Biomedical subjects

C T Noguchi

Publications and source records attributed to C T Noguchi.

At least 37 records · Page 2Linked to original sources

PubMed1996

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Adult↗

PubMed1996

Transgenic mice containing the human erythropoietin receptor gene exhibit correct hematopoietic and neural expression.

The erythropoietin receptor (EpoR), known for its role in the proliferation and differentiation of erythroid cells, has been detected in nonhematopoietic tissues. We have reported previously that in addition to hematopoietic expression, EpoR is expressed at high levels in embryonic mouse brain and decreased to nondetectable levels by birth. While using transgenic mice to characterize human EpoR expression, we observed that a 15-kb human EpoR transgene (expressed in hematopoietic tissues but at a reduced level) also exhibited in the embryonic brain low levels of expression that persisted through adulthood. To examine further the basis of tissue and developmental specificity of the human EpoR gene, we produced additional transgenic mice using an 80-kb human EpoR genomic fragment isolated from a P1 phagemid human library. We found that this transgene is expressed appropriately in hematopoietic tissues (including yolk sac, fetal liver, adult spleen, and bone marrow) at levels comparable to the endogenous murine EpoR. The 80-kb transgene also provided high-level expression in the embryonic brain that paralleled the levels of the endogenous murine EpoR and was no longer detectable after birth. These data suggest that the high level of embryonic brain expression may be relevant to the human EpoR gene and that the transgenic mouse with an 80-kb fragment is a suitable model for studying the regulation and possible functional importance of human EpoR expression in the developing embryo.

Animals↗

PubMed1995

Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs.

Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors AP2, Sp1 and the erythroid-specific GATA-1. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive hEpoR expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that Sp1 and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the Sp1 binding motif. By increasing GATA-1 levels via co-transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although GATA-1 mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the Sp1 site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing GATA-1 levels in OCIM1 cells. These data suggest that while GATA-1 can transactivate the EpoR promoter, the level of hEpoR gene expression does not depend on GATA-1 alone. Rather, hEpoR transcription activity depends on coordination between Sp1 and GATA-1 with other cell-specific factors, including possibly other Sp1-like binding proteins, to provide high level, tissue-specific expression.

Cell Line↗

PubMed1995

Hemin-induced acceleration of hemoglobin production in immature cultured erythroid cells: preferential enhancement of fetal hemoglobin.

The effects of heme, when added as the ferric chloride salt, hemin, on human erythroid cells grown in a two-phase liquid culture system were studied. When added together with erythropoietin, on initiation of the second phase of the culture, hemin greatly accelerated hemoglobin (Hb) accumulation in these cells. The effect was greater during their early stages of maturation, suggesting that heme availability is then a rate-limiting step for Hb synthesis. Hemin increased preferentially the production of fetal Hb (HbF) compared with adult Hb; this was associated with a selective twofold elevation in gamma-mRNA levels. Using succinylacetone, a potent inhibitor of heme synthesis, we showed that exogenously supplied hemin could be incorporated into the de novo formed Hb. Therefore, the mechanism of hemin action may be several fold, including effects on globin gene transcription and posttranslational events, eg, supplying the prosthetic group for Hb assembly. Hemin increased HbF of cells derived from patients with sickle cell anemia and beta-thalassemia as well as that of cells from normal donors. Moreover, when added in combination with other HbF-augmenting agents such as the cytotoxic drug, hydroxyurea, a synergistic effect was obtained, with considerably less cytotoxicity than with hydroxyurea alone. These results have clinical potential in light of the ameliorating effect that increased HbF has in patients with genetic diseases of the beta-globin chain and raise the possibility of combined treatment with hemin and other drugs now being used to treat these diseases.

Anemia, Sickle Cell↗

PubMed1995

Sickle cell rheology is determined by polymer fraction--not cell morphology.

Sickle cell disease pathophysiology is mediated by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle hemoglobin (Hb S) polymer as cells are partially deoxygenated in the microcirculation. We have recently developed a method to measure the relationship between the formation of intracellular polymerized Hb S and cell filtration. In this study, we have used this method to examine whether sickle cell morphology, independent of Hb S polymer fraction, had an effect on cell rheology. We primarily use sickle trait (AS) and Hb S-beta(+)-thalassemia (S-beta(+)-thal) erythrocytes with low hemoglobin F levels, which have normal membranes and few or no dense cells, to remove these confounding effects. We find that the relationship between filtration and the percentages of each "type" of morphological deformation of AS erythrocytes was different from that of the S-beta(+)-thal erythrocytes. In addition, we find that while the filtration of AS erythrocytes as a function of oxygen saturation was similar, whether measured during deoxygenation or reoxygenation, the relationship between the percentages of each type of deformed erythrocyte and oxygen saturation demonstrated hysteresis during oxygenation-deoxygenation experiments. Transmission electron microscopy, for both elongated and irregularly shaped cells, showed that similarly distorted cells could have very different amounts and alignment of polymer. These results suggests that cell morphology per se is not strongly related to filtration, whereas calculated intracellular Hb S polymer fraction predicts loss of filtration of AS and S-beta(+)-thal erythrocytes well. Measured or calculated polymer fraction values would appear to be a better parameter for the study of sickle cell disease pathophysiology and response to treatment than cell morphology studies.

Anemia, Sickle Cell↗

PubMed1995

Filterability of mixtures of sickle and normal erythrocytes.

We investigated the deformability of sickle (SS) cells from 25 patients and mixtures of these SS cells with blood type-compatible normal (AA) cells, using a nickel mesh filtration system, with the aim of determining optimal goals for exchange therapy. We found that for air-equilibrated SS/AA cell mixtures the fraction of dense cells (MCHC > 37 g/dl) is the determinant factor in filterability and that the dense cells contribute in a linear fashion to the loss of filtration up to 15% dense cells (y = -4.41x + 98.23, r = 0.945, P < 0.0001). The slope of this effect is approximately 25 times steeper than that of the relationship between filtration and percent nondense (MCHC < 37/g/dl) SS cells (y = -0.17x + 106.53, r = 0.772, P < 0.0001). A comparison of the proportion of high fluorescence reticulocytes to total reticulocytes (HFR ratio), indicating an elevation of immature reticulocytes, between six nontransfused patients and six exchange-transfused patients showed significant higher values in the nontransfused individuals (0.154 +/- 0.051 versus 0.070 +/- 0.054, P < 0.003). These results may have implications regarding targets for exchange transfusion therapy. Further studies of the effect on transfusion, both simple and exchange, on the numbers of dense cells and the proportions and populations of reticulocytes and the rheological characteristics of the erythrocyte subpopulations seems warranted.

Anemia, Sickle Cell↗

PubMed1995

Contributions of sickle hemoglobin polymer and sickle cell membranes to impaired filterability.

Sickle cell anemia is a disease of abnormal rheology caused by acute and reversible, as well as chronic and irreversible, changes in the properties and deformability of sickle erythrocytes. Deformability is determined by several factors, including intracellular sickle hemoglobin polymerization, the abnormal membrane properties of sickle cells, and the abnormal rheological properties of the soluble concentrated hemoglobin solution within dense sickle red blood cells. In this study, we used a 5-microns pore nickel mesh filter to evaluate quantitatively the effects of these factors on the filterability of erythrocytes containing sickle hemoglobin. We used sickle trait and sickle/beta(+)-thalassemia cells, because they have minimal membrane abnormalities or density heterogeneity, to investigate the effects of polymer formation on rheological properties. We found that filterability of these cells is sensitive to small amounts of intracellular polymer and that impaired filtration is linearly related to oxygen-dependent polymer formation, up to a polymer fraction of 0.3. By increasing the proportion of dense cells in populations of normal cells or cells from individuals with sickle syndromes and equilibrating these cells with gas ligands, we estimate that polymerization, even at 95% saturation, contributes twice as much to impaired filterability of sickle erythrocytes as the abnormal membranes in homozygous sickle cell disease. At lower saturation values, the effects of polymer are even greater. The viscosity of the concentrated hemoglobin in dense cells had the smallest effect, over physiologically relevant saturation values. These results emphasize the importance of sickle hemoglobin polymerization in the pathogenesis of sickle cell disease and should help define its pathophysiology and responses to therapy in quantitative terms.

Anemia, Sickle Cell↗

PubMed1994

Variation in fetal hemoglobin parameters and predicted hemoglobin S polymerization in sickle cell children in the first two years of life: Parisian Prospective Study on Sickle Cell Disease.

Intracellular hemoglobin S (HbS) polymerization is most likely to be the primary determinant of the clinical and biologic manifestations of sickle cell disease (SCD). Fetal hemoglobin (HbF) does not enter the HbS polymer and its intracellular expression in sickle erythrocytes inhibits polymerization. HbF levels, high at birth but decreasing thereafter, protect the newborn from the clinical manifestations of this hemoglobinopathy. We have measured the sequential changes in HbF, F reticulocytes, and F cells in the first 2 years of life in 25 children with SCD and compared the results with those obtained in 30 normal children (AA). We have also calculated HbF per F cell (F/F cell), the preferential survival of F cells versus non-F cells, as measured by the ratio F cells versus F reticulocytes (FC/FR) and polymer tendency at 40% and 70% oxygen saturation. HbF levels decreased from about 80.4% +/- 4.0% at birth to 9.2% +/- 2.9% at 24 months. During this time, we observed a regular decrease of the F reticulocytes and the F cells. The kinetics of the decline of F/F cell was comparable with the decline of HbF, rapid from birth (mean, 27.0 +/- 3.6 pg) to 12 months of age (mean, 8.5 +/- 1.5 pg) and then slower from 12 to 24 months of age (mean, 6.2 +/- 1.0 pg) in the SCD children. In the AA children, the decrease in HbF, due to changes in both numbers of F cells and F/F cell, was more precipitous, reaching steady-state levels by 10 months of age. Calculated values for mean polymer tendency in the F-cell population showed that polymerization should begin to occur at 40% oxygen saturation at about 3 months and increase progressively with age, whereas polymerization at 70% oxygen saturation would not occur until about 24 months. These values correspond to HbF levels of 50.8% +/- 10.8% and 9.2% +/- 2.9%, respectively, and F/F cell levels of 15.6 +/- 4.5 pg and 6.2 +/- 1.0 pg, respectively. In the non--F-cell population, polymerization was expected at birth at both oxygen saturation values. Three individuals had significantly greater predicted polymerization tendency than the remainder of the group because of early decreases in HbF. These individuals in particular, the remainder of the cohort, as well as other recruited newborns, will be studied prospectively to ascertain the relationship among hematologic parameters, which determine polymerization tendency and the various clinical manifestations of SCD.

Anemia, Sickle Cell↗

PubMed1994

Erythropoietin receptor mRNA expression in human endothelial cells.

A previous report demonstrated that endothelial cells have erythropoietin receptors and respond to this hormone with enhanced proliferation. The present study demonstrates the existence of mRNA for erythropoietin receptor in human umbilical vein endothelial cells. We have reverse transcribed mRNA of endothelial cells and then used different PCR primers to amplify erythropoietin receptor target cDNA between exons 5 and 6 as well as 3-5 in addition to an internal standard DNA fragment. Correspondence of size as well as location of restriction endonuclease scission (Ava II) was used in comparing the amplified fragments of human endothelial cell erythropoietin receptor to those of two human erythroleukemia cell lines, OCIM1 and K562. No alpha- or gamma-globin mRNA was detected in endothelial cells but was readily demonstrable in OCIM1 cells. In addition, to determine whether the expression of human erythropoietin receptor on endothelial cells occurs in vivo, sections of umbilical cord and placenta were immunostained with antibodies against the extracellular portion of the receptor; the results showed strong positive staining of the vascular endothelium.

Base Sequence↗

PubMed1994

Tissue specific expression of human erythropoietin receptor in transgenic mice.

We have made transgenic mice using the human erythropoietin receptor (hEpoR) encoding gene contained within a 15-kb DNA fragment. The transgenic mice that incorporated the hEpoR transgene into the genome were analyzed for tissue specific expression of hEpoR mRNA using reverse transcriptase and DNA amplification. In the control animals, endogenous EpoR transcripts were identified in bone marrow and spleen; no transcripts were detected in heart, kidney, liver, or brain. In the transgenic mice, hEpoR transcripts were detected in bone marrow and spleen but not in heart, kidney, or liver, suggesting that the transgene contains sufficient genetic information to direct appropriate expression in hematopoietically active tissues. The hematological parameters of the transgenic mice were within normal limits, consistent with the relatively low level of hEpoR transcripts detected. Surprisingly, hEpoR transcripts but not mouse EpoR transcripts were detected in the brains of the transgenic mice. Brain hEpoR transcripts were observed in all transgenic mice assayed, indicating that transgene expression in the brain did not result from the effects of aberrant integration sites. Comparable expression of the transgene was also observed in the embryonic brain. Interestingly, we observed significant expression of the endogenous EpoR gene in the early embryonic brain (Day 10) of normal mice at levels comparable to that observed in the adult spleen and bone marrow. The level of endogenous EpoR expression in the brain decreased during embryonic development to nondetectable levels prior to birth preceding the decrease of endogenous EpoR expression in the fetal liver, while hEpoR expression in the brains of transgenic mice persisted throughout embryonic development into adulthood. These data suggest that the hEpoR transgene contains appropriate regulatory sequences to direct tissue specific expression in tissues associated with hematopoietic activity and in the embryonic brain, but lacks the control elements to provide levels of expression comparable to that of the endogenous gene or to selectively silence brain expression in the adult mouse.

Animals↗

PubMed1994

Reducing erythropoietin in cultures of human erythroid precursors elevates the proportion of fetal haemoglobin.

In order to clarify the mechanism of the effect of erythropoietin (Epo) on the fetal haemoglobin (HbF) phenotype of peripheral erythrocytes, we studied the dose-response effect of Epo on HbF production by erythroid precursors derived from the peripheral blood of normal adult individuals and grown in a two-phase liquid culture system. The proportion of HbF out of the total haemoglobin (Hb) content (%HbF) was dependent on the duration of exposure to Epo; on day 6 it comprised up to 15%, but dropped to < 2% on day 14. Both cell yield and cellular Hb content were markedly increased by high (1 U/ml) Epo, compared to normal physiological (20-50 mU/ml) levels, but neither the initial nor final %HbF were dependent on the increased Epo dose. However, when cells grown with high Epo were transferred on day 7 to low Epo, their progeny contained by day 14 a higher %HbF as compared to cells that were continuously exposed to high Epo. This was accompanied by acceleration and synchronization of their maturation process, as evidenced by their morphology, density and size, and restriction on cell multiplication, as indicated by the lower cell yield. These results are consistent with the following model. As early erythroid precursors, with relatively high HbF, mature under steady-state levels of Epo, HbA production predominates and HbF is diluted. However, when such precursors are switched from high to low levels of Epo they undergo a synchronized, accelerated maturation which shortens the period of HbA production, leading to a decreased Hb content and a relatively high proportion of HbF. This mechanism may contribute to the elevated HbF observed following Epo administration (due to short half-life of Epo in vivo), and might also explain the HbF-augmenting effect of Epo administered together with hydroxyurea observed in patients with sickle cell anaemia.

Cell Division↗

PubMed1993

Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S at physiologic ligand saturations.

Recent interest in therapies for sickle cell anemia based on elevating fetal Hb has made accurate estimates of the sparing effect of fetal Hb (Hb F) and other non-sickle Hbs on sickle Hb (Hb S) polymerization essential. We have developed a technique, using HbCO as surrogate for HbO2, that enables us to assess the solubility of Hb S as a function of ligand saturation under conditions that mimic those of the sickling disorders. Equimolar mixtures of unliganded Hb S with Hb F or normal Hb A2 were isosoluble. Solubilities for equimolar mixtures with normal (Hb A) or abnormal (Hb C) Hbs were also identical but were lower than in the prior case. Thus, the sparing effect of both Hb F and Hb A2 should be considered in therapeutic strategies designed to modify Hb S polymerization. Hemolysates, stripped of 2,3-bisphosphoglycerate, from sickle cell disease patients with Hb (F + A2) levels varying from 6 to 25%, as well as from a sickle trait individual, were used to evaluate equilibrium solubility as a function of ligand saturation over the range of pathophysiologic interest (25-70%). Our results show that the sparing effect of Hb (F + A2) increases relative to that of Hb A as ligand saturation increases, and that in the absence of ligand, approximately 30% Hb (F + A2) is essentially isosoluble with the 60% Hb A of sickle trait. Although detailed knowledge of expected therapeutic benefits is confounded by the heterogeneity of Hb F distribution and other variables, these data should provide a framework for estimating likely clinical benefit from pharmacologic efforts to modulate globin gene expression.

Anemia, Sickle Cell↗

PubMed1993

Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or beta-thalassemia.

Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood-derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5-fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta-thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.

Anemia, Sickle Cell↗

PubMed1993

Protein-DNA interactions in the epsilon-globin gene silencer.

The developmental control of expression of the human epsilon-globin gene appears to be mediated, at least in part, by a transcriptional silencer in the DNA 5' to the cap site of this gene. We have used site-directed mutagenesis and DNA-protein binding assays to define the active motifs of this epsilon-globin silencer. DNase I foot-printing of the silencer region with K562 cell nuclear extracts defined a sequence, which we designate as the epsilon-globin silencer motif or epsilon GSM (epsilon -278 to -258 base pairs (bp)) containing a region (epsilon -270 to -258) with 90% homology to the yeast mating type silencer, ABF-1 (autonomous replicating sequence binding factor one) and which also overlaps at (epsilon -269 to -262) with the human YY1 consensus sequence, an element which mediates transcription repression and activation of viral, mouse, and human genes. The DNase I footprint extended 5' in the silencer region to include an inverted repeat of a six-nucleotide motif (epsilon -267 to -278 bp) which shares 5 of 6 bases with the GATA-1 consensus sequence. In gel mobility shift assays, two specific proteins (A and B) in nuclear extracts from erythroleukemia K562 cells bound to the DNase I-footprinted region. Protein B, associated with epsilon-globin silencer activity in vitro, required an intact epsilon GSM sequence for binding. Mutation of 5 bases within the epsilon GSM in an epsilon-globin promoter-containing fragment extending upstream to 1400 bp in transient transfection assays increased activity by 3.0-fold compared with the native sequence, suggesting that the silencer activity was mediated by the epsilon GSM sequence. We found that protein A could be displaced by a competitor containing the GATA-1 consensus sequence, suggesting that protein A is a GATA-like protein. The region from -267 to -271 within the epsilon GSM and GATA-1 homology region was important for binding of both proteins A and B. These data suggest that protein binding to the epsilon GSM and GATA motifs mediate the negative effect of the silencer on transcription, possibly via direct competition for binding to this DNA region. Recombinant yeast ABF-1 and human YY1 bound to the epsilon GSM. Mutating three bases (epsilon -259, -262, -264) in the epsilon GSM decreased the binding affinity of protein B and recombinant human YY1 but increased the binding affinity of recombinant yeast ABF-1. Furthermore, competitor containing the YY1 consensus sequence competed for protein B binding, whereas competitor containing a perfect yeast ABF-1 consensus sequence did not.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1993

Augmentation by erythropoietin of the fetal-hemoglobin response to hydroxyurea in sickle cell disease.

BACKGROUND: Hydroxyurea increases the production of fetal hemoglobin in patients with sickle cell anemia, inhibiting the polymerization of hemoglobin S and potentially improving vaso-occlusive manifestations and hemolysis. Recombinant erythropoietin increases the number of reticulocytes containing fetal hemoglobin in laboratory animals and in humans. We studied whether hydroxyurea and erythropoietin might have a potentiating effect on the production of fetal hemoglobin in patients with sickle cell disease. METHODS: We treated four patients who were receiving hydroxyurea for sickle cell disease (three who were homozygous for sickle cell anemia and one with sickle beta zero-thalassemia) with escalating doses of intravenous erythropoietin for seven weeks, along with oral iron sulfate. Doses of hydroxyurea on four consecutive days were alternated with doses of erythropoietin on three consecutive days. RESULTS: There was a 28 percent increase in the number of reticulocytes containing fetal hemoglobin and a 48 percent increase in the percentage of fetal hemoglobin, as compared with the maximal values obtained with hydroxyurea alone. The percentage of erythrocytes containing fetal hemoglobin (F cells) increased from 64 to 78 percent. As compared with hydroxyurea alone, treatment with hydroxyurea and erythropoietin decreased the mean (+/- SD) serum indirect bilirubin level from 0.8 +/- 0.2 to 0.5 +/- 0.1 mg per deciliter (13.3 +/- 2.9 to 8.9 +/- 2.2 mumol per liter) (P = 0.02), suggesting a further decrease in hemolysis. Red-cell filterability improved. CONCLUSIONS: Intravenous recombinant erythropoietin with iron supplementation alternating with hydroxyurea elevates fetal-hemoglobin and F-cell levels more than hydroxyurea alone. Such increases decrease intracellular polymerization of hemoglobin S and improve the overall rheologic characteristics of erythrocytes. A reduced dosage of hydroxyurea alternating with erythropoietin may prove less myelotoxic than hydroxyurea given daily or in pulsed-dose regimens. It may also increase levels of fetal hemoglobin in patients with sickle cell disease who have not been helped by hydroxyurea alone.

Adult↗

PubMed1993

Sickle cell disease pathophysiology.

The primary pathophysiological event in the erythrocytes of individuals with the various sickle syndromes is the intracellular aggregation or polymerization of sickle haemoglobin (HbS). The extent of polymerization is determined by the intracellular haemoglobin composition (% HbS and % HbS A, A2 and F), concentration (MCHC and % of dense cells) and oxygen saturation, as well as minor factors such as intracellular pH and DPG concentration. Intracellular HbS polymerization leads to a marked decrease in the flexibility or rheological properties of the sickle erythrocytes and obstruction in various microcirculatory beds, as well as chronic anaemia. Other abnormalities in the properties of the sickle erythrocytes, including membrane abnormalities, changes in ion fluxes and volume and endothelial adhesion, result from acute and chronic oxygen-linked polymerization events and may, in turn, modify polymerization. However, within a good approximation, many aspects of sickle cell disease pathophysiology--for example variations in anaemia among the different sickle syndromes--can be explained in terms of differences in polymerization tendency. Thus, the effects of alpha-thalassaemia can be explained with reference to changes in MCHC and syndromes with high HbF are understandable in terms of the sparing effect of HbF on polymerization. Recent therapeutic approaches to sickle cell disease focus on attempts to reduce intracellular HbS polymerization by altering the haemoglobin molecules, erythrocyte properties, or the distribution of intracellular haemoglobin species. The last, through pharmacological elevation of HbF, has become the central focus of much laboratory and clinical research in recent years. Agents such as hydroxyurea (with or without recombinant erythropoietin) and butyrate compounds elevate HbF (and reduce HbS) in a majority of sickle erythrocytes, thus decreasing intracellular polymerization. Current prospective protocols are designed to see if these changes cause clinical improvement at acceptable doses. Other treatment strategies, including bone marrow transplantation and possible gene replacement therapies, are also under active clinical or laboratory investigation.

Anemia, Sickle Cell↗

PubMed1992

The epsilon-globin gene silencer. Characterization by in vitro transcription.

K562 human erythroleukemia cells constitutively express epsilon- and gamma- but not beta-globin genes. We have previously shown that the differential expression of globin genes observed in intact K562 cells could be simulated in vitro as K562 nuclear extract (NE) actively transcribes the epsilon-globin (with 2 kilobases of 5'-flanking sequence) and gamma-globin gene DNA templates but not beta-globin gene templates. We have now used the K562 in vitro transcription system to examine a silencer transcriptional control element which has been reported to be localized between -177 and -392 base pairs (bp) 5' of the canonical cap site for the epsilon-globin gene. We find that K562 NE has markedly reduced synthesis of RNA in vitro from epsilon-globin gene DNA deletion templates which contain the silencer sequence, or part thereof, but not the adjacent 5'-positive regulatory region (-453 to -535 bp). Furthermore, those transcripts generated in vitro from DNA templates extending to -453 bp or less of the epsilon-globin gene were not correctly initiated at the canonical cap site. Separating the K562 NE by ion exchange chromatography, we isolated a fraction (F175) transcriptionally active for all tested globin genes including the epsilon-globin gene containing the silencer sequence and a fraction (F50) which contains the trans-acting factors associated with the silencer activity. F50 showed a strong dose-dependent inhibitory effect on correctly initiated epsilon-globin gene transcription directed by either unfractionated K562 NE or F175. This suppression by F50 was not observed on transcriptional activity of the permissive adenovirus 2 major late promoter. In electrophoretic mobility shift assays using the epsilon-globin gene silencer region as probe, F50 and F175 exhibited different DNA binding protein patterns; a specific protein band in F50 appears to be associated with the silencer activity. These studies suggest that this protein may be specifically responsible for the activity of the silencer element of the epsilon-globin gene. The expression and silencing of the epsilon-globin gene during development may be modulated by the interactions of this protein with the cis-acting DNA silencer.

Amanitins↗

PubMed1991

Cloning of the human erythropoietin receptor gene.

We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5' region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5' of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5' flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5' of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5' to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

Amino Acid Sequence↗