Elevation of Fetal Hemoglobin (HbF) by Novel Alkylating Agents in Human Erythroid Cells; Synergistic Effects with Established HbF-Inducing Agents.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3815-3815
Author(s):  
I. Chiotoglou ◽  
M. Samara ◽  
S. Likousi ◽  
S. Samara ◽  
Z. Iakovidou ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Alkylating Agents ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Cell Number ◽  
Mrna Levels ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Mrna Transcripts

Abstract Pharmacological induction of fetal hemoglobin (HbF) is beneficial for some patients with β-thalassemia and also ameliorates the severity of pain episodes in sickle cell anemia, mainly by hydroxyurea (HU). However, refractoriness or poor response of some patients treated with HU triggered research for other drugs. In the present study, we evaluated the effects of novel steroidal alkylating agents EA80, XK4 and CS on HbF induction in CD34+ cell cultures from normal donors. Furthermore, we examined the effects of these agents combined with HU, hemin (HE) and butyric acid (BU) on HbF modulation in adult erythroid cells. CD34+ cells from normal donors cultured in serum-free StemSpan medium were exposed to EPO (4 u/ml) + SCF (100ng/ml). Different concentrations of EA80 (0.1–1μM), XK4 (0.1–10 μM) and CS (0.1–1μM) were added to the cultures at day 6 (proerythroblast stage), then cells were washed and harvested 1– 3 days later. The effect of the drugs on cell number was measured by the trypan blue exclusion technique. The number of Hb-containing cells were determined using the benzidine-HCl procedure. EA80, over a wide range of concentrations (0.1–0.8μM), did not compromise cell survival. Continuous exposure of CD34+ to EA80 had a dose (up to 0.4μM)- and-time dependent effect on cell number as well as on globin mRNA levels. Treatment of CD34+ cells with EA80 at 0.4μM for 3 days was followed by a two-fold increase in cell number and a 1.5-fold in benzidine-positive cells. Qualitative and quantitative RT-PCR evaluation of globin-mRNA transcripts in CD34+ demonstrated that EA80 (0.4μM) caused a time- and dose-dependent increase in gamma globin mRNA (1– 3 days: 1.5 to 2.0-fold). The addition of HU and HE in combination with EA80 in normal CD34+ cell cultures led to a 20–30% increase in cell number by day 9. Furthermore, the combination of EA80 with either HU or HE was accompanied by an increase in γ-mRNA content (1.5 and 2.5-fold, respectively).No significant difference was detected in the level of both adult globin mRNAs. In contrast, BU addition had no effect either on erythroid cell proliferation or γ-mRNA levels. The addition of XK4 had a dose dependent effect on cell number and γ-globin mRNA transcripts [highest effect (2.0–5.0-fold) at 5 and 10μM]. Addition of EA80 (0.4μM) concurrently with varied concentrations of XK4 (0.1–10μM) caused a x2–4-fold increase in gamma globin content (highest effect at 5/10μM). For both drugs, the levels of β-and α-mRNAs in normal CD34+ cell cultures were not affected by dose. The addition of the third drug, CS at concentrations between 0.1 and 1μM proved toxic (reduced cell number and γ-globin mRNA transcripts). Our findings suggest that the beneficial effect of EA80 and XK4 might be threefold: increasing cell number affecting preferentially the rate of transcription of γ-globin mRNA, acting synergisticallly with HE and HU, most likely through transcriptional and posttranscriptional mechanisms. These results indicate that novel alkylating agents EA80 and XK4, either alone or in combination with other HbF-augmenting drugs, might provide a potentially useful treatment for patients with β-hemoglobinopathies with poor or no response to established Hb-F inducing agents.

scholarly journals Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1862-1869 ◽  
Author(s):  
P Constantoulakis ◽  
B Nakamoto ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Cell Cultures ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Fetal Sheep ◽  
Gamma Globin

Abstract Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady- state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units- erythroid (BFU-E) colonies. There was a high correlation of gamma- globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma- globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma- globin expression in adult erythroid progenitor cell cultures.

Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2161-2161
Author(s):  
Jaira F. de Vasconcellos ◽  
Y. Terry Lee ◽  
Colleen Byrnes ◽  
Laxminath Tumburu ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Hemoglobin ◽  
Cell Counting ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Empty Vector ◽  
Gene And Protein Expression

Abstract HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

Induction of Fetal Hemoglobin by Inactivation of HDAC1 or HDAC2 without Altering Cellular Proliferation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 354-354
Author(s):  
Erica B. Esrick ◽  
Jian Xu ◽  
Katherine Lin ◽  
Marie Ellen McConkey ◽  
Alyse Frisbee ◽  
...  
Keyword(s):  
Hdac Inhibitor ◽  
Cell Cycle Progression ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Hydroxyurea Treatment

Abstract Abstract 354 Histone deacetylase (HDAC) inhibitors are effective inducers of fetal hemoglobin, and prior studies have shown that selective inactivation of HDAC1 or HDAC2 is sufficient to induce fetal hemoglobin in vitro. In our current work, we demonstrate that HDAC1 and HDAC2 are attractive targets for clinical translation for two reasons: 1) Selective inhibition will decrease off-target effects that currently limit the use of hydroxyurea and pan-HDAC inhibitors, and 2) HDAC inhibitors induce fetal hemoglobin in various preclinical models, and they can be combined with hydroxyurea to achieve further fetal hemoglobin induction. To investigate off-target effects, we selectively inactivated HDAC1, HDAC2 or HDAC3 in human erythroid progenitor cells, and examined the effect of each knockdown on cellular cytotoxicity and cell cycle progression. Although knockdown of HDAC3 negatively influenced growth, selective knockdown of HDAC1 or HDAC2 had no effect on expansion of erythroid progenitors. In addition, knockdown of HDAC2 does not block cell cycle progression. These data support the possibility that an HDAC1- or HDAC2-specific inhibitor may offer a therapeutic advantage by reducing side effects, while maintaining robust HbF induction. Armed with this knockdown data, we are now investigating HDAC inhibitor compounds of various selectivity in in vitro and in vivo models. To perform optimal clinical trials, and ultimately to benefit the most sickle cell disease patients, it would be ideal to combine HDAC inhibitor treatment with hydroxyurea. A combination treatment approach may ameliorate some of the limitations of hydroxyurea use, such as the unpredictable effect on fetal hemoglobin levels, and the lack of benefit in beta thalassemia patients. First, we combined HDAC2 inactivation with hydroxyurea treatment in vitro. Human bone marrow-derived CD34+ cells were infected with lentiviruses containing an shRNA targeting either HDAC2 or a luciferase control gene. The cells were then treated on day 4 of erythroid differentiation with hydroxyurea (10–20 uM dose). Compared to the untreated luciferase control samples, we observed a 7–9-fold increase in gamma-globin expression in the untreated HDAC2-knockdown samples, a 2.5-fold increase in the hydroxyurea-treated luciferase control samples, and a trend toward an additive effect on gamma-globin induction in the cells where HDAC2 knockdown was combined with hydroxyurea treatment. To investigate the effects of HDAC inhibitors in vivo, we administered compounds to BCL11A conditional knockout transgenic mice (by erythroid-selective EpoR-GFP Cre) containing the human beta-globin locus. As reported previously, BCL11A inactivation powerfully de-repressed gamma-globin expression, and administration of an HDAC inhibitor, SAHA, led to a further elevation of gamma-globin mRNA. We now demonstrate that administration of another pan-HDAC inhibitor, panobinostat (LBH589), results in an additional 1.5- to 2.5-fold increase in gamma-globin mRNA relative to pre-treatment baseline. We are currently evaluating the combination of panobinostat and hydroxyurea in these mice to confirm that the compounds have an additive effect in vivo as well as in vitro. Taken together, these experiments indicate that inhibiting HDAC1 or HDAC2 is a promising therapeutic approach to increasing fetal hemoglobin levels in patients with beta-hemoglobinopathies, both alone and in combination with hydroxyurea. Disclosures: Bradner: Acetylon: .

scholarly journals Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1862-1869
Author(s):  
P Constantoulakis ◽  
B Nakamoto ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Cell Cultures ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Fetal Sheep ◽  
Gamma Globin

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady- state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units- erythroid (BFU-E) colonies. There was a high correlation of gamma- globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma- globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma- globin expression in adult erythroid progenitor cell cultures.

Targeted Reduction of Let-7a miRNA Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Jaira F. de Vasconcellos ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Megha Kaushal ◽  
Joshua M. Allwardt ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mirna Family ◽  
Fetal Hemoglobin ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Total Hemoglobin ◽  
Cd34 Cells ◽  
Mrna Expression Levels

Abstract MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.

scholarly journals Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1150-1157 ◽  
Author(s):  
AR Migliaccio ◽  
G Migliaccio ◽  
M Brice ◽  
P Constantoulakis ◽  
G Stamatoyannopoulos ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Gm Csf ◽  
Gamma Globin ◽  
Fetal Bovine ◽  
Globin Synthesis

Abstract We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437 ◽  
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 555-555 ◽  
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

The Role of Nitric Oxide in γ-Globin Induction by Hydroxyurea.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3794-3794
Author(s):  
Tzu-Fang Lou ◽  
Ashley Williams ◽  
Wei Li ◽  
Betty S. Pace
Keyword(s):  
Nitric Oxide ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Fold Increase ◽  
Heme Iron ◽  
Guanosine Monophosphate ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
No Donor

Abstract Hydroxyurea (HU) has been shown to induce fetal hemoglobin (HbF) synthesis through activation of the soluble guanosine cyclase/cyclic guanosine monophosphate signaling pathway. The release of NO from HU by heme iron is thought to be involved in this mechanism of HbF induction. Studies completed in sickle cell patients confirmed increased serum NO levels after oral HU therapy but NO generation in red blood cells and the effect on γ-gene transcription have not been extensively investigated. Therefore, we performed studies to quantify NO generated by HU in K562 cells and normal erythroid progenitors as a mechanism for γ-globin activation. NO levels were measured after drug treatments using the Nitric Oxide Assay Kit (Calbiochem) and γ-globin mRNA was measured using quantitative PCR. HU (100μM) increased NO 1.4 to 1.8-fold at 24–72 hrs in K562 cells compared to a 2.0 to 2.5-fold increase in NO produced by the known NO donor, deta-nonoate (DE; 400μM). NO levels were also measured in erythroid progenitors grown in liquid cultures; a 1.6-fold increase in NO was produced by 30μM HU after 48 hrs with comparable increases produced by 200μM DE. To understand the effects of HU on normal NO synthesis from L-arginine through the action of NO synthase (NOS), we performed studies with the NOS inhibitor, NG-Monomethyl-L-arginine (L-NMMA). Interestingly, HU increased NO levels 2.5-fold at 24 hrs when combined L-NMMA compared a 1.4-fold increase produced by HU alone; this pattern persisted up to 72 hrs. Parallel with these findings γ-globin activation by HU was augmented approximately 25% by L-NMMA; DE combined with L-NMMA did not produce the same effect. These data suggest a novel mechanism for NOS regulation by HU compared to DE. Subsequent studies were completed to determine if HbF synthesis could be augmented by combining NO donors since they have different mechanisms of action. HbF levels in K562 cells were measured by ELISA (Bethyl Laboratories) and normalized by total hemoglobin and protein. Treatment with HU or DE increased HbF 3.6-fold and 4.6-fold respectively; when HU was combined with DE an additive 7.6-fold increase in HbF was produced. These data confirm that HU treatment lead to NO generation in K562 cells and normal erythroid progenitors which plays a role in its mechanism of γ-globin activation. HU combined with DE had an additive effect on HbF synthesis. These findings are relevant to current research efforts to develop novel HbF inducers for therapy in sickle cell patients.

Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4102-4102
Author(s):  
Vladan P. Cokic ◽  
Bojana B. Beleslin-Cokic ◽  
Constance Tom Noguchi ◽  
Alan N. Schechter
Keyword(s):  
Progenitor Cells ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

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