Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited ...diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039‐thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well‐described NMD (nonsense‐mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base‐pairing, inducing a ribosomal read‐through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read‐through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039‐thalassaemia. In this context, we started the development of a cellular model of the β039‐thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039‐thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β‐globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039‐globin mutation causing β‐thalassaemia.
Ineffective erythropoiesis (IE) in β-thalassemia has been attributed to erythroid cell death mediated by apoptosis or hemolysis during the maturation process. Historically, ferrokinetic studies in ...this disease suggested that 60%–80% of erythroid precursors die in the marrow or extramedullary sites. However, several observations have challenged this view. First, the number of apoptotic erythroid cells in patients is low compared to net expansion of the erythroid cell pool. Second, hemolytic markers in β-thalassemic patients are normal or only slightly increased, unless additional pathological conditions appear. Third, our most recent study (Blood, Gardenghi et al, 2007 Jun 1) demonstrated that GI iron absorption in β-thalassemia is increased by the dysregulation of genes such as hepcidin and ferroportin that control iron absorption, resulting in iron levels that exceed the amount required for erythropoiesis. We have undertaken a detailed investigation using cohorts of mice (n>30 per genotype) with β-thalassemia intermedia (th3/+) and major (th3/th3). Using these models, we have previously shown that the severity of anemia (as low as 1 g/dL) inversely correlates with the total number of nucleated erythroid cells (»100 fold compared to wild-type (wt) mice). Cytological analysis has clearly shown that thalassemic spleen specimens were comprised of a homogeneous pre-erythroblastic population. In contrast, the percentage of apoptotic cells and the level of hemolytic markers, such as bilirubin and lactic acid dehydrogenase, slightly increased or were not different compared to wt mice. While not excluding a role for apoptosis, our observations suggest that control of the cell cycle and maturation of erythroid precursors play an important role in IE. We then explored whether the erythroid cell cycle was dysregulated in our model system. We found that erythropoietin (Epo) levels were raised in thalassemic animals by as much as three orders of magnitude. Binding of Epo to its receptor (EpoR), activates antiapoptotic and cell cycle promoting genes, through activation of Jak2 and Stat5. By Western blot we demonstrated up-regulation of EpoR, Stat5 and the antiapoptotic protein BclXL, as well as that of proliferation promoting genes, such as CycA and Cdk2, in purified thalassemic erythroid cells compared to those of wt animals. This data was confirmed by staining both wt and thalassemic liver and spleen sections using the proliferation markers Ki67 and Mcm3, by clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after BrdU injection. In the latter case, we observed 22%, 30% and 44% BrdU+ cells from wt, th3/+ and th3/th3 mice, respectively. In addition, freshly purified thalassemic erythroid cells proliferate faster in vitro than normal cells, a phenomenon blocked by AG490, a Jak2 inhibitor. Significantly, we have been able to reproduce results from our animal studies in humans, comparing normal and thalassemic blood and spleen specimens. In conclusion, we propose that IE in β-thalassemia is likely to be the result of altered cell proliferation and impaired cell differentiation, which in turn limit apoptosis, thereby mimicking tumor-like behavior.
β-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β-thalassaemia we observed that the liver expresses relatively ...low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. We hypothesized that despite the overt iron overload, a putative plasma factor found in β-thalassaemia might suppress liver hepcidin expression. We therefore compared sera from β-thalassaemia and haemochromatosis (C282Y mutation) patients with those of healthy individuals in terms of their capacity to evoke changes in expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β-thalassaemia major patients evoked a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared to sera from healthy individuals. Significant correlation was found between the degree of downregulation of HAMP and HFE2 evoked by b-thalassaemia major sera (r=0.852, p<0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera collected from β-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients evoked an increase in HAMP expression, which correlated with their transferrin saturation (r=0.765, p<0.0099). Our results suggest that in β-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption.
Ineffective erythropoiesis (IE) in β-thalassemia is described as increased expansion of erythroid progenitor cells in combination with accelerated apoptosis and intramedullary hemolysis. However, ...evidence for this assumption is not particularly strong. In this study we evaluated the kinetics of red blood cell proliferation and survival in thalassemic mice that exhibit levels of anemia consistent with thalassemia intermedia (th3/+) and major (th3/th3), as we described previously. Th3/+ mice show anemia and increased reticulocyte counts (8.9±1.1 g/dL and 17.7±2.6x105/ul compared to 14.9±1.1 g/dL and 2.6±0.4x105/ul in +/+), whereas th3/th3 mice show more severe anemia than th3/+ mice and reduced production of reticulocytes (3.0±1.2 g/dL and 1.8±0.7x105/ul). As soon as two months of age, EPO levels in thalassemic mice are significantly increase over normal mice, 10 times in th3/+ and up to three orders of magnitude in th3/th3 mice. The total number of nucleated erythroid cells (spleen + bone marrow) increased from 3.3±0.9x108 in +/+ to 1.2±0.5x109 (or 3.6 folds) in th3/+ and 1.6±0.6x109 (or 4.8 folds) in th3/th3 mice (N=5 per group), whereas the level of apoptotic cells increased only 2 to 3 folds in percentage in th3/+ and th3/th3, respectively, as observed using the apoptotic Annexin-V and erythroid specific Ter119 markers (9.9±5.0, 14.3±2.5, 24.2±6.7 in BM and 10.6±3.5, 14.8±6.5, 25.4±6.2 in spleen of +/+, th3/+ and th3/th3, respectively; n=3 per genotype). This data was confirmed by TUNEL, cleaved caspase-3/7 and by immunostaining assays. Bilirubin and LDH levels were not different between thalassemic and +/+ mice. Altogether these observations indicated that in thalassemia there is a disproportion between number of proliferating and dying cells with a net increase of erythroid cells. Furthermore, microarray analysis on erythroid cells indicated increased expression of cell cycle promoting genes such as Ki67, Mcm3, Cyclin-A, CDK2 and BclXL (2 to 6 folds compared to +/+ mice, n=5 per genotype). This data was confirmed by Q-PCR, Western blot, immunostaining, clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after in vivo BrdU injection (22, 30 and 44% BrdU+ in +/+, th3/+ and th3/th3, respectively). On the other hand, in th3/th3, which show more apoptosis than th3/+ mice, the cyclin-dependent kinase inhibitor p21 was upregulated both at the RNA (50-folds) and protein level. P53 was also analyzed in th3/th3 mice, showing no expression. In order to investigate the function of p21 in thalassemic erythroid cells, its expression was analyzed on purified erythroid cells isolated from th3/th3 that were transfused (10.4±0.4 g/dL of Hb) or thalassemic mice showing different levels of anemia (6.2±0.2 and 2.1±0.8 g/dL of Hb, respectively; N=3 per each group). We observed that the level of p21 increased with anemia and the severity of the pathology. However, in th3/th3 mice that were injected with BrdU, immunostaining analysis indicated that a large amount of p21+ erythroid cells were also BrdU+. In conclusion, we propose that erythropoiesis in β-thalassemia is characterized by enhanced expression of cell cycle promoting and survival factors that are able to overcome or mitigate p21 cell cycle block and, probably, apoptosis.
beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing ...their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ- PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb super(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb super(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia. Am. J. Hematol. 81:479-483, 2006.
Progressive iron overload occurs in β-thalassemia as a result of increased gastrointestinal absorption. Our goal is to investigate the relationship between ineffective erythropoiesis (IE), ...iron-related genes and organ iron distribution in mice that exhibit levels of anemia consistent with thalassemia intermedia (th3/+) and major (th3/th3), as we described previously. The th3/th3 mice die in 8 weeks due to severe anemia but can be rescued by transfusion therapy. We analyzed up to 90 animals at 2, 5 and 12 months, as appropriate. We monitored various hematological parameters, tissue iron content and quantitative-PCR levels of Hamp, Fpn1, Smad4, Cebpa, Hfe, Tfr1 and other genes involved in iron metabolism in liver, spleen, kidney, heart and duodenum. At 2 months, th3/th3 mice had the highest total body iron content and highest degree of IE. The total iron was 53.6±21.0, 406.1±156.1, 657.7±40.3 μg in the spleen, and 107.5±35.7, 208.5±24.9 and 1298.7±427.5 μg in the liver of +/+, th3/+ and th3/th3, respectively (n≥5 per genotype). However, if the organ size was not taken in account, the iron concentration in the spleen of th3/+ was higher, in average, than that of th3/th3 mice (3.8±1.5 and 2.9±0.5 μg/mg), while in the liver was the opposite (0.6±0.1 and 5.1±2.0 μg/mg of dry weight, P<0.001). Heme and non-heme iron analyses provided similar results. Surprisingly, the distribution of iron within organs also differed. In th3/+ mice, the hepatic iron was almost exclusively located in Kupffer cells, whereas in th3/th3 mice in parenchymal cells. Our data suggest that Hamp is responsible for the increased iron absorption, being reduced to 20% and 70% in 2 month-old th3/+ and th3/th3 mice compared to +/+ animals (P<0.001). Hfe was reduced by 50% (P<0.05) in the liver of the animals that expressed low Hamp levels, indicating that Hfe could be directly responsible for Hamp regulation or share the same regulatory pathway. Low levels of Smad4 and Cebpa were observed only in the liver of mice with the lowest Hamp expression (P<0.05), indicating that these proteins might contribute to further decreased Hamp synthesis. In addition, while Tfr1 in th3/+ mice was 40% lower in the liver, it was up-regulated (400%) in th3/th3 mice (P<0.001), which may explain why iron is increased more in the liver of th3/th3 mice. In 5 and 12 month-old th3/+ mice, the surprising observation was the normal expression level of Hamp. However, in the duodenum, the Fpn1 RNA and protein levels were augmented (300%, P<0.001). In transfused th3/+ and th3/th3 animals, Hamp, Hfe, Cbpa and Smad4 expression levels were normalized or increased, while Tfr1 was down-regulated in both groups, which may explain the increased splenic iron deposition in these animals. Our data suggest that IE, together with the relative expression levels of Hamp and Tfr1, is largely responsible for the organ iron overload observed in young thalassemic mice. However, in older mice, it is the increase of Fpn1 levels in the duodenum that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the genesis of iron overload in β-thalassemia.
We generated the first transplantable adult mouse models of beta-thalassemia intermedia and major by infusing mouse hematopoietic-fetal-liver cells (HFLC) heterozygous or homozygous for a deletion of ...the beta-globin gene (respectively with th3/+ and th3/th3 cells) into lethally irradiated congenic C57BL/6 mice. Six to 8 weeks post transplantation, mice transplanted with th3/+ HFLCs show 7 to 9 g/dL of hemoglobin levels, splenomegaly, abnormal red cells and increased iron overload. Mice transplanted with th3/th3 HFLCs, unless blood transfused, die 8 to 10 weeks after engraftment showing profound anemia, massive splenomegaly and very rapid and dramatic iron overload. For this reason, we began a systematic study to compare iron content and the expression level of iron related genes in normal and thalassemic mice of varying ages and sex in different organs (liver, duodenum, spleen, kidney and heart). In liver, we observed that iron content increases proportionally with the level of anemia, age and if the blood transfusion is included. We are currently analyzing the other organs. The expression of hepcidin, ferroportin, Hfe, ferritin, transferrin, transferrin-receptor 1 and 2, ceruloplasmin, divalent metal transporter 1 and hemojuvelin are being tested also in all these organs. In particular, we observed that hepcidin is dramatically downregulated in liver of beta-thalassemic animals. Our hypothesis is that low expression of this gene leads to high iron content in these animals. We intend to demonstrate that administration or increasing hepcidin levels of this peptide can prevent iron absorption in beta-thalassemia. We developed two alternative strategies to test our hypothesis. In the first one, we synthesized the active form of the mouse hepcidin peptide that will be administered intraperitoneally to mice affected by beta-thalassemia. In the second, lentiviral vectors have been generated in order to constitutively secrete hepcidin in the bloodstream of animals affected by beta-thalassemia. These vectors were introduced into hematopoietic stem cells derived from mouse embryos of normal and mice affected by beta-thalassemia and engrafted in myeolablated normal mice. The engrafted mice express hepcidin 6 weeks post transplantation by RT PCR. These animals, along with the animals in which hepcidin will be administrated intraperitoneally, will be analyzed at the endpoint of the experiment (> 4 months) for their hematological values and iron content to see if the use of hepcidin can be used to prevent excessive iron absorption in beta-thalassemia.
The ability to genetically modify embryonic stem cells (ESC) expands the potential of ESC to correct hereditary disorders and deliver gene products for specific therapies. Integrating gene transfer ...vectors, such as lentivirus vectors, have been used to deliver genes to ESC, but the transduction efficiencies are far from 100% and selection of transduced cells using drug resistance is often unpredictable and toxic. A surface marker delivered by a lentivirus vector that could be employed in a magnetic-bead purification strategy would be a powerful tool in selecting transduced from untransduced ESC. To address these issues, we hypothesized that: (1) lentivirus transfer of the truncated human low-affinity nerve growth factor (LNGFR), a non-toxic, non-tumorigenic and non- functional protein expressed on the surface of transduced cells, could be used to isolate transduced ESC; (2) LNGFR expression would not alter the characteristics of the undifferentiated ESC; and (3) LNGFR expression would not interfere with the response of ESC to differentiation signals. To assess LNGFR as a purification strategy for ESC, we utilized a lentivirus vector with a human phosphoglycerate kinase 1 (PGK) promoter to drive expression of LNGFR. Murine ESC (C57BL/6 strain) were transduced with this vector and cultured on a murine embryonic fibroblast (MEF) feeder layer. Flow cytometry analysis (FACS) 48 hr after transduction revealed LNGFR expression in 44% of the ESC and 2 % in the corresponding mouse IgG1-PE control. On day 4, LNGFR+ ESC were purified with magnetic beads using an anti-LNGFR antibody. After single purification with magnetic beads, 95% of the transduced cells were LNGFR+. Several different assessments demonstrated that LNGFR expression did not alter the characteristics of the purified undifferentiated ESC. First, LNGFR+ cells plated onto a MEF feeder layer remained undifferentiated for 2 wk and the expression pattern of SSEA1 (FACS analysis), a marker for undifferentiated ESC, was identical in mock and LNGFR+ ESC (p=0.7). Second, embryoid body formation was identical in LNGFR+ and mock transduced ESC. Third, after subcutaneous administration of purified LNGFR+ or mock transduced ESC, teratomas, tumors arising from undifferentiated ESC, developed within 2 wk. Finally, to determine whether LNGFR expression would interfere with the response of ESC to differentiation signals, differentiation to endoderm was induced by Activin A. After 5 days, FACS analysis for CXCR4, a marker for definitive endoderm, showed similar levels of expression in LNGFR+ ESC and the control group (20.4% vs 21.2%), suggesting that LNGFR expression does not interfere with differentiation to endoderm. Together, these observations demonstrate that lentivirus- mediated transfer of LNGFR is a useful tool in purifying transduced ESC without deleterious or toxic effects on maintenance or differentiation.
We have recently shown that hepcidin expression undergoes a significant down regulation in the liver of a thalassaemia intermedia mouse model (TIM) C57Bl/6 Hbbth3/+. (Adamsky K. et al. BJH 2004; ...124(1):123–4). We extended these studies to a b-thalassemia major mouse model (TMM) generated via engraftment with beta-globin-null (Hbbth3/th3) fetal liver cells. The resulting phenotype displayed considerably more severe symptoms than the TIM: the TMM succumbed to ineffective erythropoiesis within 60 days, developed massive splenomegaly, severe anemia, extramedullary hematopoiesis and hepatic iron overload. The expression levels of various iron metabolism-related genes (normalized to b-actin) were analyzed by quantitative RT-PCR on RNA extracted from the livers of adult mice. When compared to wild-type (WT) C57Bl/6 mice, the liver mRNA expression levels of TMM were markedly reduced for hepcidin and TfR2 (16 and 3 fold respectively), markedly increased for the lipocalin NGAL and transferrin receptor 1 (TfR1) (2.7 and 3.6 fold respectively), moderately increased for the ferroportin transporter (IREG1) (1.4 fold) and unaltered for the hemochromatosis gene (HFE). A possible mechanism that could explain the decreased expression of liver hepcidin in thalassaemia is one based on a putative regulatory serum factor that is associated with enhanced erythropoietic activity. In order to assess this hypothesis we compared the hepcidin inductive capacity of sera from iron-overloaded patients that either had or had not enhanced erythropoiesis, namely thalassemia and hemochromatosis, respectively. These included the following individuals: 14 with β-thalassemia major, 22 with hereditary hemochromatosis (HFE 282C mutation) and 3 healthy. The human sera were analyzed in terms of their capacity to modulate expression of iron-related genes in human hepatoma HepG2 cells, using quantitative RT-PCR. Hepcidin expression evoked by thalassemic sera was an average of 3 fold lower than that evoked by normal human serum, whereas hemochromatotic sera evoked an average of 7.83 fold increase. The down regulating effect of thalassemic sera on hepcidin expression, suggests the possible involvement of an upstream factor whose serum levels might increase in thalassemia due to ineffective erythropoiesis, i.e. an “erythropoietic regulator”. The effect of such an “erythropoietic regulator” is assumed to override the expected increase in hepcidin expression that results from the “stores regulator” which is responsive to iron overload such as in hemochromatosis (where there is no ineffective erythropoiesis). The reduced hepcidin expression found in thalassaemia might explain the increased enteric iron absorption whose extent could be moderated either by factors that increase hepcidin expression or by administration of hepcidin itself.
Anemias are a numerous and diverse group of disorders, ranging from limited erythroid precursor production to premature senescence of RBCs. In many cases, anemia is associated with hypoproliferative ...erythropoiesis (HE), while in other forms, with ineffective erythropoiesis (IE). HE leads to a reduction of the erythron and consequent anemia by absent or limited proliferation of the erythroid precursors. In contrast, the anemia in IE occurs despite expansion of the erythron. In some cases, IE is triggered by impaired maturation of the erythroid precursors while, in others, by hemolysis and/or premature senescence of RBCs. In some forms of hypoproliferative anemia, administration of iron and an erythropoiesis-stimulating agent (ESA), such as recombinant human erythropoietin (rhEPO), might be required. In aplastic and IE-associated anemias, blood transfusion and iron chelation may be necessary as supportive therapies.
PDF
