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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. IC 50 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.

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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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 17β-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) β, but not ERα, as evaluated by the mimetic effect of ERβ-specific agonist DPN and by the blockage of E₂ effect in ERβ-silenced SK-N-BE cells. Furthermore, both rapid (15 min) ERβ-dependent activation of p38/MAPK and transcriptional ERβ 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., (Copyright © 2011 S. Karger AG, Basel.)

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.

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.

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)., ((c) 2007 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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., (Copyright 2003 International Society for Experimental Hematology)

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.

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.

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.

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.

Sickle transgenic mice expressing exclusively human globins are desirable for studying pathophysiology and testing gene therapy strategies, but they must have significant pathology and show evidence of amelioration by antisickling hemoglobins. Mice were generated that expressed exclusively human sickle hemoglobin with 3 levels of HbF using their previously described sickle constructs (cointegrated human miniLCRalpha2 and miniLCRbeta(S) [PNAS 89:12150, 1992]), mouse alpha- and beta-globin-knockouts, and 3 different human gamma-transgenes. It was found that, at all 3 levels of HbF expression, these mice have balanced chain synthesis, nearly normal mean corpuscular hemoglobin, and, in some cases, F cells. Mice with the least adult HbF expression were the most severe. Progressive increase in HbF from less than 3% to 20% to 40% correlated with progressive increase in hematocrit (22% to 34% to 40%) and progressive decrease in reticulocyte count (from 60% to 30% to 13%). Urine concentrating ability was normalized at high HbF, and tissue damage detected by histopathology and organ weight were ameliorated by increased HbF. The gamma-transgene that produces intermediate levels of HbF was introduced into knockout sickle mice described by Pàszty and coworkers that express the miniLCRalpha1(G)gamma(A)gammadeltabeta(S) transgene and have fetal but not adult expression of HbF. It was found that the level of HbF required to ameliorate low hematocrit and normalize urine concentrating defect was different for the miniLCRalpha2beta(S) and miniLCRalpha1(G)gamma(A)gammadeltabeta(S) mice. We conclude that knockout mice with the miniLCRalpha2beta(S) transgene and postnatal expression of HbF have sufficiently faithful sickle pathology to serve as a platform for testing antisickling interventions.

The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid differentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes differentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6 cells, EPO-induced differentiation was inhibited. Conversely, a dominant negative ras mutant (ras17N) induced differentiation even in the absence of EPO, suggesting that the basal Ras activity is essential for the maintenance of the undifferentiated phenotype and proliferative potential in this cell line. Rapid inactivation of ERK was observed after expression of ras17N. Slow but significant inactivation of ERK was also observed during EPO-induced differentiation. Furthermore, overexpression of a constitutively active mutant of ERK-activating kinase (MAPKK) was found to suppress erythroid differentiation, while pharmacological inhibition of MAPKK induced differentiation. These findings suggest that down-regulation of Ras/ERK signaling pathway may be an essential event in EPO-induced erythroid differentiation in this system.

To evaluate the role of the erythropoietin (Epo) receptor (R) in erythropoiesis in more primitive stem cells, we assessed the influence of retrovirus-mediated gene transfer of human (h) EpoR complementary DNA (cDNA) into murine embryonic stem (ES) cells on erythroid differentiation of these cells. The hEpoR cDNA was efficiently transduced into ES cells, forming hEpoR that stably expressed ES (ES-hEpoR) cells. Expression of hEpoR cDNA was confirmed in ES-hEpoR cells by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Colony assays demonstrated that definitive erythroid and primitive erythroid colonies were significantly increased from ES-hEpoR cells, when compared with mock virus-transduced ES (ES-Neo) cells, during the time course of differentiation induced by withdrawal of leukemia inhibitory factor, in either the presence or the absence of Epo. Multipotential colony-forming units (CFU-Mix) were also increased in ES-hEpoR cells at different stages of differentiation, but no changes were detected for CFU-granulocyte-macrophage colonies (CFU-GM). Time course studies by Northern blot analysis demonstrated elevated levels of expression of beta-H1 and beta-Major globin genes in embryoid bodies derived from ES-hEpoR cells stimulated with Epo, when compared with similar expression from ES-Neo cells. Expression of the GATA-1 gene was enhanced in ES-hEpoR cells, when compared with ES-Neo cells, beginning immediately after initiation of the cultures until 8 days of differentiation. These data indicate that primitive and definitive erythropoiesis in differentiating embryoid bodies can be enhanced by retrovirus-mediated gene transfer of an hEpoR gene.

Acetaldehyde is the first oxidation product of ethanol in vivo. Our earlier work showed that with sufficient acetaldehyde, five of the six possible sites of the peptide pentalysine were modified as a Schiff base (Braun KP, et al: J Biol Chem 270:11263-11266, 1995). However, we were unable to deduce unequivocally which site was unmodified. Lysine residues, as well as the amine terminal valine residues, in hemoglobin have been implicated as target structures for acetaldehyde adducts resulting from ethanol consumption. Hemoglobin adducts of acetaldehyde have been used clinically as a marker of ethanol consumption, but the chemical nature of these adducts remains undefined. As part of our continuing structural characterization of acetaldehyde-protein adduct formation, we studied the peptides Val-His-Leu-Thr-Pro and Val-His-Leu-Thr-Pro-Val-Glu-Lys, from the amine terminus of the beta-globin chain of hemoglobin, in vitro. Both peptides have at least one potential site for adduct formation. In the octapeptide, the N-terminal amine group of Val as well as the epsilon-amine group of the lysine sidechain can potentially be modified by acetaldehyde. We used mass spectrometry, carbon-13 nuclear magnetic resonance, and Raman spectroscopy and characterized stable Schiff base acetaldehyde adducts of these two peptides at both reactive sites. The identification of stable Schiff base adducts with the N-terminal peptides of the beta-chain of hemoglobin as well as with epsilon-amino groups of lysine provides another possible means of monitoring ethanol consumption. The functional implications of these stable Schiff bases remains undefined.

Short-chain fatty acids, such as butyrate and propionate, are under investigation as therapeutic stimulants of fetal hemoglobin production in the beta-hemoglobin disorders. Significant limitations to these fatty acids and derivatives as optimal therapeutics are their rapid metabolism in vivo and their induction of cell growth arrest in the G1 phase of the cell cycle. This antiproliferative activity is related to their inhibition of metabolic transport pumps which are essential for cell proliferation. Other small carbon compounds, the phenylalkyl acids, phenoxyacetic acids, and phenylacetic acids, which are structurally resistant to oxidative metabolism, are shown here to induce fetal globin production in human erythroid cultures at concentrations of 0.2 mM, lower than those required for most other fatty acids. Certain of these compounds were found not to inhibit cellular neutral amino acid transport function in erythroid cells, nor to inhibit erythroid colony (Bfu-e) growth. Certain of these compounds even stimulated human Bfu-e proliferation in vitro beyond that induced by optimal concentrations of hematopoietic growth factors. The combination of increased fetal globin chain production by these compounds and their stimulatory effects on erythropoiesis result in an increase in Hb F-expressing erythroid cells in culture several-fold greater than that achieved by the butyrates. These new compounds thus have the potential to provide superior therapy for the beta-hemoglobinopathies and other anemias.

Thymidine kinase (TK) gene expression is controlled in normal cells at both the transcriptional and posttranscriptional levels. Together, these regulatory systems mediate the 20-50-fold induction of TK mRNA observed as cells traverse the G1-S boundary of the cell cycle. Previously, we have reported that a "Yi" protein complex was observed to bind the mouse TK promoter in a cell cycle-dependent manner in nontransformed cells (Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 88: 1157-1161, 1991) and bound constitutively in transformed cells (D. W. Bradley, Q-P. Dou, J. L. Fridovich-Keil, and A. B. Pardee, Proc. Natl. Acad. Sci. USA, 87: 9310-9314, 1990). Nonetheless, TK mRNA levels in these cells continue to exhibit a marked S-specific induction (> 10 fold), raising the question: what is the status of TK promoter-mediated, as opposed to posttranscriptional, gene regulation in these transformed cells? To address this question, we have used cell synchrony experiments involving both transformed and nontransformed cells stably transfected with a TK promoter-beta-globin reporter gene construct. We have found that, in marked contrast to the tight regulation of reporter gene expression observed in nontransformed cells (J. L. Fridovich-Keil, J. M. Gudas, Q-P. Dou, I. Bouvard, and A. B. Pardee, Cell Growth & Differ., 2: 67-76, 1991), reporter gene expression in the transformed cells is constitutive and, therefore, closely parallels the presence of Yi DNA-binding activity. These data are fully consistent with other recently published observations concerning differential controls of TK transcriptional and posttranscriptional regulation (J. M. Gudas, J. L. Fridovich-Keil, and A. B. Pardee, Cell Growth & Regul., 4: 421-430, 1993) and support the hypothesis that, in transformed cells, endogenous TK is regulated predominantly at the posttranscriptional level.

The effects of cylindrospermopsin isolated from a blue-green alga Umezakia natans on mice were examined morphologically and biochemically. The main target of the phycotoxin was the liver. The thymus, kidneys and heart were also affected. There were four consecutive phases of the pathological changes in the liver. The initial phase was that of inhibition of the protein synthesis, the second phase of membrane proliferation followed, and then the third phase of fat droplet accumulation and finally the phase of cell death. Using globin synthesis in the rabbit reticulocytes system, it was clearly demonstrated that cylindrospermopsin is a potent inhibitor of the protein synthesis. Protein in microsomes from the mouse livers treated by cylindrospermopsin decreased in amount more significantly than that of phospholipid in microsomes. Furthermore, the amount of total P450 was extensively diminished in the toxin treated with hepatic microsomes.

An HPLC method for analysis of blood protein adducts of methyl bromide was developed. With this method, the alkylated amino acid S-methylcysteine can be quantified both in globin and in serum albumin. The determination of these adducts was implemented in a field study on fumigators who use methyl bromide for the control of insects, nematodes and fungi. Sister chromatid exchange (SCE) was determined in the lymphocytes of the fumigators as an additional biomonitoring parameter. Exposure of persons living in the vicinity of fumigated objects to methyl bromide has been repeatedly reported in the past. The new method for determination of blood protein adducts can be applied for evaluation of such environmental exposure.

Six sickle cell/beta-thalassemia patients (3 males and 3 females) were treated with 500 U/kg body weight human recombinant erythropoietin (h-rEPO) along with 300 mg/day iron sulfate in two phases, for a period of 90 days. Fetal hemoglobin (HbF) was assayed every 2 weeks and the gamma-chain ratio at three successive intervals during the treatment. All patients showed a moderate to high increase in their HbF values (1.25- to 12-fold). The gamma-chain ratio, as determined by high performance liquid chromatography was found to be unaffected by the HbF increase. Two patients with the newborn gamma-chain ratio, responded faster to the h-rEPO treatment and achieved higher HbF values than the rest of the group. The h-rEPO treatment was very well tolerated and had a positive effect on the general clinical condition of all the patients.

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB has been detected in hydrolysates of hemoglobin from smokers and snuff dippers and has been proposed as a dosimeter of exposure to and metabolic activation of NNK in people exposed to tobacco products. In this study, labeling experiments were carried out with [18O]NaOH that provide strong evidence that the globin adduct that releases HPB upon hydrolysis is a carboxylic ester. Globin was isolated from rats treated with [5-3H]NNK. This globin was reacted with NaCNBH3, followed by hydrolysis at room temperature with 0.2 N NaOH. Analysis of the products demonstrated the presence of 4-hydroxy-1-(3-pyridyl)-1-butanol, but not HPB. These results demonstrate that the adduct in globin has a free carbonyl group and is not a Schiff base. This sequence of reactions was then carried out with [18O]NaOH under conditions that were shown to result in incorporation of 18O if nucleophilic displacement at C-4 had occurred. Analysis by GC-MS of the 4-hydroxy-1-(3-pyridyl)-1-butanol formed in this experiment demonstrated that there was no incorporation of 18O. These results are consistent only with the hydrolysis of an ester by a BAC2 mechanism. Therefore, the adduct releasing HPB upon mild base hydrolysis must be a 4-(3-pyridyl)-4-oxobutyl ester of aspartate, glutamate, or a terminal carboxylate.(ABSTRACT TRUNCATED AT 250 WORDS)

Damaged or old erythrocytes are cleared rapidly from circulation. Because several common biochemical lesions can induce the clustering of integral membrane proteins, we have proposed that formation of microscopic protein aggregates in the membrane might constitute a cell surface marker that promotes removal of the defective/senescent cells. We demonstrate here that treatments that cluster integral membrane proteins in erythrocytes (1 mM ZnCl2, 1 mM acridine orange, and 0.35 microM melittin) induce autologous IgG binding, complement fixation, and phagocytosis by human monocytes in vitro. Removal of the clustering agents prior to incubation in autologous serum or cross-linking of cell surface proteins before addition of clustering agents prohibited the above response, while cross-linking after treatment with the clustering agents preserved the response even if the clustering agents were later removed. Furthermore, subsequent reversal of the chemical cross-link maintaining the clustered distribution also reversed the induction of IgG binding, complement deposition, and phagocytosis. Finally, by deleting or inactivating different steps in the phagocytosis pathway, the chronology of steps was shown to be: (i) integral protein clustering, (ii) IgG binding, (iii) complement deposition, and (iv) phagocytosis.

Globin chain synthesis was studied in the reticulocytes of 30 patients with various myelodysplastic syndromes (MDS) to determine the alpha:beta globin chain synthetic ratio and its probable prognostic value. The mean (SD) value of the total alpha:beta ratio was 0.82 (0.45) ranging from 0.05 to 1.73. The same ratio in 10 normal controls was 1.01 (0.04). This difference was significant. Furthermore, the alpha:beta ratios were lower than normal in 14 patients (alpha-thalassaemia-like) (group I), almost within normal limits in 11 (group II), and higher than normal in five (beta-thalassaemia-like) (group III). In each group almost all the FAB subtypes were represented. The addition of exogenous haem in several of the test samples resulted in a slight to pronounced increase in the alpha:beta ratios, particularly in group I. In 92% of the high risk cases (refractory anaemia with excess blasts (RAEB), chronic myelomonocytic leukaemia (CMML] or 87.5% of patients who finally developed acute non-lyphoid leukaemia (ANLL) low or normal alpha:beta ratios were found. No significant correlation was noticed between alpha:beta ratios and various haematological variables or survival. It is concluded that in MDS the alpha:beta ratio varied enormously across the entire population of patients, as well as within each FAB subtype, thereby restricting its prognostic value. Although haem deficiency may be implicated in some cases of MDS, why this should be remains unclear.

In contrast to normal human erythroid tissues where the alpha 2:alpha 1-globin mRNA ratio is about 72:28, in human erythroleukemia K562 cells this ratio was found to be quite low, i.e. about 8:92. The ratio was moderately increased by hemin induction and approached almost normal levels after chromosomal transfer from K562 to the mouse erythroleukemia (MEL) cells. We suggest that operationally positive regulatory factors may exist in erythroleukemia cells, modifying the relative alpha 1- and alpha 2-globin gene expression by events following induction and by the adult MEL environment. These factors may act by altering the relative rate of alpha 1- and alpha 2-globin mRNA synthesis.