Adult Hemoglobin Synthesis Research Articles

Prostaglandin E 2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E 2 (PGE 2) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of 35S-methione labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE 2 treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE 2 was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE 2 to culture dishes presumably constitutes an environmental change to promote the functional seen in the blood erythroid bursts in terms of Hb synthesis and switching.

Genetic disorders of human haemoglobin as models for analysing gene regulation.

The genetic and acquired disorders of human haemoglobin provide a diverse group of naturally occurring models for analysing the regulation of protein synthesis. They include structural haemoglobin variants, thalassaemias, which are conditions in which there is a reduced rate of globin chain production, and hereditary persistence of foetal haemoglobin (HPFH) in which there is an inherited abnormality in the switch from foetal to adult haemoglobin synthesis. The thalassaemias result from a diverse series of cis acting lesions of the globin genes which include deletions, insertions, frame shift mutations, and point mutations involving transcription, messenger RNA processing, initiation, termination, poly A addition and globin chain stability. Many forms of HPFH are due to deletions of the beta-like gene cluster; it has been suggested that they may involve regions of the cluster which are involved in the regulation of the foetal to adult globin chain switch. So far, however, no regions of this type have been identified with certainty. The varieties of HPFH not associated with major gene deletions, or those caused by genetic determinants that are not linked to the globin gene clusters, and some of the acquired forms of alpha thalassaemia associated with mental retardation or leukaemia, may be more useful models for studying the regulation of the globin genes, particularly their developmental control.

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10 −7–10 −8 M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10 −4 M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.

Adult hemoglobins are synthesized in murine fetal hepatic erythropoietic cells

The hemoglobins present in murine fetal hepatic erythroblasts on days 12-15 of gestation were studied by biochemical and immunocytologic techniques. In addition, fetal hepatic hemopoietic progenitor cells obtained from normal and mutant f/f mouse fetuses on days 11-13 of gestation were cultured in vitro with added erythropoietin and adult spleen cell conditioned medium to form large erythroid colonies. In all instances, adult hemoglobin synthesis was detected in the fetal hepatic erythroblasts and in the erythroid cell cultures in vitro. The tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, enhanced the fetal hepatic erythroid colony growth in vitro, but did not alter the hemoglobin phenotypic expression.

Adult Hemoglobins Are Synthesized in Murine Fetal Hepatic Erythropoietic Cells

Abstract The hemoglobins present in murine fetal hepatic erythroblasts on days 12–15 of gestation were studied by biochemical and immunocytologic techniques. In addition, fetal hepatic hemopoietic progenitor cells obtained from normal and mutant f/f mouse fetuses on days 11–13 of gestation were cultured in vitro with added erythropoietin and adult spleen cell conditioned medium to form large erythroid colonies. In all instances, adult hemoglobin synthesis was detected in the fetal hepatic erythroblasts and in the erythroid cell cultures in vitro. The tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, enhanced the fetal hepatic erythroid colony growth in vitro, but did not alter the hemoglobin phenotypic expression.

The synthesis of adult hemoglobins during hepatic erythropoiesis in the calf fetus.

We investigated hemoglobin synthesis in suspension cultures of liver erythroid cells obtained from calf fetuses of 103 to 198 days. A significant amount of radioactivity was associated with adult hemoglobins which were separated from the fetal hemoglobins by isoelectric focusing, even after purification of the cell hemolysates by chromatography on Sephadex G-100. A radioactive beta-globin fraction was isolated by chromatography on carboxymethyl-cellulose from hemolysates, which were first fractionated on Sephadex G-100. Fingerprint analysis of peptides obtained by trypsinolysis of radioactive beta-globin chains revealed that its structure was closely related to that of beta-globin, isolated from cow bone marrow cells. The amount of beta-globin which was synthesized by calf liver cells varied from 0.3 to 3.5% of the non-alpha globin chains and remained at a low level for all the fetuses which were studied. Our results indicate that the bovine fetal liver is a valuable model to investigate the switch from fetal to adult hemoglobin synthesis.

Erythrocyte differentiation during the metamorphic hemoglobin switch of Rana catesbeiana.

Anurans (frogs and toads) switch from tadpole to adult hemoglobin synthesis during metamorphosis. A number of workers have attempted to determine whether tadpole and adult Hb types are expressed in the same or different erythroid cells during the switch. If the different Hb types are found in different cells during the transition, the switch in globin gene expression occurs at an early stage of cellular differentiation. Previous studies, in which immunocytochemical techniques were used to approach this question, are in conflict in regard to the metamorphic Hb switch of the North American bullfrog Rana catesbeiana. We have purified newly differentiating erythroid cells from the blood of metamorphosing tadpoles by using Percoll gradients. These new cells have an immature morphology, are very active in the synthesis of adult Hb, and contain no detectable tadpole Hb. The tadpole cells have no detectable adult Hb, are synthetically inactive, increase in density during the switch, and are then cleared from the circulation. Thus, only adult Hb expression is detected in newly differentiating erythroid cells during metamorphosis.

Postnatal fetal and adult hemoglobin synthesis is preterm infants whose birth weight was less than 1,000 grams.

To determine if environmental factors could effect the switchover from fetal hemoglobin (HbF) to adult hemoglobin (HbA) synthesis, studies were carried out on blood samples from eight infants born at less than 1,000 g, when they had reached their postconceptional age corresponding to term. All of these infants required prolonged intensive care, multiple blood transfusions, and two required exchange transfusions. Several were ventilated mechanically for 60 d and two infants had bronchopulmonary dysplasia at the time of the study. The blood samples were incubated in an amino acid mixture containing [14C]leucine followed by column chromatography on DEAE Sephadex for separation of radioactive HbA and HbF. In spite of the extreme prematurity and poor growth of these sick infants, the proportional synthesis of HbF and HbA, as determined by the incorporation of [14C]leucine during the erythrocyte incubations, was characteristic of the period of human development from which the erythrocytes were obtained.

Adult hemoglobins are synthesized in erythroid colonies in vitro derived from murine circulating hemopoietic progenitor cells during embryonic development.

Circulating peripheral blood cells and disaggregated yolk sac cells were obtained from normal mouse embryos as early as day 9 of gestation, prior to the formation of the fetal liver. These were cultured in vitro in plasma clots or methylcellulose, in the presence of either embryonic fluid or adult spleen cell-conditioned medium, with or without added erythropoietin. Large erythroid colonies were observed by the sixth day of culture. In all instances, these erythroid colonies synthesized adult hemoglobins. These results indicate that erythroid progenitor cells committed to adult hemoglobin synthesis are present in early embryonic circulation.

Haemoglobin synthesis in CFU‐E‐derived erythroid colonies during sheep ontogeny

The patterns of haemoglobin synthesis in erythroid colonies grown from sheep marrow CFU-E of different ontogenic stages have been reported previously (Darbre et al, 1979) but only at a single fixed time point in culture. Culture of adult and late fetal progenitors enhanced gamma chain synthesis whereas culture of earlier progenitors during the 'fetal switch' seemed to increase beta-chain synthesis. In order to try to explain these findings, patterns of growth and of haemoglobin synthesis in the colonies were examined over a time-course of 6 d for all developmental stages. Colonies assessed later in vitro made proportionately less gamma chain at every age. It was concluded that because fetal colonies mature more quickly in vitro than later and adult counterparts, and also because gamma and beta chain synthesis appeared to be asynchronous during sheep colony maturation, the results of assay at a single time point are not strictly comparable. The relevance of these findings to the study of the switch from fetal to adult haemoglobin synthesis is discussed.

Effect of thyroid hormones on the switch from larval to adult hemoglobin synthesis in the salamander Pleurodeles waltlii

The evolution of globin chain synthesis was studied in larvae treated either with thyroxine or with an antithyroid substance. Thyroxine treatment accelerated the rate of Hb switching; it induced a preferential synthesis of adult globins while the synthesis of larval globins decreased rapidly. Treatment with thiourea did not prevent the Hb transition, which occurred even at concentrations of thyroid hormones that did not permit induction of anatomical metamorphosis.

Haemoglobin synthesis by fetal erythroid cells in an adult environment.

Although the developmental switch from fetal to adult haemoglobin production has been well characterized in terms of protein synthesis, very little is known about its control at the cellular level. In order to determine whether the switch is controlled by environmental factors or programmed into the haemopoietic cell population, we have studied the effects of transplantation of fetal and adult haemopoietic cells into lethally irradiated lambs on haemoglobin synthesis by the transplanted cells. In two lambs which were irradiated but not transplanted, two lambs which were grafted with autologous marrow, and one lamb which was grafted from an allogenic twin, haemoglobin synthesis showed an adult pattern following transplantation, with gamma-chain synthesis never exceeding 7% of non-alpha chain production. Of 11 lambs transplanted with fetal haemopoietic cells, only two showed evidence of engraftment. During the 24-26 d that these animals survived, haemoglobin synthesis showed a predominantly fetal pattern, though there was a gradual increase in beta-chain production of donor origin. This increase occurred earlier than would be expected from the gestational age of the transplanted cells, but more slowly than might be expected if environmental factors were entirely responsible for expression of adult or fetal haemoglobin synthesis.

Interaction between homozygous beta (0) thalassaemia and the Swiss type of hereditary persistence of fetal haemoglobin.

The interaction between beta(0) thalassemia and an heterocellular form of hereditary persistence of fetal haemoglobin (HPFH), presumably of the Swiss type, has been studied in three generations of a family in which both traits occur. The haematological parameters and the segregation of the two characters in the family suggest that the propositus, a 52-year-old male from southern Sardinia, is homozygous for beta(0) thalassemia and carrier of the HPFH. In spite of the complete suppression of adult haemoglobin synthesis, the patient is not anaemic and shows only morphological abnormalities of the red cells associated with a moderate decrease of the erythrocyte life span. Studies of the synthesis of haemoglobin chains in vitro have revealed only a mild degree of unbalance in the propositus, with a gamma/alpha ratio of 0.67, and a very slight unbalance in a 3-year-old child heterozygous for beta thalassaemia and HPFH. Preliminary analysis of the linkage between this kind of heterocellular HPFH and the beta Hb locus has been performed, utilizing all the suitable families reported in the literature. Although positive lod scores (1.535) have been obtained at a recombination fraction of 0.20, the data available are not sufficient to conclude in favour or against the linkage between the beta Hb locus and the heterocellular type of HPFH.

Control of the simian fetal hemoglobin switch at the progenitor cell level.

This investigation was designed to define the cellular level at which the gamma to beta globin switch is established in the developing simian fetus in order to determine whether the switch is controlled by environmental influences within differentiating erythroid precursors or predetermined by the genetic program of erythroid progenitors. Samples of marrow and liver were obtained from rhesus fetuses throughout the switch period, and marrow was obtained from adult rhesus monkeys. Globin chain synthesis was then measured in differentiated erythroblasts and in erythroid progenitor-derived colonies grown in semisolid media. The relative rates of synthesis of gamma and beta chains were determined by the uptake of [(3)H]leucine into the respective chains separated by Triton gel electrophoresis and in some cases by urea carboxymethyl cellulose chromatography. Four periods of the switch were defined during fetal development. In the preswitch period both erythroblasts and progenitor-derived colonies produced <5% beta globin. In the early switch erythroblasts produced 5-15% beta globin, while progenitor-derived colonies produced 10-35% beta globin. In mid-switch erythroblasts synthesized 50% beta globin, whereas progenitor-derived colonies produced only 15-35% beta. At the completion of the switch erythroblasts produced 100% beta globin, while progenitor-derived colonies produced as little as 40% beta chains. We conclude that the program of globin synthesis that characterizes the fetal switch is established at the level of erythroid progenitors. Fetal erythroid burst-forming units (BFU-E) dominate the marrow prior to the switch. The early switch period is heralded by the appearances of adult erythroid burst-forming units programmed to express increasing beta chain synthesis in colonies. By mid-switch a second class of adult erythroid progenitors capable of giving rise to fetal and adult hemoglobin synthesis in in vitro colonies becomes apparent. These shifting populations of erythroid progenitors with unique globin synthesis programs give rise to the erythroblasts that create the sigmoid pattern of the fetal to adult hemoglobin switch in the developing simian fetus.

Embryonic erythroid differentiation in the human leukemic cell line K562.

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.

Effect of Burst-Promoting Activity (BPA) and Erythropoietin on Hemoglobin Biosynthesis in Culture

Abstract The influence of erythropoietin (Ep) and burst-promoting activity (BPA) on the relative rates of synthesis of fetal and adult hemoglobins (HbF and HbA) were investigated in clonal cell culture. In the case of crude Ep, a positive dose-response relationship between the concentration and the relative rate of HbF biosynthesis by circulating erythropoietic precursors was clearly observed. In contrast, increasing the concentration of purified Ep enhanced the synthesis of HbF and HbA equally. BPA from bone marrow conditioned media and T-cell conditioned media enhanced the relative rate of HbF biosynthesis and burst number at limiting serum concentrations (10%-15%). We, therefore, conclude that BPA is important in the modulation of HbF biosynthesis in culture.

Effect of burst-promoting activity (BPA) and erythropoietin on hemoglobin biosynthesis in culture

The influence of erythropoietin (Ep) and burst-promoting activity (BPA) on the relative rates of synthesis of fetal and adult hemoglobins (HbF and HbA) were investigated in clonal cell culture. In the case of crude Ep, a positive dose-response relationship between the concentration and the relative rate of HbF biosynthesis by circulating erythropoietic precursors was clearly observed. In contrast, increasing the concentration of purified Ep enhanced the synthesis of HbF and HbA equally. BPA from bone marrow conditioned media and T-cell conditioned media enhanced the relative rate of HbF biosynthesis and burst number at limiting serum concentrations (10%-15%). We, therefore, conclude that BPA is important in the modulation of HbF biosynthesis in culture.

Overview: mechanisms of the regulation of hemoglobin synthesis at the cellular level.

The concept that has emerged from our experiments and those of others is that erythroid stem cells are committed to undergo a program of erythroid differentiation with respect to the ultimate hemoglobin phenotype of their progeny erythrocytes. A clear distinction can be drawn between the switch from Hb A (or Hb F) to Hb C in sheep and the switch from Hb F to adult hemoglobin in humans. The former appears to be regulated in a relatively late erythroid stem cell with characteristics of CFU-E. In contrast, the CFU-E found in adult sheep bone marrow from animals that lack the beta C gene appear to be preprogrammed to produce only adult hemoglobin. Fetal stem cells may be induced to synthesize Hb C within a time frame that is similar to that seen in cultures of adult bone marrow. Thus, a common mechanism modulating the potential for expression of this gene and commitment of erythroid stem cells with respect to Hb C production in progeny erythroblasts seems quite likely. Again fetal CFU-E and BFU-E in animals lacking the beta C gene appear to be, for the most part, committed toward producing erythroblasts making Hb F. Further analysis will be required to determine at exactly which stage of stem cell differentiation this programming occurs and also the factors that are important in modulating the potential for fetal and adult hemoglobin synthesis.

Fetal hemoglobin (HbF) synthesis in baboons, Papio cynocephalus. Analysis of fetal and adult hemoglobin synthesis during fetal development

Fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis was studied in fetal baboons, Papio cynocephalus, to determine the normal pattern of hemoglobin production during fetal development. Fetuses ranging from 53 to 180 days gestation (term gestation 184 days) were used. Erythroid cells were incubated with 3H-L-leucine, and the rates of globin chain synthesis and the distribution of radioactivity into hemoglobin intermediates and completed hemoglobin molecules were determined. Gamma chain synthesis accounted for approximately 97% of the total nonalpha chain synthesis up to 140 days gestation; beta chain synthesis accounted for the remainder. After 140 days gestation, approximately equal quantities of gamma and beta chain were synthesized in the bone marrow. Prior to 140 days gestation, total alpha chain synthesis was 30% greater than total non-alpha chain synthesis, while there was balanced chain synthesis after 140 days gestation. During the period of excess alpha chain synthesis, fetal erythrocytes contained a large pool of alpha-hemoglobin (alpha chain with heme attached) molecules uncombined with beta or gamma chains. In view of the possibility that alpha chains may have a lower affinity for gamma chains than beta chains, excess alpha chain synthesis may be required to maintain low levels of free gamma chains.

Fetal Hemoglobin (HbF) Synthesis in Baboons, Papio cynocephalus. Analysis of Fetal and Adult Hemoglobin Synthesis During Fetal Development

Fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis was studied in fetal baboons, Papio cynocephalus, to determine the normal pattern of hemoglobin production during fetal development. Fetuses ranging from 53 to 180 days gestation (term gestation 184 days) were used. Erythroid cells were incubated with 3H-L-leucine, and the rates of globin chain synthesis and the distribution of radioactivity into hemoglobin intermediates and completed hemoglobin molecules were determined. Gamma chain synthesis accounted for approximately 97% of the total nonalpha chain synthesis up to 140 days gestation; beta chain synthesis accounted for the remainder. After 140 days gestation, approximately equal quantities of gamma and beta chain were synthesized in the bone marrow. Prior to 140 days gestation, total alpha chain synthesis was 30% greater than total non-alpha chain synthesis, while there was balanced chain synthesis after 140 days gestation. During the period of excess alpha chain synthesis, fetal erythrocytes contained a large pool of alpha-hemoglobin (alpha chain with heme attached) molecules uncombined with beta or gamma chains. In view of the possibility that alpha chains may have a lower affinity for gamma chains than beta chains, excess alpha chain synthesis may be required to maintain low levels of free gamma chains.