The dominant negative ß isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRß isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRß was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRß mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and ß-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and ß-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRß. These data indicate that GRß expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Dynamic regulation of Gata1 expression during the maturation of conventional dendritic cells.

OBJECTIVES: To identify the regulatory sequences driving Gata1 expression in conventional dendritic cells (cDC). MATERIALS AND METHODS: The number and expression levels of Gata1, Gata1-target genes and hypersensitive site (HS) 2 (the eosinophil-specific enhancer)-driven green fluorescent protein (GFP) reporter of cDCs from mice lacking HS1 (the erythroid/megakaryocytic-specific enhancer, Gata1(low) mutation) and wild-type littermates, as well as the response to lipopolysaccharide of ex vivo-generated wild-type and Gata1(low) DCs were investigated. RESULTS: cDC maturation was associated with bell-shaped changes in Gata1 expression that peaked in cDCs precursors from blood. The Gata1(low) mutation did not affect Gata1 expression in cDC precursors and these cells expressed the HS2-driven reporter, indicating that Gata1 expression is HS2-driven in these cells. By contrast, the Gata1(low) mutation reduced Gata1 expression in mature cDCs and these cells did not express GFP, indicating that mature cDCs express Gata1 driven by HS1. In blood, the number of cDC precursors expressing CD40/CD80 was reduced in Gata1(low) mice, while CD40(pos)/CD80(pos) cDC precursors from wild-type mice expressed the HS2-GFP reporter, suggesting that Gata1 expression in these cells is both HS1- and HS2-driven. In addition, the antigen and accessory molecules presentation process induced by lipopolysaccharide in ex vivo-generated wild-type DC was associated with increased acetylated histone 4 occupancy of HS1, while ex vivo-generated Gata1(low) cDCs failed to respond to lipopolysaccharide, suggesting that HS1 activation is required for cDC maturation. CONCLUSION: These results identify a dynamic pattern of Gata1 regulation that switches from an HS1 to an HS2-dependent phase during the maturation of cDCs associated with the antigen-presentation process in the blood.

Compensated variability in the expression of globin-related genes in erythroblasts generated ex vivo from different donors.

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced. STUDY DESIGN AND METHODS: Immature erythroblasts (IEs) were expanded in human erythroid massive amplification cultures from blood mononuclear cells of 19 normal donors and four beta(0)-thalassemia patients (for comparison) and induced to mature for 4 days in the presence of erythropoietin. mRNA was prepared from IEs and mature erythroblasts to evaluate the expression of alpha-, beta-, and gamma-globin genes and of adult hemoglobin-stabilizing protein (AHSP) and BCL11A, two proteins directly controlling globin function and/or production. Results were analyzed using Pearson's correlation coefficient, the Wilcoxon signed rank, and the Mann-Whitney rank sum tests. RESULTS: The absolute levels of globin, AHSP, and BCL11A mRNA expressed by erythroblasts generated ex vivo from normal donors were distributed along a 2-log range. With maturation, the levels of gamma-globin and BCL11A mRNA did not decrease while those of alpha-globin, gamma + beta-globins, and AHSP mRNA greatly increased. In normal cells, the modest imbalance (two- to fourfold) observed between alpha- and gamma + beta-globin mRNA was fully compensated by AHSP expression. Thus, the levels of alpha-globin mRNA were correlated with those of gamma + beta-globin (R(2) = 0.93, p < 0.0001) and AHSP (R(2) = 0.86, p < 0.0001). CONCLUSIONS: Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of alpha-globin and gamma + beta-globin fully compensated by AHSP expression, likely ensuring normal function and survival.

Identification of two new synthetic histone deacetylase inhibitors that modulate globin gene expression in erythroid cells from healthy donors and patients with thalassemia.

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.

Protein kinase Calpha is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the (A)gamma globin-promoter in cellular models of hemoglobin switching.

PKCalpha was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

Variegation of the phenotype induced by the Gata1low mutation in mice of different genetic backgrounds.

All mice harboring the X-linked Gata1low mutation in a predominantly CD1 background are born anemic and thrombocytopenic. They recover from anemia at 1 month of age but remain thrombocytopenic all their life and develop myelofibrosis, a syndrome similar to human idiopathic myelofibrosis, at 12 months. The effects of the genetic background on the myelofibrosis developed by Gata1low mice was assessed by introducing the mutation, by standard genetic approaches, in the C57BL/6 and DBA/2 backgrounds and by analyzing the phenotype of the different mutants at 12 to 13 (by histology) and 16 to 20 (by cytofluorimetry) months of age. Although all the Gata1low mice developed fibrosis at 12 to 13 months, variegations were observed in the severity of the phenotype expressed by mutants of different backgrounds. In C57BL/6 mice, the mutation was no longer inherited in a Mendelian fashion, and fibrosis was associated with massive osteosclerosis. Instead, DBA/2 mutants, although severely anemic, expressed limited fibrosis and osteosclerosis and did not present tear-drop poikilocytes in blood or extramedullary hemopoiesis in liver up to 20 months of age. We propose that the variegation in myelofibrosis expressed by Gata1low mutants of different strains might represent a model to study the variability of the clinical picture of the human disease.

Isolation of TPO-dependent subclones from the multipotent 32D cell line.

Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.

Ex vivo amplification of T cells from human cord blood.

The current status of immunotherapy for cancer is here summarized with particular attention to the new methodologies developed for ex vivo expansion of T cells from neonatal cord blood. Umbilical cord blood (UCB) mononuclear cells (MNC) generate CD45RA naive T lymphocytes when cultured under serum-deprived conditions with appropriate combinations of growth factors. These ex vivo generated T cells resemble precursors for the lymphoid lineage present in adult bone marrow in terms of active transcription of RAG-2 and pTalpha.

A pathobiologic pathway linking thrombopoietin, GATA-1, and TGF-beta1 in the development of myelofibrosis.

Idiopathic myelofibrosis (IM) is a disease characterized by marrow fibrosis, abnormal stem/progenitor cell trafficking, and extramedullary hematopoiesis frequently associated with alterations in megakaryocytes (Mks). Mice harboring genetic alterations in either the extrinsic (ectopic thrombopoietin expression, TPO(high) mice) or intrinsic (hypomorphic GATA-1 mutation, GATA-1(low) mice) control of Mk differentiation develop myelofibrosis, a syndrome similar to IM. The relationship, if any, between the pathobiologic mechanism leading to the development of myelofibrosis in the 2 animal models is not understood. Here we show that plasma from GATA-1(low) mice contained normal levels of TPO. On the other hand, Mks from TPO-treated wild-type animals (TPO(high) mice), as those from GATA-1(low) animals, had similar morphologic abnormalities and contained low GATA-1. In both animal models, development of myelofibrosis was associated with high transforming growth factor beta1 (TGF-beta1) content in extracellular fluids of marrow and spleen. Surprisingly, TPO treatment of GATA-1(low) mice restored the GATA-1 content in Mks and halted both defective thrombocytopoiesis and fibrosis. These data indicate that the TPO(high) and GATA-1(low) alterations are linked in an upstream-downstream relationship along a pathobiologic pathway leading to development of myelofibrosis in mice and, possibly, of IM in humans.

Expression of signal transduction proteins during the differentiation of primary human erythroblasts.

The high number (>10(8-10)) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72-96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC beta4 was not expressed, PLC beta2, delta1, and gamma2 were expressed at constant levels throughout the maturation process, while expression of PLC beta3 and of PLC gamma1 decreased, as PI-3K, by 24 h and that of PLC beta1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC beta1, beta3, and gamma1) might be involved in the control of erythroid differentiation in humans.