βMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in β thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2213-2220 ◽  
Author(s):  
Selda Samakoglu ◽  
Elena Fattori ◽  
Stefania Lamartina ◽  
Carlo Toniatti ◽  
Daniel Stockholm ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Globin Chain ◽  
Dna Encoding ◽  
Globin Mrna ◽  
Naked Dna ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
Fetal Erythropoiesis

Abstract Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in β thalassemic C57Bl/6Hbbth mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to β thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound α chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect βminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased βminor/α-globin chain synthesis ratio, reduced levels of α chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased βminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with β thalassemia.

scholarly journals Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 1-10
Author(s):  
EJ Jr Benz ◽  
PS Swerdlow ◽  
BG Forget
Keyword(s):  
Messenger Rna ◽  
Globin Chain ◽  
Globin Mrna ◽  
Free System ◽  
Gamma Globin ◽  
Globin Chains ◽  
Mouse Ascites ◽  
Chain Synthesis ◽  
A Chain ◽  
Globin Chain Synthesis

Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, andone patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.

scholarly journals Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 1-10 ◽  
Author(s):  
EJ Jr Benz ◽  
PS Swerdlow ◽  
BG Forget
Keyword(s):  
Messenger Rna ◽  
Globin Chain ◽  
Globin Mrna ◽  
Free System ◽  
Gamma Globin ◽  
Globin Chains ◽  
Mouse Ascites ◽  
Chain Synthesis ◽  
A Chain ◽  
Globin Chain Synthesis

Abstract Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, andone patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.

Studies of Globin Chain Synthesis and Globin mRNA Content in a Patient Homozygous for Hemoglobin Lepore

Hemoglobin ◽  
1978 ◽  
Vol 2 (2) ◽  
pp. 117-128 ◽  
Author(s):  
B. G. Forget ◽  
C. Cavallesco ◽  
E. J. Benz ◽  
P. D. McClure ◽  
D. G. Hillman ◽  
...  
Keyword(s):  
Globin Chain ◽  
Globin Mrna ◽  
Mrna Content ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

The LSD1 Inhibitor RN-1 Recapitulates the Fetal Pattern of Hemoglobin Synthesis in Anemic and Normal, Non-Anemic Baboons

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 642-642
Author(s):  
Angela Rivers ◽  
Kestis Vaitkus ◽  
Vinzon Ibanez ◽  
Maria Armila Ruiz ◽  
Ramaswamy Jagadeeswaran ◽  
...  
Keyword(s):  
Post Treatment ◽  
Globin Genes ◽  
Globin Chain ◽  
Cell Disease ◽  
Globin Mrna ◽  
F Cells ◽  
Globin Promoter ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
Dose Dependent

Abstract Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease (SCD). Hydroxyurea (HU), the only drug approved for the treatment of sickle cell disease, increases HbF levels, reduces pain crises, and increases the lifespan of patients. Because HU is not effective in a large proportion of patients, new pharmacological agents that increase HbF levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in SCD mice. As the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and baboon, the baboon remains the best predictor of the activity of HbF-inducing agents in man. Therefore, experiments were performed to test the effect of RN-1 on HbF in anemic baboons. Anemia (Hct=20) was induced by repeated phlebotomies for 14 days prior to administration of RN-1. Five anemic baboons were treated with varying doses of RN-1(0.125-2.5 mg/kg/d; 5d; sc). Dose-dependent increases in HbF, F cells, F retics, and γ-globin chain synthesis in reticulocytes were associated with decreased neutrophils and increased monocytes. Globin chain synthesis analysis following RN-1 treatment showed that the 5' Iγ and 3' Vγ-globin genes were expressed in the ratio characteristic of fetal development (5'Iγ/3'Vγ>2) rather than that induced in adults by erythropoietic stress (5'Iγ/3'Vγ<0.5) or decitabine treatment where changes in the Iγ/Vγ ratio are observed but the fetal ratio is not attained. RT-PCR analysis of pre-and post-treatment BM showed that RN-1 increased γ-globin mRNA nearly 5 fold (p<0.05) with no effect on ε-globin mRNA. ChIP analysis of pre- and post-treatment BM erythroid cells showed increased levels of pol II, H3K9ac, H3K4me2 and H3K4me3 associated with the γ-globin but not with the ε- or β-globin promoter and IVS II regions consistent with increased γ-globin transcription. Levels of H3K9me2 were increased at the β-globin promoter and IVSII regions. Bisulfite sequence analysis showed a small decrease in level of DNA methylation of the γ-globin promoter in post-treatment (0.68+0.07% total cytosine) compared to pre-treatment (0.79+0.05%; p<0.05). To investigate whether induction of γ-globin expression was dependent on erythropoietic stress, four normal, non-anemic baboons were treated with varying doses and schedules of RN-1 (0.2-0.5mg/kg/d; 5d/wk; 1-10wks; sc). The effect of RN-1 was measured by analysis of F cells and globin chain synthesis in peripheral blood. The results (Table) show that RN-1 increases γ-globin expression and F cells in normal, non-anemic baboons in a dose-dependent manner. Analysis of globin chain synthesis showed predominant synthesis of the Iγ-globin chain with an Iγ/Vγ ratio exceeding the fetal ratio in all individuals. Effect of RN-1 in Normal, Non-Anemic Baboons Table.AnimalRN-1 Dose (mg/kg/d)ScheduleGlobin chain synthesis (γ/γ+β)Iγ/Vγ chain synthesisF cells (%)ANC (/μl)Plt(X103/μl)Mono(%)PrePostPrePostNadirNadirPeak85490.55d0.030.714.63NDND191016921.685490.255d0.010.264.47NDND191021914.586980.255d/wk/2 wks0.010.2020.12.09.21210687.986960.2-0.255d/wk/10 wksND0.106.633.526.81440646.1 Neutropenia following RN-1 treatment of anemic baboons (mean ANC nadir=746±156) was reduced in non-anemic animals (2 of 4 baboons >1500; mean ANC nadir=1617±350; p<0.02). Increased monocytes and decreased platelet counts were observed. Differences between anemic and non-anemic baboons may reflect perturbation of hematopoietic differentiation by phlebotomy. We conclude that RN-1 is a powerful in vivo inducer of HbF in both anemic and non-anemic baboons that preferentially increases synthesis of the 5' Iγ-globin gene and recapitulates the fetal pattern of hemoglobin synthesis. Our data predicts that RN-1 treatment will induce clinically relevant levels of HbF in SCD patients, although careful titration of dose may be required to minimize effects on hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Butyrate increases the efficiency of translation of γ-globin mRNA

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1807-1809 ◽  
Author(s):  
Rona S. Weinberg ◽  
Xinjun Ji ◽  
Millicent Sutton ◽  
Susan Perrine ◽  
Yelena Galperin ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Peak Level ◽  
Mrna Levels ◽  
Globin Genes ◽  
Globin Mrna ◽  
Multiple Treatment ◽  
Rapid Induction ◽  
Polysomal Fraction ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

AbstractFetal hemoglobin (Hb F) levels increase in most patients with sickle cell disease following intermittent butyrate therapy. Although the full effects of butyrate on Hb F levels usually require multiple treatment cycles, in some patients a peak level is achieved after a few days of butyrate therapy. Our investigation of the mechanism(s) responsible for this rapid induction of Hb F by butyrate showed that reticulocyte γ-globin chain synthesis markedly increased within 24 hours of butyrate exposure, without concomitant changes in reticulocyte γ-globin mRNA levels. This suggests that butyrate might induce Hb F by increasing the efficiency of translation of γ-globin mRNA. This hypothesis was confirmed by ribosome loading studies that demonstrated enrichment of the polysomal fraction of reticulocytes with γ-globin mRNA following butyrate exposure. Thus, the induction of Hb F by butyrate may be mediated by translational effects in addition to its well-known effects on transcription of the γ-globin genes. (Blood. 2005;105:1807-1809)

γ-Globin Gene (HBG) Promoter Methylation and Gene Expression Is Affected by the Micro-Environment In Adult but Not Fetal Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4271-4271
Author(s):  
Donald Lavelle ◽  
Yogenthiran Saunthararajah ◽  
Nadim Mahmud ◽  
Vinzon Ibanez ◽  
Maria Armila Ruiz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Globin Gene ◽  
Feeder Layer ◽  
Erythroid Cells ◽  
Globin Chain ◽  
Globin Mrna ◽  
Feeder Layers ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract Abstract 4271 Background: During adult erythroid differentiation, there is a period of transient γ-globin (HBG) gene expression that coincides with transient DNA hypomethylation of the HBG gene promoter. This precedes the final pattern of high β-globin (HBB) gene expression in terminally differentiated adult erythroid cells (the ‘maturational switch’). Possibly, the micro-environment can modulate the period of transient HBG promoter hypomethylation and gene expression, which could have implications for efforts to sustain HBG expression for therapeutic objectives. To evaluate the responsiveness of the maturational switch to external factors, CD34+ hematopoietic progenitor cells from adult baboon bone marrow were cultured with and without AFT024 murine fetal liver stromal cell line feeder layers, as the micro-environmental variable. Methods: All cultures contained Iscove's media with 30% fetal bovine serum, 200ng/ml stem cell factor, 2U/ml erythropoietin, and 1μM dexamethasone. Globin chain synthesis was measured by biosynthetic radiolabelling of globin chains on d11 and d14 followed by HPLC separation, HBG transcript levels were determined by real time PCR, and F cells were analyzed by flow cytometry. Results: γ-globin chain synthesis was significantly lower in the presence of AFT024 feeder layers (0.07±0.05 γ/γ+β, n=4 versus 0.34±0.09 γ/γ+β, n=4, mean±SD, p<0.01). HBG mRNA was also significantly lower in the presence of AFT024 feeder layers (0.09±0.05 γ/total β-like globin mRNA, n=3, versus 0.32±0.10 γ/total β-like globin mRNA, n=3, p<0.025). Accordingly, F cell numbers were substantially decreased in the presence of feeder layers (38 versus 64%). Cell growth was similar in the presence and absence of feeder layers (115±48 versus 86±8.5 fold expansion, n=3). To test whether decreased γ-globin expression in the presence of ATF024 feeder cells is secondary to preferential expansion of more differentiated progenitors less capable of HBG expression, BM cells were fractionated into CD34+ CD36+ and CD34+ CD36- subpopulations prior to culture with and without feeder layers. Expression of HBG in both subpopulations was similarly reduced by the feeder layers, suggesting that decreased HBG expression was due to a direct effect of the feeder layer rather than to selective expansion of a more differentiated subpopulation. Bisulfite sequence analysis was performed to determine if differences in HBG expression were associated with differences in DNA methylation. DNA methylation of 5 CpG residues in the HBG promoter in purified erythroid cells from two independent cultures was 97.6 and 97.7% in the presence of feeder layers and 68.6 and 69.7% without feeder layers. Addition of the DNA methyltransferase inhibitor decitabine (0.5 × 10-6 M), to cells cultured in the presence of feeder layers increased γ-globin synthesis nearly tenfold (0.60 γ/γ+β) compared to untreated controls (0.063 γ/γ+β), consistent with mechanistic role for DNA methylation in HBG repression associated with feeder layer culture. In contrast to the results with adult-derived CD34+ BM cells, cord blood derived CD34+ cells sustained high levels of HBG expression (0.84 γ/γ+β)in the presence of feeder layers. Conclusion: The switch from HBG to HBB gene expression that occurs during the differentiation of adult erythroid cells is responsive to the micro-environment. Furthermore, this switch depends on DNA methylation, and depleting DNMT1 counteracts the micro-environmentally induced switch to sustain γ-globin expression in adult erythroid cells. In contrast to adult erythroid cells, fetal erythroid cells were resistant to micro-environmental induction of a HBG to HBB switch, consistent with previous studies showing that intrinsic differences between adult and fetal erythroid cells are a critical component in developmental stage-specific differences in globin gene expression. Disclosures: No relevant conflicts of interest to declare.

Globin Chain Synthesis in Haemoglobin New York (β 113 Valine→Glutamic acid

1980 ◽  
Vol 46 (4) ◽  
pp. 557-564 ◽  
Author(s):  
D. Todd ◽  
Vivian Chan ◽  
Rose G. Schneider ◽  
Andree M. Dozy ◽  
Y. W. Kan ◽  
...  
Keyword(s):  
New York ◽  
Glutamic Acid ◽  
Globin Chain ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

scholarly journals Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor

Blood ◽  
1978 ◽  
Vol 51 (4) ◽  
pp. 653-658 ◽  
Author(s):  
RS Franco ◽  
JW Hogg ◽  
OJ Martelo
Keyword(s):  
Globin Chain ◽  
Free System ◽  
Globin Chains ◽  
Reticulocyte Lysate ◽  
Chain Imbalance ◽  
Globin Synthesis ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.

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