

Erythroid differentiation involves the transcription factor GATA-1 that positively regulates promoters of erythroid genes (including haemoglobin, glycophorin, erythropoietin receptor) and of erythropoietin. Terminal erythroid differentiation is characterized by major morphological changes that include chromatin condensation and cell size reduction. The morphological changes are partially similar at least to those observed during apoptosis. The production of red cells depends on the apoptosis rate of erythroid progenitors and precursors. Upon erythropoietin starvation or engagement of the death receptor Fas, caspases are activated in erythroid precursors and cleave GATA-1, thus inducing maturation arrest and apoptosis of immature erythroblasts. We have recently demonstrated that, upon erythropoietin stimulation, caspase-3 was also activated, an event required for human terminal erythroblast maturation. Proteins cleaved by caspases in erythroid cells undergoing terminal differentiation include Lamin B and Acinus, which are involved in chromatin condensation. In contrast, despite caspase-3 activation neither GATA-1 degradation nor apoptosis was observed. Thus, the fate of erythroid precursors is determined downstream of caspase activation by the pattern of cleaved targets. Therefore, there are some mechanisms underlying the selective protection of caspase-3 targets during erythropoiesis. This model in which caspases activation is required for differentiation may apply to other haematopoietic or non haematopoietic cellular systems which are described in this review.

Modulation of the expression and activity of cyclooxygenases in normal and accelerated erythropoiesis.

OBJECTIVE: The present study was aimed at characterizing the expression and activity of cyclooxygenase (COX) isoenzymes in erythropoiesis. METHODS: The expression and activity of cyclooxygenase (COX) and prostaglandin (PG) synthases were investigated in: 1) erythroblasts developed in culture from human CD34(+) hematopoietic progenitors, 2) erythroblasts in bone marrow specimens, and 3) peripheral erythrocytes isolated from healthy donors and from patients with a high regeneration rate of erythrocytes. RESULTS: While COX-1 protein was observed at each stage of erythroblast development, COX-2 protein was induced at later stages through a p38/MAPK-dependent pathway. Both COX isoforms were also observed in mature erythroblasts of the bone marrow. Erythroblasts developed in culture synthesized significantly more PGE(2) than TXB(2) and indomethacin delayed erythroid maturation. COX-1 and COX-2 were also observed in erythrocytes by immunostainings, although COX expression was confined to a fraction of circulating erythrocytes. Peripheral erythrocytes synthesized low but detectable amounts of PGE(2) and TXB(2). Similarly to erythroblast progenitors, PGE(2) was the prevalent prostanoid released by erythrocytes. This biosynthetic capacity was significantly increased in erythrocytes from patients with accelerated erythropoiesis as compared to controls. CONCLUSIONS: Both COX isoforms are present and enzymatically active during human erythropoiesis, although with different kinetics, and COX-derived prostanoids may play a role in erythroid maturation. Furthermore, peripheral erythrocytes retain in part the capacity of expressing COX and synthesizing prostanoids, which may contribute to the hemostatic/thrombotic response to vascular injury in different diseases, including congenital hemolytic disorders.

Aberrant iron accumulation and oxidized status of erythroid-specific delta-aminolevulinate synthase (ALAS2)-deficient definitive erythroblasts.

Alas2 encodes the erythroid-specific delta-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with the Alas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking the Alas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype of Alas2-null definitive erythroblasts was examined. Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes in Alas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate that Alas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron, Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.

Caspase activation is required for terminal erythroid differentiation.

The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.

Increased apoptosis in acquired sideroblastic anaemia.

Idiopathic acquired sideroblastic anaemias (IASAs) form a subgroup of the myelodysplastic syndromes and are characterized by mitochondrial iron accumulation, bone marrow erythroid hyperplasia and decreased peripheral red blood cell counts. Increased intramedullary apoptosis of erythroid precursors is presumed to constitute the pathophysiological mechanism explaining this ineffective erythropoiesis, but if and how mitochondrial dysfunction is implicated in this process is currently unknown. We therefore studied bone marrow precursor cells obtained from nine patients with IASA for (i) caspase 3 activity, (ii) numbers of Annexin V- and 7-amino-actinomycin-positive cells, (iii) numbers of cells with diminished mitochondrial membrane potential, Delta Psi(m), and (iv) numbers of cells producing reactive oxygen species (ROS), and we compared the results with those of five normal bone marrow samples. Compared with controls, we found increased caspase 3 activity in all IASA samples, which correlated with increased numbers of Annexin-V-positive cells (r = 0.7). Analysis of different subpopulations showed increased apoptosis in erythroid populations compared with myeloid and/or lymphoid populations in five out of nine cases, and increased apoptosis in the last two populations in four out of nine cases. As evidence of mitochondrial dysfunction, Delta Psi(m) was found to be diminished in the erythroid subpopulations of all cases of IASA (66.6 +/- 17% vs. 34.6 +/- 12% in normals). Delta Psi(m) decrease was correlated to Annexin V positivity (r = 0.7). Astonishingly, no difference was found between IASA and normal bone marrows with regard to the number of ROS-producing cells. In fact, both groups exhibited a similar low proportion of ROS production (10.3 +/- 7% in normals vs. 6.8 +/- 5% in IASA). Taken together, our results show that mitochondria are clearly implicated in the apoptotic process in IASA patients. Whether this is a result of an intramitochondrial defect (e.g. Fe accumulation, secondary to mitochondrial or nuclear DNA mutations) or is secondary to an extracellular stimulus [e.g. tumour necrosis factor (TNF), Fas ligand (FasL)] remains to be determined.

The thyroid hormone receptor functions as a ligand-operated developmental switch between proliferation and differentiation of erythroid progenitors.

The avian erythroblastosis virus (AEV) oncoprotein v-ErbA represents a mutated, oncogenic thyroid hormone receptor alpha (c-ErbA/ TRalpha). v-ErbA cooperates with the stem cell factor-activated, endogenous receptor tyrosine kinase c-Kit to induce self-renewal and to arrest differentiation of primary avian erythroblasts, the AEV transformation target cells. In this cooperation, v-ErbA substitutes for endogenous steroid hormone receptor function required for sustained proliferation of non-transformed erythroid progenitors. In this paper, we propose a novel concept of how v-ErbA transforms erythroblasts. Using culture media strictly depleted from thyroid hormone (T3) and retinoids, the ligands for c-ErbA/TRalpha and its co-receptor RXR, we show that overexpressed, unliganded c-ErbA/ TRalpha closely resembles v-ErbA in its activity on primary erythroblasts. In cooperation with ligand-activated c-Kit, c-ErbA/ TRalpha causes steroid-independent, long-term proliferation and tightly blocks differentiation. Activation of c-ErbA/ TRalpha by physiological T3 levels causes the loss of self-renewal capacity and induces synchronous, terminal differentiation under otherwise identical conditions. This T3-induced switch in erythroid progenitor development is correlated with a decrease of c-ErbA-associated histone deacetylase activity. Our results suggest that the crucial role of the mutations activating v-erbA as an oncogene is to 'freeze' c-ErbA/ TRalpha in its non-liganded, repressive conformation and to facilitate its overexpression.