LncRNA STXBP5-AS1 suppresses stem cell-like properties of pancreatic cancer by epigenetically inhibiting neighboring androglobin gene expression.

Previous studies suggest the tumor suppressor role of long non-coding RNA (lncRNA) STXBP5-AS1 in cervical and gastric cancer, but its expression pattern and functional mechanism are still elusive in pancreatic cancer (PC). Relative expression of STXBP5-AS1 in PC both in vivo and in vitro was analyzed by real-time PCR. IC50 of Gemcitabine was determined by the MTT assay. Cell proliferation in response to drug treatment was investigated by colony formation assay. Cell apoptosis was measured by both caspase-3 activity and Annexin V/PI staining. Cell invasion capacity was scored by the transwell assay in vitro, and lung metastasis was examined with the tail vein injection assay. Cell stemness was determined in vitro by sphere formation and marker profiling, respectively, and in vivo by limited dilution of xenograft tumor incidence. Subcellular localization of STXBP5-AS1 was analyzed with fractionation PCR. Association between STXBP5-AS1 and EZH2 was investigated by RNA-immunoprecipitation. The binding of EZH2 on ADGB promoter was analyzed by chromatin immunoprecipitation. The methylation was quantified by bisulfite sequencing. We showed downregulation of STXBP5-AS1 in PC associated with poor prognosis. Ectopic STXBP5-AS1 inhibited chemoresistance and metastasis of PC cells. In addition, STXBP5-AS1 compromised stemness of PC cells. Mechanistically, STXBP5-AS1 potently recruited EZH2 and epigenetically regulated neighboring ADGB transcription, which predominantly mediated the inhibitory effects of STXBP5-AS1 on stem cell-like properties of PC cells. Our study highlights the importance of the STXBP5-EZH2-ADGB axis in chemoresistance and stem cell-like properties of PC.

Evaluation of neuroglobin and cytoglobin expression in adult rats exposed to silver nanoparticles during prepubescence.

Silver nanoparticles (AgNPs) are clusters of silver atoms with diameters that range from 1 to 100 nm. Due to the various shapes and large surface areas, AgNPs have been employed in the food and textile industries and medical fields. Therefore, because of the widespread use of these compounds, the aim of this study was to evaluate the effect of AgNP exposure on the gene and protein expression levels of Neuroglobin (Ngb) and Cytoglobin (Cygb), in the rat cortex, hippocampus and cerebellum. Post-natal day (PND) 21 male Wistar rats were randomly divided into three groups. One group received 15 µg/kg body weight of AgNP by gavage another group received 30 µg/kg and the control group that received saline, from PND23 to PND58. On PND102 the animals were euthanized and the cortex, hippocampus and cerebellum were isolated and evaluated for gene and protein expression levels of Nbg and Cygb. The results demonstrated that the 30 µg/kg AgNP group displayed increased gene and protein expression of Cygb in the cortex. In the Hippocampus, AgNP exposure did not modulate gene or protein expression levels of Ngb and Cygb. In cerebellum the Ngb gene and protein expression was increased with both doses of AgNP. AgNP exposure during prepubescence can modulate the gene and protein expression levels of Ngb and Cygb in adulthood. Furthermore, the observed modulation was specific to the cerebellum, and cortex, and was dose dependent.

Hemin supports the survival of photoreceptors injured by N-Methyl-N-nitrosourea: The contributory role of neuroglobin in photoreceptor degeneration.

Retina is a critical component of the central nerve system that is responsible for the conversion of light stimulus into electrical spikes. Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited retinal dystrophies leading to blindness. We examined retinal neuroglobin (Ngb) expression in a pharmacologically induced RP animal model, the N-Methyl-N-nitrosourea (MNU) administered mice. The retinal Ngb expression in MNU administered mice attenuated following a time dependent manner, suggesting Ngb was involved in the photoreceptor degeneration. Conversely, the intravenous delivery of Hemin, a Ngb up-regulator, enhanced the Ngb expressions in the retinas of MNU administered mice. Optokinetic behavioral tests and Electroretinogram (ERG) examination suggested that the Hemin treatment could improve the visual function of MNU administered mice. The retinal morphology of the Hemin treated group was much more intact than the MNU group as evidenced by retinal sections and optical coherence tomography (OCT) examinations. Moreover, immunostaining experiments showed the cone photoreceptors in the MNU administered mice were also rescued by Hemin treatment. Furthermore, mechanism studies suggested the Hemin treatment not only alleviated the oxidative stress, but also rectified the apoptotic changes in the retinas of MNU administered mice. In conclusion, the intraperitoneally delivery of Hemin can enhance the Ngb expressions in the MNU administered retinas, thereby ameliorating the photoreceptor degeneration and associated visual impairments. These findings would shed light on the opportunity to develop Ngb into a therapeutic molecular against RP.

Crystallographic studies with xenon and nitrous oxide provide evidence for protein-dependent processes in the mechanisms of general anesthesia.

BACKGROUND: The mechanisms by which general anesthetics, including xenon and nitrous oxide, act are only beginning to be discovered. However, structural approaches revealed weak but specific protein-gas interactions. METHODS: To improve knowledge, we performed x-ray crystallography studies under xenon and nitrous oxide pressure in a series of 10 binding sites within four proteins. RESULTS: Whatever the pressure, we show (1) hydrophobicity of the gas binding sites has a screening effect on xenon and nitrous oxide binding, with a threshold value of 83% beyond which and below which xenon and nitrous oxide, respectively, binds to their sites preferentially compared to each other; (2) xenon and nitrous oxide occupancies are significantly correlated respectively to the product and the ratio of hydrophobicity by volume, indicating that hydrophobicity and volume are binding parameters that complement and oppose each other's effects; and (3) the ratio of occupancy of xenon to nitrous oxide is significantly correlated to hydrophobicity of their binding sites. CONCLUSIONS: These data demonstrate that xenon and nitrous oxide obey different binding mechanisms, a finding that argues against all unitary hypotheses of narcosis and anesthesia, and indicate that the Meyer-Overton rule of a high correlation between anesthetic potency and solubility in lipids of general anesthetics is often overinterpreted. This study provides evidence that the mechanisms of gas binding to proteins and therefore of general anesthesia should be considered as the result of a fully reversible interaction between a drug ligand and a receptor as this occurs in classical pharmacology.

17ß-estradiol--a new modulator of neuroglobin levels in neurons: role in neuroprotection against H2O2-induced toxicity.

Although discovered in 2000, neuroglobin (Ngb) functions are still uncertain. A contribution to the role played by Ngb in neurons could certainly derive from the identification of Ngb endogenous modulators. Here, we evaluate the possibility that Ngb could be regulated by 17ß-estradiol (E2) signaling in both SK-N-BE human neuroblastoma cell line and mouse hippocampal neurons. 1 nM E2 rapidly induced a 300% increase in Ngb levels in both models. The E2 effect was specific, being not induced by testosterone or dihydrotestosterone. The E2-induced Ngb increase requires estrogen receptor (ER) ß, but not ERα, as evaluated by the mimetic effect of ERß-specific agonist DPN and by the blockage of E2 effect in ERß-silenced SK-N-BE cells. Furthermore, both rapid (15 min) ERß-dependent activation of p38/MAPK and transcriptional ERß activity were required for the estrogenic regulation of Ngb. Finally, E2 exerted a protective effect against H2O2-induced neuroblastoma cell death which was completely prevented in Ngb-silenced cells. Overall, these data suggest that Ngb is part of the E2 signaling mechanism that is activated to exert protective effects against H2O2-induced neurotoxicity.

Reduction of AHSP synthesis in hemin-induced K562 cells and EPO-induced CD34(+) cells leads to alpha-globin precipitation, impairment of normal hemoglobin production, and increased cell death.

OBJECTIVE: alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression. MATERIALS AND METHODS: Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. RESULTS: RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. CONCLUSION: These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Tyrosine as a redox-active center in electron transfer to ferryl heme in globins.

A wide range of organic reductants, including many iron chelators, reduce ferryl myoglobin to its ferric states in exponential time courses whose rate constants display double hyperbolic dependencies on the reductant concentration. This concentration dependence is consistent with a mechanism in which electron transfer to the heme takes place at two independent sites where reductants appear to bind. We propose that the low-affinity site is located close to the heme edge, within the heme pocket; the maximum rate of electron transfer is highly variable depending on the nature of the reductant (0.005 to >10 s(-1)). The other site has higher apparent affinity (K(D) 0.2-50 microM) but a low maximum rate of electron transfer (0.005 to 0.01 s(-1)). By examining native and engineered proteins we have determined that the high-affinity pathway represents a through-protein electron transfer pathway that involves a specific tyrosine residue. The low apparent rate constant for electron transfer from the tyrosine to the heme (approximately 5 A) is accounted for by proposing that electron transfer occurs only in a very poorly populated protonated state of ferryl heme and tyrosine. Hemoglobin shows similar kinetics but only one subunit exhibits double rectangular hyperbolic concentration dependency. The consequence of a high-affinity through-protein electron transfer pathway to the cytotoxicity of ferryl heme is discussed.

Oral decitabine reactivates expression of the methylated gamma-globin gene in Papio anubis.

The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis).

Plasmodium berghei: in vitro and in vivo activity of dequalinium.

Bisquinoline compounds have exhibited remarkable activity in vitro and in vivo against Plasmodium parasites by inhibition of heme detoxification. We have tested the ability of dequalinium 1,1'-(1,10-decanediyl)bis(4-amino-2-methylquinoline), a known antimicrobial agent, to inhibit beta-hematin synthesis using a non-emzymatic colorimetric assay and globin proteolysis by electrophoretic analysis (SDS-PAGE-15%). Dequalinium was able to inhibit both processes in vitro with close correlation to a murine malaria model, reducing parasitemia levels, prolonging the survival time post-infection and curing 40% of infected mice using a combination therapy with a loading dose of chloroquine. These results confirm that dequalinium is a promising lead for antimalarial drug development.

Short-chain fatty acids induce gamma-globin gene expression by displacement of a HDAC3-NCoR repressor complex.

High-level induction of fetal (gamma) globin gene expression for therapy of beta-hemoglobinopathies likely requires local chromatin modification and dissociation of repressor complexes for gamma-globin promoter activation. A novel gamma-globin-inducing short-chain fatty acid derivative (SCFAD), RB7, which was identified through computational modeling, produced a 6-fold induction in a reporter assay that detects only strong inducers of the gamma-globin gene promoter and in cultured human erythroid progenitors. To elucidate the molecular mechanisms used by high-potency SCFADs, chromatin immunoprecipitation (ChIP) assays performed at the human gamma- and beta-globin gene promoters in GM979 cells and in erythroid progenitors demonstrate that RB7 and butyrate induce dissociation of HDAC3 (but not HDAC1 or HDAC2) and its adaptor protein NCoR, specifically from the gamma-globin gene promoter. A coincident and proportional recruitment of RNA polymerase II to the gamma-globin gene promoter was observed with exposure to these gamma-globin inducers. Knockdown of HDAC3 by siRNA induced transcription of the gamma-globin gene promoter, demonstrating that displacement of HDAC3 from the gamma-globin gene promoter by the SCFAD is sufficient to induce gamma-globin gene expression. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes associated with SCFAD-induced activation of the gamma-globin gene and provide a specific molecular target for potential therapeutic intervention.

Effects on erythroid differentiation of platinum(II) complexes of synthetic bile acid derivatives.

In this study, we compared some bile acid derivatives and their platinum(II) complexes with respect to their ability to induce erythroid differentiation of human leukemic K562 cells. The complexes analyzed were cis-[(3-dehydrocholanoyliden-L-tartrate)-diammineplatinum(II)] (compound 1) and cis-[di(dehydrocholanoate)-bis(triphenylphosphine)-platinum(II)] (compound 3), together with their free ligands, respectively, 3-dehydrocholanoyliden-L-tartaric acid (compound 2) and dehydrocholanoic acid (4), and their parent compounds, respectively, cisplatin and cis-[dichloride-bis(triphenylphosphine)-platinum(II)] (5). We found that compound 1 stimulates erythroid differentiation of K562 cells and an increase of fetal hemoglobin (HbF) production in erythroid precursor cells isolated from peripheral blood of human subjects. This increase is similar to that obtained by hydroxyurea, a potent inducer of HbF production both in vitro and in vivo. Another important conclusion of this study is related to the evaluation of the effects of compound 1 on production of gamma-globin mRNA in human erythroid precursors grown in the two-stage liquid culture system. We demonstrated that compound 1 induces preferential accumulation of gamma-globin mRNA. The results presented in this manuscript could have practical impact, since it is well known that an increase in HbF production could ameliorate the clinical status of patients with beta-thalassemia and sickle cell anemia.

Establishment and characterization of a new erythroblastic leukemia cell line, EEB: phosphatidylglucoside-mediated erythroid differentiation and apoptosis.

A new erythroblastic leukemia cell line (EEB) was established from a patient with early erythroblastic leukemia. The cells had features of immature erythroblasts, including an agranular basophilic cytoplasm and CD36, CD71, CD175s (sialyl-Tn) and CD235a (glycophorin A) expression without CD41 expression, myeloperoxidase activity and platelet-peroxidase activity. The cells were confirmed to be of the erythroid lineage based on expression of the gamma-globin message. They were induced to differentiate into benzidine-positive cells by hemin and delta-amino levulinic acid (delta-ALA). An analysis of cell membrane lipids showed that EEB cells contain a type of glycerolipid, phosphatidylglucose (PhGlc), but not unbranched type 2 chains, i antigens. GL-7 which is a recombinant Fab fragment of GL-2 and binds to PhGlc, induced production of hemoglobin F (HbF) associated with accumulation of the gamma-globin (gamma-globin) message in EEB cells. The GL-7-mediated erythroid differentiation was associated with apoptosis. These results suggest that direct signaling to PhGlc mediates erythroid differentiation and apoptosis in EEB cells.

Investigations of the induction of the goat beta(C) globin gene by erythropoietin: studies in transgenic mice.

The genes of the beta globin locus of sheep and goats undergo three developmental switches, from embryonic epsilon to fetal gamma to juvenile beta(C) and from beta(C) to adult beta(A). The juvenile beta(C) gene of adult goats and sheep are strikingly induced by erythropoietin (Epo). To obtain insights on the mechanism of beta(C) induction by Epo, we produced transgenic mice carrying various beta(C) gene constructs and examined the inducibility of the beta(C) gene following administration of high doses of erythropoietin. None of the treatments resulted in reproducible induction of the beta(C) gene by erythropoietin. We conclude that the Epo inducibility elements are not contained in the 4.7 kb (which included 0.88 kb of the beta(C) promoter and 2.3 kb downstream sequence) beta(C) gene we used or that a trans-acting environment specific to the goat and sheep erythroid cell lineage is required for induction of the beta(C) globin gene by erythropoietin.

Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin.

Reactivation of fetal hemoglobin (HbF) expression using pharmacological agents represents a potential strategy for the therapy of beta-thalassemia, sickle cell disease, HbE and other beta-hemoglobinopathies. However, the drugs currently available have low efficacy and specificity and are associated with high toxicity. We describe the development of stable cellular genomic reporter assays (GRAs) based on the green fluorescence protein (EGFP) gene under the Ggamma-globin promoter in the intact human beta-globin locus. We show that human erythroleukemic cell lines stably transfected with a Ggamma-EGFP beta-globin locus construct can maintain a uniform basal level of EGFP expression over long periods of continuous culture and that induction of EGFP expression parallels the induction of the endogenous globin genes. We compared the EGFP-induction potency of a number of chemotherapeutic agents, including histone deacetylase inhibitors and DNA-binding agents. We show that hydroxyurea and butyrate result in moderate levels of induction (70-80%) but with an additive inductive effect. Among the DNA-binding agents tested, cisplatin was the most potent inducer of HbF expression, (442+/-32%), a level which is comparable to hemin (764+/-145%). These results indicate that cellular GRAs containing Ggamma-EGFP-modified beta-globin locus constructs can be used to develop novel inducers of HbF synthesis for the therapy of beta-hemoglobinopathies.

Effect of hydroxyurea on G gamma chain fetal hemoglobin synthesis by sickle-cell disease patients.

Hydroxyurea is used for sickle-cell disease patients in order to increase fetal hemoglobin synthesis and consequently decrease the severity of pain episodes. Fetal hemoglobin, which is formed by gamma-globin chains A and G, is present in a constant composition throughout fetal development: about 75% of Ggamma and 25% of Agamma. In contrast, adult red cells contain about 40% of Ggamma and 60% of Agamma. In the present study, we analyzed the effect of hydroxyurea induction on the gamma chain composition of fetal hemoglobin in 31 sickle-cell disease patients treated with hydroxyurea. The control group was composed of 30 sickle-cell disease patients not treated with hydroxyurea in clinical steady state. The patients were older than 13 years and were not matched for age. All patients were seen at Hemocentro/UNICAMP and Boldrini Infantile Center, Campinas, SP, Brazil. The levels of total hemoglobin were significantly higher in patients treated with hydroxyurea (mean +/- SD, 9.6+/-2.16 g/dl) than in untreated patients (8.07+/-0.91 g/dl). Fetal hemoglobin levels were also higher in treated patients (14.16+/-8.31%) than in untreated patients (8.8+/-4.09%), as was the Ggamma/Agamma ratio (1.45+/-0.78 vs 0.98+/-0.4, P < 0.005). The increase in the Ggamma/Agamma ratio in patients treated with hydroxyurea suggests the prevalence of a pattern of fetal hemoglobin synthesis, whereas patients not treated with hydroxyurea maintain the adult pattern of fetal hemoglobin synthesis. Because no correlation was observed between the Ggamma/Agamma ratio and total hemoglobin or fetal hemoglobin levels, the increase in Ggamma chain synthesis may not imply a higher production of hemoglobin.

Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression.

OBJECTIVE: To examine whether hydroxamic acids are inducers of fetal hemoglobin expression, we tested the effects on gamma gene expression of butyric and propionic hydroxamic acids and of two other hydroxamic acids (SBHA and SAHA), which are potent inhibitors of histone deacetylase (HDAC). We also investigated whether there is a correlation between HDAC inhibitory activity of the compounds and their ability to induce gamma-globin gene expression. MATERIALS AND METHODS: Effects on gamma-globin expression were assessed by two methods: 1) a screening assay in which specific gamma-globin gene inducers are recognized by their ability to increase gamma firefly luciferase activity significantly more than beta-renilla luciferase activity; and 2) measurements of gamma-globin mRNA and the frequency of fetal hemoglobin-positive erythroblasts in cultures of burst-forming unit erythroid (BFU-E) from normal individuals. HDAC in vitro activity was measured with a partially purified rat liver HDAC and a fluorogenic substrate. RESULTS: All compounds tested increased gamma firefly luciferase activity, gamma/gamma+beta mRNA ratios, and percentage of fetal hemoglobin-containing erythroblasts in BFU-E cultures, in a dose-dependent fashion. Butyryl-hydroxamic acid 100 microM increased the gamma/gamma+beta mRNA ratios by 5.8-fold and the frequency of fetal hemoglobin-containing erythroblasts by 4.1-fold. Propionyl-hydroxamic acid 150 microM increased the gamma/gamma+beta ratios by 6.3-fold and the fetal hemoglobin-containing erythroblasts by 3.9-fold. SBHA induced gamma-globin gene expression at very low concentrations, 5 to 20 microM in the luciferase system and 2 to 8 microM in BFU-E cultures; SAHA at 1 to 7.5 microM in the luciferase system and 1 to 2.5 microM in the BFU-E cultures. HDAC in vitro inhibition was observed in the millimolar range for propionate and butyrate. IC(50) determinations led to values of 384 microM for propionyl-hydroxamate, 47 microM for butyryl-hydroxamate, 0.93 microM for SBHA, and 0.26 microM for SAHA. CONCLUCION: Our data indicate that hydroxamic acid-based HDAC inhibitors are potent gamma-globin gene inducers and that the concentration range of their effects on gamma gene expression can be correlated roughly with their HDAC inhibitory potencies.

Functional cross-antagonism between transcription factors FLI-1 and EKLF.

FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the beta-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on beta-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.

Progesterone upregulates GATA-1 on erythroid progenitors cells in liquid culture.

Steroids hormones modify the hematological features of homozygous sickle cell disease, including the levels of fetal hemoglobin. We used semi-quantitative RT-PCR analysis of GATA-1, GATA-2, NF-E2, and gamma-globin mRNA levels in a two-phase liquid culture system of human adult erythroid cells in order to assay the effect of progesterone upon gene expression. The levels of expression of GATA-1 and gamma-globin mRNA were significantly increased in cells treated with progesterone compared to untreated cells (1.7- to 2.0-fold). Progesterone treatment did not produce any stimulatory effect upon GATA-2 and NF-E2 mRNA expression. Differences in the synthesis of HbF protein could not be detected by flow cytometry, although we observed a small difference in mean intensity fluorescence between cells treated and cells untreated with progesterone on days 7 and 9. Using anti-transferrin receptor and anti-glycophorin A antibodies, we verified that addition of progesterone did not cause any change in erythroid proliferation and differentiation. In conclusion, it is possible that the increased expression of gamma-globin mRNA after progesterone treatment observed in this study may be related to the increased GATA-1 mRNA expression. Interactions of the steroid receptors with the basal transcriptional machinery and with transcription factors might mediate their transcriptional effects.

Post-transcriptional effects of interleukin-3, interferon-gamma, erythropoietin and butyrate on in vitro hemoglobin chain synthesis in congenital hemolytic anemia.

BACKGROUND AND OBJECTIVES: Various agents modulate hemoglobin synthesis. In vitro modulation of translation in hemoglobin chain synthesis was analysed in patients with congenital hemolytic anemia (n=32) and healthy controls (n=17). DESIGN AND METHODS: Enriched reticulocytes were co-incubated with (3)H-leucine and cytokines or butyrate. Reversed-phase chromatography enabled separation of alpha-, beta- and gamma-globin chains. Globin chain synthesis was calculated from measured (3)H-leucine incorporation. Transferrin, erythropoietin, interleukin-3 and interferon-gamma receptors were detected by flow cytometry. Reverse-transcription polymerase chain reaction (RT PCR) was used to demonstrate changes of RNA stability. RESULTS AND DISCUSSION: Interleukin-3, interferon-gamma and butyrate caused a significant 2-fold increase (range 1.8-2.4; p<0.01) of the alpha- and beta-chain synthesis in congenital hemolytic anaemias. Analysis of gamma-globin chain synthesis revealed a lower, i.e. 1.4 fold increase (range 1.32 to 1.41; p<0.03). The absolute amount of globin synthesis was calculated to be 2.9 x 10(-12) g/reticulocyte/24h. After incubation with interleukin-3 the absolute additional synthesis of the alpha-globin chain reached 1.31 x 10(-12) g/reticulocyte/24h, of the beta-globin chain, 1.15 x 10(-12) g/reticulocyte/24h and of the gamma-globin chain, 0.26 x 10(-12) g/reticulocyte/24h. Butyrate and interferon-gamma had no or even an inhibiting effect on reticulocytes from normal controls, while interleukin-3 stimulated alpha- and gamma-chain synthesis (1.4 and 2.4 fold, respectively; p<0.03) suggesting an increase of fetal hemoglobin (HbF). Erythropoietin showed no stimulating influence. Membrane associated interleukin-3 receptors were detected in 0.78+/-0.14%, and interferon-gamma receptors in 0.1+/-0.015% of the red cells. Erythropoietin receptors were extremely rare (0.05+/-0.015%). The expression of transferrin receptors (CD71) correlated with the extent of globin chain stimulation. The alpha-, and beta-globin mRNA content of the reticulocytes after interleukin-3 incubation, as measured by RT-PCR, increased. INTERPRETATION AND CONCLUSIONS: Hemoglobin chain synthesis could be modulated post-transcriptionally by interleukin-3, interferon-gamma and butyrate. Transferrin receptor and globin RNA stability might be involved in this phenomenon.

Valproic acid, trichostatin and their combination with hemin preferentially enhance gamma-globin gene expression in human erythroid liquid cultures.

BACKGROUND AND OBJECTIVES: In addition to conventional therapy, current treatment of thalassemia and sickle cell anemia includes inducers of hemoglobin F synthesis (hydroxyurea, erythropoietin, azacytidine and butyrate). However, because of concerns about the dose-limiting myelotoxicity, potential carcinogenicity and high cost of the above agents, an intensive search for less toxic or more effective drugs is ongoing. In this study we tested the effect of valproic acid and trichostatin, alone or in combination with hemin, on gamma chain synthesis in human erythroid liquid cultures. DESIGN AND METHODS: The agents were tested on erythroid human liquid cultures derived from normal peripheral blood, peripheral blood from beta(s)/beta(thal) patients, normal cord blood and normal bone marrow samples. The effect of the agents was expressed as increase of gamma/gamma+beta m-RNA, measured with competitive reverse transcriptase-polymerase chain recation (RT-PCR), or as increase of HbF, measured by high performance liquid chromatography (HPLC). RESULTS: Addition of valproic acid or trichostatin to human erythroid cell cultures preferentially enhanced gamma mRNA synthesis in all blood samples (2.9 to 3.5-fold). The addition of hemin enhanced the effect up to 10-fold. INTERPRETATION AND CONCLUSIONS: Valproic acid, trichostatin and their combination with hemin (all three FDA-approved drugs) preferentially increase gamma-globin chain synthesis and may be helpful for the treatment of hemoglobinopathies.