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The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution., (© 2020 by The American Society of Hematology.)

Objective: Hypertensive cerebral hemorrhage leads to greater mortality and worse functional outcomes at high altitudes. Experimental studies have suggested that hemoglobin can lead to increased perihemorrhagic edema after intracerebral hemorrhage., Methods: Patients were divided into a high-hemoglobin (H-H) group (>180 g/L) and a low-hemoglobin (L-H) group (≤180 g/L). The distance from the cortex to the midline was used to indicate the degree of edema. At 1, 7, 14, and 21 days, the patients' status was scored using the Glasgow coma scale, and survival was plotted using Kaplan-Meier survival curves. Pearson correlation analysis showed that the difference between the postoperative and preoperative Glasgow coma scale score correlated with the hemoglobin concentration. The Glasgow outcome scale was used to assess neurological recovery after 6 months., Results: On days 7, 14, and 21, the edema of the H-H group was significantly greater than that of the L-H group (P < 0.01 and P < 0.001, respectively). The edema of the H-H group peaked at 14 and 21 days, but that of the L-H group peaked at 7 days. The hemoglobin concentration and postoperative neurological recovery had a linear relationship in the H-H group. The L-H group had greater survival compared with the H-H group (P < 0.05). The L-H group had higher Glasgow outcome scale scores compared with the H-H group (P < 0.05)., Conclusion: The hemoglobin concentration affects the mortality and morbidity from hypertensive cerebral hemorrhage in high-altitude regions, and a linear relationship exists between hemoglobin concentration and neurological recovery in the H-H group., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Re-expression of fetal hemoglobin (HbF) was proposed as a possible therapeutic strategy for β-haemoglobinopathies. Although several inducers of HbF were tested in clinical trials, only hydroxyurea (HU) received FDA approval. Despite it produced adequate HbF levels only in half of HU-treated SCD patients, and was ineffective at all in β-thalassemia patients, beneficial effects of this approach suggested to continue in this direction identifying further molecules capable of inducing HbF. We tested the potential of essential oil isolated from Ocimum basilicum L. leaves (ObEO) in inducing hemoglobin biosynthesis. Initially, dose-dependent effect and kinetics of hemoglobin accumulation in K562 cells after treatment with ObEO were evaluated. ObEO induced dose-dependent hemoglobin accumulation superior to hydroxyurea and rapamycin and a strongest γ-globin mRNA expression. Terpenes composition of ObEO was studied by GC-MS. Three main constituents, linalool, eugenol and eucalyptol, represented about 75% of total. A blend of these three terpenes fully replicated the ObEO's biological effect, thus indicating that one of them or all together could be the active ingredients. When terpenes were tested individually, eugenol was the only one inducing stable hemoglobin accumulation, while eucalyptol and linalool produced only a small transient response. However, eugenol potential was strongly enhanced in the presence of eucalyptol and linalool, suggesting a synergistic effect on hemoglobin accumulation. By these results, the discovery of a new inducer and the interesting activity of a blend of major terpenes from ObOE on Hb accumulation could have positive fallouts on β-thalassemia and sickle cells anemia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The iron metabolism of malignant cells, which is altered to ensure higher acquisition and utilization, motivates the investigation of iron chelation strategies in cancer treatment. In a prochelation approach aimed at increasing intracellular specificity, disulfide reduction/activation switches are incorporated on iron-binding scaffolds resulting in intracellularly activated scavengers. Herein, this strategy is applied to several tridentate donor sets including thiosemicarbazones, aroylhydrazones and semicarbazones. The novel prochelator systems are antiproliferative in breast adenocarcinoma cell lines (MCF-7 and metastatic MDA-MB-231) and do not result in the intracellular generation of oxidative stress. Consistent with iron deprivation, the tested prochelators lead to cell-cycle arrest at the G 1 /S interface and induction of apoptosis. Notably, although hemoglobin-synthesizing blood cells have the highest iron need in the human body, no significant impact on hemoglobin production was observed in the MEL (murine erythroleukemia) model of differentiating erythroid cells. This study provides new information on the intracellular effects of disulfide-based prochelators and indicates aroylhydrazone (AH1-S) 2 as a promising prototype of a new class of antiproliferative prochelator systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Increased hemoglobin (Hb) accumulation was reported in abdominal aortic aneurysms (AAAs). CD163 is a macrophage receptor involved in tissue Hb clearance, however its role in AAA has not been reported. We investigated the role of Hb on monocyte recruitment and differentiation towards CD163 expressing macrophages ex vivo, in vitro and in human AAA., Methods and Results: CD163 mRNA and protein expression was significantly higher in human AAA (n=7) vs. healthy wall (n=6). CD163 was predominantly found in adventitia of AAA, coinciding with areas rich in hemosiderin and adjacent to neoangiogenic microvessels. Dual CD14/CD163 expression was observed in recently infiltrated monocytes surrounding microvessels. A higher release of soluble CD163 was observed in the conditioned medium from AAA (AAA-CM, n=10), mainly in the adventitial layer. Similar to Hb, AAA-CM induced CD163-dependent monocyte chemotaxis, especially on circulating monocytes from AAA patients. Hb or AAA-CM promoted differentiation towards CD163(high)/HLA-DR(low)-expressing macrophages, with enhanced Hb uptake, increased anti-inflammatory IL-10 secretion and decreased pro-inflammatory IL-12p40 release. All these effects were partially suppressed when Hb was removed from AAA-CM. Separate analysis on circulating monocytes reported increased percentage of pre-infiltrating CD14(++)CD16(+) monocytes in patients with AAA (n=21), as compared to controls (n=14). A significant increase in CD163 expression in CD14(++)CD16(+) monocyte subpopulation was observed in AAA patients., Conclusions: The presence of Hb in the adventitial AAA-wall promotes the migration and differentiation of activated circulating monocytes in AAA patients, explaining the existence of a protective CD163-macrophage phenotype that could take up the Hb present in the AAA-wall, avoiding its injurious effects., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Astrocytes and microglia become reactive in many neurological disorders resulting in phenotypic and functional alterations. Both cell types might also display functional changes during normal aging. To identify gene signatures and changes in basal cellular functions of astrocytes and microglia in relation to aging, we isolated viable astrocytes and microglia from young adult and aged mouse cortices and determined their gene expression profile. Aged astrocytes, compared with young astrocytes, showed an increased inflammatory phenotype and increased 'zinc ion binding.' Young astrocytes showed higher expression of genes involved in 'neuronal differentiation' and hemoglobin synthesis. Astrocyte expression of genes involved in neuronal signaling remains high throughout age. Aged microglia had higher expression of genes involved in 'vesicle release,' 'zinc ion binding,' and genes within the tumor necrosis factor-ligand family and young microglia had increased transcript levels of C-C motif chemokines. These data provide a transcriptome database of cell-type enriched genes of astrocytes and microglia from adult mice and give insight into the differential gene signature of astrocytes and microglia in relation to normal aging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this issue of Blood, Cooke et al demonstrate the potential of a fully human anti-hepcidin antibody as a novel therapeutic for iron-restricted anemias such as anemia of inflammation, cancer, or chronic kidney disease (formerly known as “anemia of chronic diseases”).

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Changes in the endocannabinoid system are implicated in numerous diseases, making it an attractive target for pharmaceutical development. The endocannabinoid receptors have traditionally been thought to act through the effects of lipophilic messengers called cannabinoids. The exciting finding of endocannabinoid system modulation by the nonapeptide hemopressin and its N-terminal extensions has highlighted the complexity of cannabinoid biology and pharmacology and sparked interest for therapeutic purposes. However, many questions surrounding the generation and regulation of the hemopressin peptides, the self-assembly of hemopressin and the potential for drug development based on hemopressin remain and are discussed in this review., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Since the approval of recombinant insulin from Escherichia coli for its clinical use in the early 1980s, the amount of recombinant pharmaceutical proteins obtained by microbial fermentations has significantly increased. The recent advances in genomics together with high throughput analysis techniques (the so-called-omics approaches) and integrative approaches (systems biology) allow the development of novel microbial cell factories as valuable platforms for large scale production of therapeutic proteins. This review summarizes the main achievements and the current situation in the field of recombinant therapeutics using yeast Saccharomyces cerevisiae as a model platform, and discusses the future potential of this platform for production of blood proteins and substitutes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Benzene is a common occupational hazard and a ubiquitous environmental pollutant. Benzene exposure at the levels even below 1ppm still showed hematotoxicity. It is widely accepted that the metabolites of benzene play important roles in the benzene toxicity to the hematopoietic system, but little is known about the effects of benzene metabolites on erythropoiesis. In present study, erythroid progenitor-like K562 cells were used to determine the effects of phenolic metabolites of benzene, including phenol, hydroquinone and 1,2,4-benzenetriol, on the erythroid differentiation. After the treatment with these benzene metabolites at the concentrations with no obvious cytotoxicity, the hemin-induced hemoglobin synthesis in K562 cells decreased in a concentration- and time-dependent manner, and the expression of CD71 and GPA protein on the surface of K562 cells was also inhibited. The reverse transcription-PCR was used to determine the mRNA level of the erythroid related genes in the K562 cells that were treated with benzene metabolites. The hemin-induced expression of globin genes, including α-, β- and γ-globin genes, and the gene encoding the heme synthesis enzyme porphobilinogen deaminase was inhibited by benzene metabolites. When the K562 cells were pretreated with benzene metabolites, the hemin-induced expression of two transcription factor genes GATA-1 and NF-E2 was distinctly reduced, and the pre-treatment with benzene metabolites promoted the decrease of the mRNA level of transcription factor gene GATA-2 by hemin. These results indicated that benzene metabolites inhibited the hemin-induced erythroid differentiation through affecting the transcription of the erythroid related genes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

We used pulsed stable isotope labeling of amino acids in cell culture (pSILAC) to assess protein dynamics during monocyte-macrophage differentiation. pSILAC allows metabolic labeling of newly synthesized proteins. Such de novo protein production was evaluated from 3 to 7 days in culture. Proteins were identified by liquid chromatography-tandem mass spectrometry then quantified by MaxQuant. Protein-protein linkages were then assessed by Ingenuity Pathway Analysis. Proteins identified were linked to cell homeostasis, free radical scavenging, molecular protein transport, carbohydrate metabolism, small molecule chemistry, and cell morphology. The data demonstrates specific biologic events that are linked to monocyte transformation in a defined biologic system., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as alpha-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

Objective: There is limited and controversial information on whether anaemia is a risk factor for cardiovascular mortality in type 2 diabetes, and whether this risk is modified by the presence of chronic kidney disease (CKD). We assessed the predictive role of lower hemoglobin concentrations on all-cause and cardiovascular mortality in a cohort of type 2 diabetic individuals., Methods: The cohort included 1153 type 2 diabetic outpatients, who were followed for a mean period of 4.9 years. The independent association of anaemia (i.e., hemoglobin <120 g/l in women and <130 g/l in men) with all-cause and cardiovascular mortality was evaluated by Cox proportional hazards regression models and adjusted for several potential confounders, including kidney function measures., Results: During follow-up, 166 (14.4%) patients died, 42.2% (n=70) of them from cardiovascular causes. In univariate analysis, anaemia was associated with increased risk of all-cause (hazard ratio HR 2.62, 95% confidence intervals 1.90-3.60, p<0.001) and cardiovascular mortality (HR 2.70, 1.67-4.37, p<0.001). After adjustment for age, sex, body mass index, smoking, hypertension, dyslipidemia, diabetes duration, hemoglobin A1c, medication use (hypoglycemic, anti-hypertensive, lipid-lowering and anti-platelet drugs) and kidney function measures, the association of anaemia with all-cause (adjusted HR 2.11, 1.32-3.35, p=0.002) and cardiovascular mortality (adjusted HR 2.23, 1.12-4.39, p=0.020) remained statistically significant., Conclusions: Anaemia is associated with increased risk of all-cause and cardiovascular mortality in type 2 diabetic individuals, independently of the presence of CKD and other potential confounders. The advantage to treat anaemia in type 2 diabetes for reducing the risk of adverse cardiovascular outcomes remains to be demonstrated., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Radio-toxins are toxic metabolites produced by ionizing irradiation and have toxic effects similar to those caused by direct irradiation. We have investigated the effect of a quinoid radio-toxin (QRT) obtained from gamma-irradiated potato tuber on various organs in mice using ex vivo and in vivo EPR spectroscopy. Results indicate a decrease in the activity of ribonucleotide reductase enzyme in spleen of mice treated with 0.2mg QRT. A dose of 2mg QRT was fatal to mice within 45-60 min of treatment. Nitrosyl hemoglobin complexes alpha-(Fe(2+)-NO)alpha-(Fe(2+))beta-(Fe(2+))(2) were detected from spleen, blood, liver, kidney, heart, and lung tissue samples of mice treated with lethal doses of QRT. A significant decrease of pO(2) in liver and brain was observed after administration of QRT at the lethal dose. The time of the appearance of the nitrosyl hemoglobin complex and its intensity varied with the dose of QRT and the type of tissue. These results indicate that the effect of the QRT is more prominent in spleen and to a lesser extent in liver and blood. The QRT action at the lethal doses resulted in an increased hypoxia over time with disruption of compensatory adaptive response. The results indicate similar outcome of QRT as observed with gamma-irradiation.

Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In anemia of chronic disease, iron is withheld by macrophages, leaving extracellular fluids iron-depleted. In Friedreich ataxia, iron levels rise in the mitochondria of excitable cells but decrease in the cytosol. We explored the possibility of using deferiprone, a membrane-permeant iron chelator in clinical use, to capture labile iron accumulated in specific organelles of cardiomyocytes and macrophages and convey it to other locations for physiologic reuse. Deferiprone's capacity for shuttling iron between cellular organelles was assessed with organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy imaging. Deferiprone facilitated transfer of iron from extracellular media into nuclei and mitochondria, from nuclei to mitochondria, from endosomes to nuclei, and from intracellular compartments to extracellular apotransferrin. Furthermore, it mobilized iron from iron-loaded cells and donated it to preerythroid cells for hemoglobin synthesis, both in the presence and in the absence of transferrin. These unique properties of deferiprone underlie mechanistically its capacity to alleviate iron accumulation in dentate nuclei of Friedreich ataxia patients and to donate tissue-chelated iron to plasma transferrin in thalassemia intermedia patients. Deferiprone's shuttling properties could be exploited clinically for treating diseases involving regional iron accumulation.

Developing erythroid cells are dependent on transferrin (Tf) to acquire iron in amounts sufficient for hemoglobin production. Previously, we showed that although these cells cannot grow in culture in the absence of Tf, ferritin (Ft) can substitute Tf to some extent and support the development of hemoglobin-containing cells. In the current study, we investigated the ability of various iron sources to replace Tf in cultures of normal human erythroid precursors. The results showed that whereas Ft and hemin supported erythroid cell proliferation and hemoglobinization in Tf-free cultures to some extent, ferric amonium citrate and iron complexed with several chelators had little or no effect. Although salicylaldehyde-isonicotinoyl-hydrazone, which is a tridentate lipid-soluble chelator, complexed with iron increased both cytosolic and mitochondrial labile iron pools, it failed to support heme synthesis and did not decrease the surface Tf receptors, suggesting that its iron is not recognized by the cells. Moreover, this iron-chelator complex did not support erythroid precursor proliferation and hemoglobinization. Thus, although under normal conditions, Tf is the major route of iron uptake, Ft and hemin, but not iron-chelator complexes, may serve as alternative iron sources under Tf-poor conditions.

Low iron status is known to increase the uptake of dietary cadmium in both adolescents and adults and there are indications that cadmium is absorbed from the intestine by the two major iron transporters divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1). In addition, it has been suggested that duodenal metallothionein (MT) may limit the transport of cadmium across the intestinal epithelium. The present investigation was undertaken to examine whether iron status influences cadmium absorption in newborns by applying a model of suckling piglets and the possible roles of duodenal DMT1, FPN1 and MT. An oral cadmium dose (20 microg/kg body weight) was given daily for 6 consecutive days on postnatal days (PNDs) 10-15 to iron-deficient or iron-supplemented piglets. The cadmium dose was chosen to keep the cadmium level at a realistically low but still detectable level, and without inducing any adverse health effects in the piglets. As indicators of cadmium uptake, cadmium levels in blood and kidneys were measured on PND 16 by inductively coupled plasma-mass spectrometry (ICP-MS). Cadmium levels in blood were statistically significantly correlated with cadmium levels in kidneys. The cadmium uptake was not higher in iron-deficient suckling piglets; rather, we detected a higher cadmium uptake in the iron-supplemented ones. The expression and localisation of DMT1, FPN1 and MT were not affected by iron status and could therefore not explain the findings. Our results suggest that there are developmental differences in the handling of both iron and cadmium in newborns as compared to adults.

Anemia is more common among older blacks than older whites. However, it is unclear whether anemia predicts adverse events similarly in both races. Data on 1018 black and 1583 white adults aged 71 to 82 years were analyzed. Anemia, as defined by World Health Organization (WHO) criteria, was used to predict mortality over 6 years and incidence of mobility disability over 4 years. In proportional hazards models of mortality in whites, the age-adjusted hazard ratio (HR) for anemia in men was 1.96 (95% confidence interval [CI]: 1.35, 2.83) and in women was 2.86 (95% CI: 1.69, 4.82). In contrast, anemia was not associated with mortality in black men (HR = 1.15 [95% CI: 0.77, 1.72]) or women (HR = 1.39 [95% CI: 0.91, 2.14]). Higher mortality rate was observed only in black men with hemoglobin values more than 20 g/L (2.0 g/dL) below the WHO cutoff, whereas mortality rates were elevated in white men with hemoglobin values 1 to 10, 11 to 20, and more than 20 g/L below the WHO cutoff. In conclusion, anemia was significantly associated with increased risk of death and mobility disability in community-dwelling older whites. Conversely, older blacks classified as anemic by WHO criteria were not at risk for adverse events, indicating that alternative criteria are warranted.

The transcription factor PU.1 plays a central role in development and differentiation of hematopoietic cells. Evidence from PU.1 knockout mice indicates a pivotal role for PU.1 in myeloid lineage and B-lymphocyte development. In addition, PU.1 is a key player in the development of Friend erythroleukemia disease, which is characterized by proliferation and differentiation arrest of proerythrocytes. To study the role of PU.1 in erythroleukemia, we have used murine erythroleukemia cells, isolated from Friend virus-infected mice. Expression of PU.1 small interfering RNA in these cells led to significant inhibition of PU.1 levels. This was accompanied by inhibition of proliferation and restoration in the ability of the proerythroblastic cells to produce hemoglobin, i.e., reversion of the leukemic phenotype. The data suggest that overexpression of PU.1 gene is the immediate cause for maintaining the leukemic phenotype of the disease by retaining the self-renewal capacity of transformed erythroblastic cells and by blocking the terminal differentiation program towards erythrocytes.

Purpose: Angiogenesis in advanced breast cancers is highly distorted and heterogeneous. Noninvasive imaging that can monitor angiogenesis may be invaluable initially for diagnosis and then for assessing tumor response to treatment. By combining ultrasound (US) and near-infrared (NIR) optical imaging, a reliable new technique has emerged for localizing and characterizing tumor angiogenesis within the breast., Methods: This new technique employs a commercial US transducer coupled with an array of NIR optical fibers mounted on a hand-held probe. The US image is used for lesion localization and for guiding optical imaging reconstruction. Optical sensors are used for imaging tumor total hemoglobin distribution, which is directly related to tumor angiogenesis., Results: Six large breast carcinomas were studied and microvessel density count was then performed on tissue samples obtained from these cancers. Two patients had locally advanced breast cancers and received neoadjuvant chemotherapy for 3 months. In one patient, before chemotherapy, the total hemoglobin distribution showed a high concentration at the cancer periphery; the distribution was later confined to the core area after 3 months of treatment. In another patient, as treatment progressed, the maximum hemoglobin concentration decreased from 255.3, to 147.5, to 76.9 micromol/l with an associated reduction in spatial extension. The other four patients had cancers of 2.0 to 3.0 cm in size and were imaged either at the time of core biopsy or definitive surgery. The histologic microvessel density counts from these tumor samples correlate to hemoglobin distributions with a correlation coefficient of 0.64 (P < .05)., Conclusion: These initial results suggest that this new imaging technique may have great potential in imaging the heterogeneous vascular distribution of larger breast cancers in vivo and in monitoring treatment-related changes in angiogenesis during chemotherapy.

We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factor beta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 +/- 1.0%; EPO + TGF-B = 10.8 +/- 1.9%; EPO + SCF = 19.1 +/- 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 +/- 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 +/- 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 +/- 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation.

A saturated nitric oxide (NO) solution (1.88 mM) infused i.v. in the anesthetized pig at a dose of 68 nmol/kg/min for 24 min resulted in a time-dependent increase of nitrosylhemoglobin [HbFe(II)NO] as determined by electron spin resonance (ESR), reaching a C(max) of 7.99 +/- 0.42 microM at the end of the infusion, compared to 1.13 +/- 0.42 microM before (p < 0.01). This indicates that NO i.v. is efficiently bioconserved as HbFe(II)NO (approximately 34% of the NO dose) and to a greater extent than by the oxidative pathway (approximately 24% of the NO dose), as determined by measuring plasma nitrites/nitrates (chemiluminescence) and Met-Hb (ESR analysis). When the NO infusion was stopped, HbFe(II)NO declined with a t(1/2) of 15 min, indicating that it is a stable storage form of NO, able to deliver NO distally to the site of administration. No significant differences were observed in systemic and pulmonary vascular resistances during and after NO infusion, but PO(2) showed a significant decrease 15 and 30 min after the infusion. Thus, in normoxic/physiological conditions, HbFe(II)NO does not induce significant NO-dependent vasorelaxation.

Tellimagrandin I is a hydrolysable tannin compound widely present in plants. In this study, the effect of tellimagrandin I on chemically induced erythroid and megakaryocytic differentiation was investigated using K562 cells as differentiation model. It was found that tellimagrandin I not only inhibited the hemoglobin synthesis in butyric acid (BA)- and hemin-induced K562 cells with IC50 of 3 and 40microM, respectively, but also inhibited other erythroid differentiation marker including acetylcholinesterase (AChE) and glycophorin A (GPA) in BA-induced K562 cells. Tellimagrandin I also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of CD61 protein, a megakaryocytic marker. RT-PCR analysis showed that tellimagrandin I decreased the expression of erythroid genes (gamma-globin and porphobilinogen deaminase (PBGD)) and related transcription factors (GATA-1 and NF-E2) in BA-induced K562 cells, whereas tellimagrandin I induced the overexpresison of GATA-2 transcription factor that played negative regulation on erythroid differentiation. These results indicated that tellimagrandin I had inhibitory effects on erythroid and megakaryocytic differentiation, which suggested that tannins like tellimagrandin I might influence the anti-tumor efficiency of some drugs and the hematopoiesis processes.

Cardiac T2* (magnetic resonance imaging relaxation parameter) is abnormally low in approximately 40% of adults with thalassemia major (TM), suggesting myocardial iron deposition, but it is unknown at what age this occurs. To address this question, we measured cardiac T2* and function in 19 young patients (aged 7-26 years) with TM as well as 17 patients receiving long-term transfusions for sickle cell anemia (SCA) matched for age, sex, and liver iron content. Cardiac T2* was normal in all of the SCA patients but was low (high iron) in 8 of 19 TM patients. Abnormal T2* was observed only in the TM patients receiving transfusions for 13 years or longer and was correlated with ferritin but not liver iron levels. Cardiac dysfunction was present in 3 of the 8 patients with low T2*. Cardiac T2* changes have a long latency relative to liver iron accumulation. Total transfusional burden is a significant independent risk factor for low cardiac T2* and may partially account for differences observed between patients with SCA and TM.

Haemoglobin acts as an important protein for oxygen carrier in all living beings. Purification of donkey haemoglobin was done using PEG 4000 as a separating medium. Crystallization was achieved using hanging drop vapor diffusion method using 2.8 M phosphate buffer, pH 6.8. Data collection was done using mar345 image plate detector system. The crystals grown under 2.8 M phosphate buffer are monoclinic with space group C2 and cell dimensions a=107.664 A, b=63.084 A, c=54.042 A, and beta=111.747 degrees.

Insulinoma is a rare, almost always benign endocrine tumor of the pancreas, clinically characterized by hyperinsulinemic, hypoglycemic episodes. Surgical excision is the therapy of choice, which may lead to postpancreatectomy diabetes mellitus in the case of extensive pancreatic resection. We present the cases and the metabolic follow up of two patients, 81 and 73 years old, with insulinoma localized close to the main duct in the pancreatic neck. Both patients underwent an 80% left pancreatectomy, avoiding a pancreatico-enteric anastomosis. In order to prevent postpancreatectomy diabetes, the islets from the tumor-free part of the resected pancreas were isolated and injected via a right colic vein into the portal system. After a follow up of 6 and 3 years respectively, both patients remained insulin-independent without any dietary restrictions. Fasting and glucagon-stimulated C-peptide-levels and glycosylated hemoglobin remained within normal range. There were no signs of recurrent insulinoma. Liver biopsy performed in one patient at 1 year after autotransplantation, showed intact, insulin-producing islets within the portal spaces. In conclusion, autologous islet transplantation can preserve the insulin secretory reserve after extended left pancreatectomy for the treatment of benign tumors in the pancreatic neck.

Hydroxyurea (HU) enhances fetal hemoglobin (Hb) production. An increase in total Hb level has been repeatedly reported during HU treatment in patients with sickle cell disease and in several patients with beta-thalassemia intermedia. Effects in patients with beta-thalassemia major are controversial. We now report a marked elevation of total Hb levels with HU that permitted regular transfusions to be stopped in 7 children with transfusion-dependent beta-thalassemia. The median follow-up was 19 +/- 3 months (range, 13-21 months). We conclude that HU can eliminate transfusional needs in children with beta-thalassemia major, which could be particularly useful in countries such as Algeria, where supplies of blood or chelating agents are limited.

Hemoglobins in unicellular organisms, like the one here in the bacterium Vitreoscilla, have greater chemical reactivity than their homologues in multicellular organisms. They can catalyze redox reactions and may protect cells against oxidative stress. The ability of Vitreoscilla hemoglobin to complement deficiencies of terminal cytochrome oxidases in Escherichia coli also suggests that this hemoglobin can receive electrons during respiration. In this study, a recombinant strain of Enterobacter aerogenes engineered to produce the Vitreoscilla Hb was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress produced by exogenously applied hydrogen peroxide was characterized in terms of cell growth, survival and the activities of two key antioxidant enzymes (catalase and superoxide dismutase). The influence of the physiological state of the cells and different media upon these culture dynamics was determined. Results showed that the hemoglobin-expressing strain is quite distinct in terms of growth/survival properties and activity of antioxidant enzymes from that of non-hemoglobin counterparts.

Our previous assembly studies to form hemoglobin hetero-dimers and -tetramers using a coupled transcription/translation cell-free system suggested that alpha-globin chains bind to nascent non-alpha chains during and/or soon after translation to promote hemoglobin formation [Adachi et al., J. Biol Chem. 2002 (277) 13415]. In this report effects of CN-hemin on subunit assembly were studied using this cell-free system. Addition of CN-hemin and excess unlabeled heme-containing partner chains during synthesis leads to formation of radiolabeled heme-containing alpha(h)beta(h) hetero-dimers. In contrast, in the absence of added CN-hemin, unlabeled heme-containing alpha or beta chains can assemble with newly synthesized radiolabeled beta- and alpha-globin chains to form heme-containing alpha(h)beta(h) and semi-alpha (alpha(h)beta(0)) or semi-beta (alpha(0)beta(h)) hetero-dimers, respectively. These results suggest the existence of semi-hemoglobins as intermediates prior to formation of heme-containing alpha(h)beta(h) and indicate transfer of heme from alpha and/or beta chains into semi-hemoglobin hetero-dimers to form heme-containing hetero-dimers.

Bcl-X(L) is essential for the survival and normal maturation of erythroid cells, especially at the late stage of erythroid differentiation. It remains unclear whether Bcl-X(L) serves only as a survival factor for erythroid cells or if it can induce a signal for differentiation. We have previously shown that dimethyl sulfoxide (DMSO) induction of erythroid differentiation in murine erythroleukemia (MEL) cells correlates with delay of apoptosis and specific induction of Bcl-X(L). In this study, we investigate the contribution of Bcl-2 and Bcl-X(L) to survival and erythroid differentiation by generating stable MEL transfectants expressing these antiapoptotic regulators. Overexpression of Bcl-2 completely prevented apoptosis of MEL cells before and after DMSO induction, whereas overexpression of Bcl-X(L) only delayed it. Overexpression of Bcl-2 or Bcl-X(L) neither induced spontaneous erythroid differentiation nor accelerated DMSO-induced differentiation. Inhibition of Bcl-X(L) by antisense transcripts accelerated apoptosis in DMSO-treated MEL cells and blocked the synthesis of hemoglobin without altering the growth arrest associated with terminal erythroid differentiation. An antisense oligonucleotide to Bcl-X(L) did not induce apoptosis in MEL cells overexpressing Bcl-2 but greatly decreased their hemoglobin synthesis when treated with DMSO, suggesting that Bcl-X(L) is necessary for erythroid differentiation independently of its antiapoptotic function. Importantly, Bcl-X(L) antisense transcripts prevented heme synthesis but not globin mRNA induction in DMSO-treated MEL cells. Furthermore, inhibition of hemoglobin synthesis by Bcl-X(L) antisense was reversed by addition of exogenous hemin. Finally, Bcl-X(L) localized to mitochondria during MEL erythroid differentiation, suggesting that it may mediate a critical mitochondrial transport function related to heme biosynthesis.

The design and evaluation of therapies for the sickle cell and beta-thalassemia syndromes rely on our understanding of hemoglobin accumulation during human erythropoiesis. Here we report direct measurements of hemoglobin composition and messenger RNA (mRNA) levels in cultured CD34(+) cells and correlate those measurements with studies of freshly obtained bone marrow samples. Hemoglobin levels in differentiating cells were also compared with morphologic, immunophenotypic, and cell cycle assessments. A population of large preproerythroblasts was first identified within 24 hours and became the dominant population by day 5. The transition from proerythroblast to basophilic normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1% +/- 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts were the dominant and final cell type by day 13. High-performance liquid chromatography-based quantitation of fetal (HbF) and adult (HbA) hemoglobin and real-time polymerase chain reaction globin mRNA quantitation demonstrated a coordinate rise in the accumulation of both proteins and mRNA among these developmentally staged populations. Quantitative analyses on freshly sorted bone marrow populations demonstrated a similar rising pattern with beta-globin and HbA as the dominant species at both early and late stages of differentiation. We found no evidence for HbF dominant populations or switching during differentiation in adult cells. Instead, rapid increases in both HbF (heterocellular) and HbA (pancellular) content were observed, which coincided with the apex in cell cycling and the proerythroblast-basophilic normoblast transition. Based on these measurements, we conclude that HbF and HbA content are regulated with the rate of proliferation during adult erythropoiesis.

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.

We developed a method to measure hemoglobin synthesis rate (SynHb) in humans, assuming that free glycine in the red blood cell (RBC) represents free glycine in bone marrow for hemoglobin synthesis. The present rat study examines this assumption of the method and quantifies SynHb in rats. Sprague-Dawley rats (n = 9) were studied, [2-(13)C]glycine was intravenously infused over 24 h (2.5 mg kg(-1) h(-1)), blood was drawn for glycine and heme isolation, and bone marrow was harvested for glycine isolation. Isotopic enrichments of glycine and heme were measured, fractional hemoglobin synthesis rate (fSynHb% day(-1)) was calculated, and from this a value for SynHb (mg g(-1) day(-1)) was derived. Mean body weight was 446 +/- 10 g (mean +/- SE) and hemoglobin concentration was 14 +/- 0.5 g dl(-1). At 24 h, the mean isotopic enrichment, atom percentage excess (APE), of the RBC free glycine (1.56 +/- 0.18 APE) was similar to the bone marrow (1.68 +/- 0.15 APE). The rate of incorporation of (13)C into heme increased over time from 0.0004 APE/h between 6 and 12 h, to 0.0014 APE/h between 12 and 18 h, and 0.0024 APE/h between 18 and 24 h. Consequently, fSynHb (1.19 +/- 0.32, 2.92 +/- 0.66, and 4.22 +/- 0.56% day(-1), respectively) and SynHb (0.11 +/- 0.03, 0.28 +/- 0.05, and 0.42 +/- 0.05 mg g(-1) day(-1), respectively) showed similar patterns over the 24-h study period. We conclude that (1) enrichment of free glycine in the circulating RBC approximates enrichment of bone marrow free glycine for heme formation and (2) this pattern of hemoglobin synthesis rate is reflecting the characteristic release and gradual maturation of reticulocytes in the circulation., (Copyright 2001 Academic Press.)

Scalarane-type sesterterpenes, PHC-1-PHC-7, which have been isolated from a marine sponge, increased hemoglobin production in human chronic myelogenous leukemia cell line K562 at the concentration of 0.1-5 microg/ml. PHC-1, the major constituent, induced the expression of glycophorin A and the enucleation for K562 cells. These sesterterpenes were found to induce erythroid differentiation in K562 cells. In addition, PHC-1 induced G1 arrest of the cell cycle of K562 cells., (Copyright 2001 Academic Press.)

Erythroid Kruppel-like factor (EKLF) is a transcription factor of the C2H2 zinc-finger class that is essential for definitive erythropoiesis. We generated immortal erythroid cell lines from EKLF(-/-) fetal liver progenitor cells that harbor a single copy of the entire human beta-globin locus and then reintroduced EKLF as a tamoxifen-inducible, EKLF-mutant estrogen receptor (EKLF-ER) fusion protein. Addition of tamoxifen resulted in enhanced differentiation and hemoglobinization, coupled with reduced proliferation. Human beta-globin gene expression increased significantly, whereas gamma-globin transcripts remained elevated at levels close to endogenous mouse alpha-globin transcript levels. We conclude that EKLF plays a role in regulation of the cell cycle and hemoglobinization in addition to its role in beta-globin gene expression. The cell lines we used will facilitate structural and functional analyses of EKLF in these processes and provide useful tools for the elucidation of nonglobin EKLF target genes.

Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2alpha kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2alpha. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.

Terminal differentiation of erythroid cells results in terminal cell divisions followed by irreversible cell cycle withdrawal of hemoglobinized cells. The mechanisms leading to cell cycle withdrawal were assessed in stable transfectants of murine erythroleukemia cells, in which the activities of cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs) could be tightly regulated during differentiation. Cell cycle withdrawal of differentiating cells is mediated by induction of several CDKIs, thereby leading to inhibition of CDK2 and CDK4. Manipulation of CDK activity in differentiating cells demonstrates that the onset of cell cycle withdrawal can be either greatly accelerated or greatly delayed without affecting hemoglobin levels. Extending the proliferation of differentiating cells requires the synergistic action of CDK2 and CDK4. Importantly, CDK6 cannot substitute for CDK4 in this role, which demonstrates that the 2 cyclin D-dependent kinases are functionally different. The results show that differentiating hemoglobinized cells can be made to proliferate far beyond their normal capacity to divide. (Blood. 2000;96:2755-2764)

Mice lacking the erythroid Kruppel-like factor (EKLF) die in utero at embryonic day 15 (E15) from severe anemia. EKLF(-/-) embryos display a marked deficit in beta-globin gene expression. To test whether beta-globin deficiency was solely responsible for the anemia and intrauterine death, we corrected the globin chain imbalance in EKLF(-/-) embryos by breeding with a strain of mice that express high levels of human gamma-globin. Despite efficient production of hybrid malpha(2)-hgamma(2) hemoglobin in the fetal livers of EKLF(-/-) animals, hemolysis was not corrected and survival was not prolonged. We concluded that deficiency of nonglobin EKLF target genes is a major contributor to the definitive red blood cell abnormalities and prenatal death in EKLF(-/-) embryos. These results suggest that strategies designed to antagonize EKLF function in adults with hemoglobinopathy, in an attempt to reactivate gamma-globin gene expression, may adversely affect other essential aspects of red blood cell physiology. (Blood. 2000;95:1827-1833)

The transcription factor CP2 was initially identified to bind to the promoter region of the murine alpha-globin gene and known to stimulate the expression of alpha-globin by increasing CP2 transcripts 3- to 5-fold during induced differentiation of mouse erythroleukemic (MEL) cells in vitro. Here, we report that this increment of CP2 expression is crucial in erythroid-specific globin gene expression and hemoglobin synthesis. When antisense CP2 was overexpressed in MEL cells, production of endogenous CP2 protein was reduced 70-80%, and significant loss of its promoter binding activity was observed. During HMBA-induced terminal differentiation of antisense CP2 expressing MEL cells, the transcription of endogenous alpha-globin gene was suppressed as expected. Moreover, both beta-globin gene expression and hemoglobin synthesis were also severely impaired, without affecting the expression of key heme enzyme genes or HMBA-induced proliferation and viability., (Copyright 1999 Academic Press.)

Erythrocyte production in mammals is known to depend on the exposure of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red blood cell differentiation events is largely unresolved. To address this issue, mechanisms of induced globin expression in Epo-responsive SKT6 cells have been investigated. Chimeric receptors containing an epidermal growth factor (EGF) receptor extracellular domain and varied Epo receptor cytoplasmic domains first were expressed stably at physiological levels in SKT6 cells, and their activities in mediating induced hemoglobinization were assayed. While activity was exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced differentiation signaling. Moreover, mutation of a STAT5 binding site in this construct (EE372-Y343F) inhibited induced globin expression and SKT6 cell hemoglobinization, as did the ectopic expression of dominant-negative forms of STAT5 in parental SKT6 cells. As in normal CFU-e, SKT6 cells also were shown to express functional receptors for stem cell factor (SCF). To further define possible specific requirements for differentiation signaling, effects of SCF on SKT6 cell hemoglobinization were tested. Interestingly, SCF not only failed to promote globin expression but inhibited this Epo-induced event in a dose-dependent, STAT5-independent fashion. Thus, effects of Epo on globin expression may depend specifically on STAT5-dependent events, and SCF normally may function to attenuate terminal differentiation while promoting CFU-e expansion., (Copyright 1998 by The American Society of Hematology.)

To approach the goal of consistent long-term erythropoietin (Epo) expression in vivo, we developed an implantation procedure in which transduced autologous vascular smooth muscle was introduced into rats in a chamber created from a polytetrafluoroethylene (PTFE) ring placed under the serosa of the stomach. The implant became vascularized and permitted the long-term survival of smooth muscle cells expressing Epo. Hematocrits of treated animals increased rapidly and monitored over 12 months gave a mean value of 56.0 +/- 4. 0% (P < .001; n = 9), increased from a presurgery mean of 42.3 +/- 1. 6%. Hemoglobin levels rose from a presurgery mean of 15.2 +/- 0.4 g/dL and for 12 months were significantly elevated with a mean value of 19.5 +/- 1.3 g/dL (P < .001; n = 9). The hematocrit and hemoglobin levels of control animals receiving human adenosine deaminase (ADA)-expressing cells were not significantly different from baseline (P > .05; n = 5). In response to tissue oxygenation, kidney, and (to a lesser extent) liver are specific organs that synthesize Epo. Treated animals showed downregulation of endogenous Epo mRNA in kidney over a 12-month period. The PTFE implant provides sustained gene delivery, is safe, and is minimally invasive. It allows easy engraftment of transduced cells and may be applied generally to the systemic delivery of therapeutic proteins such as hormones and clotting factors., (Copyright 1998 by The American Society of Hematology.)

The product of the SCL gene is a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of hematopoietic stem cells in both the embryo and the adult. However, once the stem cell compartment is established, the function of SCL in subsequent differentiation and commitment events within normal hematopoietic cells remains undefined. The aim of the current study was to investigate this role using purified normal human hematopoietic CD34(+) cells. An SCL retrovirus was used to transduce CD34(+) cells isolated from human bone marrow, peripheral blood, and umbilical cord blood. Enforced expression of SCL increased by a median of twofold the number of erythroid colonies, with an increase in both colony size and the rate of hemoglobinization. Unexpectedly, enforced expression in CD34(+) cells also significantly increased the number of megakaryocyte colonies, but with no impact on the size of colonies. There was no consistent effect on the number nor size of granulocyte-macrophage (GM) colonies. The proliferative effect of enforced SCL expression on erythroid cells was attributed to a shortened cell cycle time; the self-renewal capacity of erythroid or GM progenitors was unchanged, as was survival of cells within colonies. These results demonstrate a role for SCL in determining erythroid and megakaryocyte differentiation from normal human hematopoietic CD34(+) cells.

We have demonstrated that iron controls hemoglobin (Hb) synthesis in erythroid differentiating K562 cells by enhancing the activity of a key enzyme of the Hb synthesis, delta-aminolevulinate synthase (ALAS). In the present study, we studied iron mobilization and the role of iron in erythroid differentiating cells by measuring the level of iron by means of high-performance liquid chromatography using electrochemical detection (HPLC-ED). After treatment of K562 cells with sodium butyrate, the expression of transferrin receptor (TfR) increased initially, followed by an increase in the levels of both total iron and Hb as well as the ALAS activity. However, no increase could be found in the levels of non-heme iron, low-molecular-mass iron (LMMFe) and ferritin. Addition of diferric transferrin (FeTf) enhanced both delta-aminolevulinic acid (ALA) and Hb synthesis. In contrast, addition of hemin elevated the levels of all iron species as well as the Hb synthesis but reduced the TfR expression and ALA contents in both butyrate treated and untreated cells. These results suggest that Hb synthesis is controlled by TfR expression, and that the ALA synthesis is suppressed by iron released from heme and/or Hb due to lowered expression of TfR.

In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.

Erythropoietin (EP) is required by late-stage erythroid progenitor cells to prevent apoptosis. Several lines of evidence suggest that it is this action of EP that regulates erythrocyte production in vivo. To study the control of apoptosis in mouse and human erythroblasts, the expression of members of the Bcl-2 family of proteins and the expression and activation of the apoptosis-linked cysteine protease Yama/CPP32/apopain were examined. These proteins have been implicated as regulators of apoptosis in several cell models. The Bcl-2 family members analyzed were Bcl-2, Bcl-X, Bax, Bad, Bak, A1, and Mcl-1. Bcl-X expression in proerythroblasts was highly EP-dependent. Bcl-X was strongly increased during the terminal differentiation stages of human and mouse erythroblasts, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression led to an apparent level of approximately 50 times the level in proerythroblasts. In contrast, neither mouse nor human erythroblasts expressed Bcl-2 transcript or protein. Bax and Bad proteins remained relatively constant throughout differentiation, but diminished near the time of enucleation. Bak protein was present in early erythroblasts, but diminished progressively during differentiation. EP deprivation in both mouse and human erythroblasts led to activation of the cysteine protease, apopain, as was indicated by cleavage of the proenzyme into its proteolytically active fragments. Apopain activation was detectable within 2 hours of EP deprivation in mouse erythroblasts. These findings suggest an important role for Bcl-X in late erythroid differentiation and for apopain in apoptosis of erythroblasts caused by deprivation of EP.

Ferritin (Ft) plays an important role in cellular iron metabolism. It can store substantial amounts of iron in a nontoxic soluble form. However, its ability to donate iron for cellular needs, in particular for hemoglobin (Hb) synthesis in human erythroid cells, is still controversial. We studied the role of intracellular Ft-iron in Hb synthesis and the involvement of lysosomal proteolysis in iron release from Ft. Ft-iron release and its subsequent incorporation into heme was investigated in normal human erythroid precursors developing in culture. Dual staining flow cytometry with antibody (Ab)-specific for Ft and for Hb showed a decrease in cellular Ft content in erythroid cells during their maturation. Cellular Ft-iron participation in heme synthesis was studied by labeling cells with 59Fe. Cells were incubated with 59Fe-labeled human diferric transferrin (Tf), then chased, and intracellular radioiron distribution between Ft and Hb was determined on subsequent days by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and/or Ft immunoprecipitation and heme extraction. On day 6, most of the 59Fe accumulated in Ft. Thereafter, a progressive decrease of radioiron in Ft and a corresponding increase of the label in Hb was observed. Inhibition of heme synthesis with succinylacetone caused radioiron to remain in Ft and prevented its redistribution. Addition of unlabeled diferric Tf to the culture medium did not prevent radioiron from appearing in Hb. Chloroquine repression of lysosomal function prevented radio-iron redistribution between Ft and Hb. Inhibition of proteolysis by chymostatin and/or leupeptin led to Ft-protein accumulation in the cells and also prevented radioiron transfer from Ft to Hb. The results of the present study suggest that intracellular Ft donates iron for heme synthesis and that proteolytic Ft degradation in a lysosomal-like compartment is necessary for iron release and its transfer to heme.

Erythropoietin (EPO) and its cell surface receptor (EPOR) play central roles in the proliferation and differentiation of mammalian erythroid progenitor cells. Recently both the tyrosine residues in the EPOR responsible for the activation of Stat5 and the role of Stat5 for EPO-dependent cell proliferation have been shown. Here, we describe the roles of Stat5 and of these tyrosine residues in the EPOR in the erythroid differentiation of murine hematopoietic cell line SKT6 which produces hemoglobin in response to EPO. Chimeric receptors carrying the extracellular domain of the EGF receptor and the intracellular domain of the EPOR were introduced into SKT6 cells. Like EPO, EGF equally activated Stat5 and induced hemoglobin. Activation of Stat5 and hemoglobin expression by EGF were markedly impaired in cells expressing the tyrosine mutated chimeric receptors. In addition, ectopic expression of the prolactin receptor, another cytokine receptor that activates Stat5, led to hemoglobin synthesis. Finally, hemoglobin synthesis was severely inhibited by overexpressing a dominant negative form of Stat5. These results collectively suggest that Stat5 plays a role in EPO-mediated hemoglobin synthesis in SKT6 cells.