Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

The regulation of fetal hemoglobin in adult erythroid cells was investigated with bone marrow cultures. Fetal hemoglobin (Hb F) was identified in individual erythroid colonies with fluorescent antibodies against Hb F and synthesis of gamma chains was determined with analyses of radioactive globins. The appearance of fetal hemoglobin in erythroid colonies was clonal. All the cells of the Hb F synthesizing colonies contained fetal hemoglobin. The frequency of erythroid colonies showing Hb F was higher than expected compared to the frequency of Hb F containing cells in the blood. Production of Hb F in culture, as shown by analysis of the radioactive globins, was 5 to 14 times higher than baseline Hb F synthesis. These results suggest that the ability for gamma chain synthesis in erythroid cells is determined at or above the level of the precursor cell from which the erythroid colonies, in vitro, derive (probably an erythropoietin responsive stem cell), and that stimulation of fetal hemoglobin synthesis in adult erythroid cells is possible.     

Early Stimulation of RNA Synthesis by Erythropoietin in Cultures of Erythroid Precursor Cells

The effect of erythropoietin on cultured erythroid precursor cells from 13-day mouse-fetal livers was examined. Within 1 hr, erythropoietin causes a 2- to 3-fold stimulation of uridine incorporation into RNA by these cells. The types of RNA preferentially stimulated by erythropoietin during the first hour of exposure of the cells to the hormone include ribosomal RNAs and their precursors, as well as 4-5S RNA. No unique RNA species, not present in control cells, could be detected by sucrose gradient sedimentation or gel electrophoresis. Inhibition of protein synthesis for up to 1 hr does not abolish the stimulatory effect of erythropoietin on RNA synthesis, suggesting that the effect of the hormone on RNA synthesis is not mediated by a newly synthesized protein.     

Effects of dexamethasone on erythroid colony and burst formation from human fetal liver and adult marrow

S ummary . Dexamethasone, a prototypic synthetic glucocorticoid, was added to cultures of human fetal liver and adult marrow cells to assess its effects on erythroid colony and burst formation. Dexamethasone decreased the number of fetal liver erythroid colonies and bursts formed in the presence of erythropoietin, and also decreased the number of cells per colony. The amount and type of haemoglobin produced per cell were unaffected by adding dexamethasone to the cultures. Dexamethasone inhibited the incorporation of ³H-thymidine into DNA in fetal liver cells stimulated with erythropoietin, supporting the hypothesis that dexamethasone inhibits the proliferation but not the differentiation of fetal liver CFU-E and BFU-E. In contrast, addition of dexamethasone to adult bone marrow cultures had a variable effect on erythroid colony and burst formation.     

Effects of insulin-like growth factor I, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on thymidine incorporation into fetal liver cells

We have studied the effect of different growth factors on thymidine incorporation in cell cultures of erythroid cells from fetal calf and rat livers, a system which has been used in the past as a bioassay for the purification of erythropoietin and erythropoietin-like factors. Insulin-like growth factor I significantly stimulated thymidine incorporation in calf and rat liver cells. Its action in both cell types was practically identical to the effect of bovine serum erythrotropin, a peptide structurally similar to insulin-like growth factor II. The synergistic effect between insulin-like growth factor I and erythropoietin could be observed with partially purified sheep plasma erythropoietin but not with recombinant human erythropoietin. The highest thymidine incorporation was observed when both erythropoietin and insulin-like growth factor I were added simultaneously. Platelet-derived growth factor had a lower thymidine incorporation-stimulating activity than insulin-like growth factor and did not have any synergistic effect with erythropoietin in rat liver cells. Fibroblast growth factor (0.4-6.0 ng/ml) was completely inactive in the thymidine incorporation assay of calf liver cells. Transforming growth factor-beta alone at a concentration of 1 ng/ml significantly inhibited thymidine incorporation into rat liver cells. It seems that from all growth factors tested so far, those belonging to the insulin family of peptides are the most likely to be detected in the thymidine incorporation assays using calf or rat liver cells.     

The Role of Erythropoietin in Prenatal Erythropoiesis of Congenitally Anaemic Flexed-tailed (f/f) Mice

The effects of the f gene on prenatal changes in liver cellularity, erythroid cell maturation, RNA, DNA, and haemoglobin synthesis, and sensitivity to erythropoietin have been investigated, f/f foetuses are smaller than f /+ but still develop a progressive deficiency in haemoglobin content/unit weight of tissue, which is 40% of normal at birth. Early f/f livers contain fewer erythroblasts, maximum numbers are reached later, and changes in relative proportions of early and late erythroblasts are retarded. Erythroid tissue and erythropoietin responsive cells persist longer in f / f than in f /+ livers. Erythropoietin induced RNA and DNA synthesis is higher in f / f than in f /+ foetal liver cell cultures. Erythropoietin causes multiphasic increments in haem synthesis in vitro . Maximum levels of synthesis by 13 day liver cells are reached later f/f but over 36 hr similar amounts are accumulated. Erythropoietin maintains the presence of early erythroid cells in both f/f and f /+ foetal liver cultures, and enhances the production of polychromatic cells.
Circulating f / f foetal reticulocytes have a reduced capacity to utilize exogenous iron for haem synthesis. The deficient haemoglobin content of f / f foetuses could be caused by this abnormality and by reductions in erythroid precursor cell numbers. The reticulocyte abnormality may be a direct or indirect consequence of a failure of erythroid colonization of the foetal liver, and of the effects of secondary compensation for this failure.     

Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

Interferons have the ability to enhance or diminish the expression of specific genes and have been shown to affect the proliferation of certain cells. Here, the effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes. This supports the concept that developmental globin gene switching can be regulated by environmental factors.     

Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation

Stem cell factor (SCF), the ligand for the c-kit tyrosine kinase receptor, markedly stimulates the accumulation of erythroid progenitor cells in vitro. We now report that SCF delays erythroid differentiation among the progeny of individual erythroid progenitors while greatly increasing the proliferation of these progeny. These effects appear to be independent of an effect on maintenance of cell viability. Highly purified day-6 erythroid colony-forming cells (ECFC), consisting mainly of colony-forming units-erythroid (CFU-E), were generated from human peripheral blood burst-forming units-erythroid (BFU-E). Addition of SCF to the ECFC in serum-free liquid culture, together with erythropoietin (EP) and insulin-like growth factor 1 (IGF-1), resulted in a marked increase in DNA synthesis, associated with a delayed peak in cellular benzidine positivity and a delayed incorporation of 59Fe into hemoglobin compared with cultures without SCF. In the presence of SCF, the number of ECFC was greatly expanded during this culture period, and total production of benzidine-positive cells plus hemoglobin synthesis were ultimately increased. To determine the effect of SCF on individual ECFC, single-cell cultures were performed in both semisolid and liquid media. These cultures demonstrated that SCF, in the presence of EP and IGF-1, acted on single cells and their descendants to delay erythroid differentiation while substantially stimulating cellular proliferation, without an enhancement of viability of the initial cells. This was also evident when the effect of SCF was determined using clones of ECFC derived from single BFU-E. Our experiments demonstrate that SCF acts on individual day-6 ECFC to retard erythroid differentiation while simultaneously providing enhanced proliferation by a process apparently independent of an effect on cell viability or programmed cell death.     

Separation of erythropoietin responsive cells in fetal mouse liver.

The erythropoietin responsive cells (ERC) in suspension cultures (ERCSC) of fetal mouse liver and the cells producing erythroid colonies in 2-day plasma clot cultures (CFU-E) sediment at similar rates. However, the sedimented fractions containing the majority of nucleated cells show minimal sensivitity to erythropoietin (Ep) and these cells sediment at a slower rate than the ERCSC. The studies suggest that in short-term suspension cultures of fetal mouse liver, Ep acts on morphologically unrecognizable cells to increase the number of hemoglobin-synthesizing cells rather than by increasing the quantity of hemoglobin synthesized per cell. This effect is similar to the principal in vivo action of Ep.     

Suppression of DNA synthesis in hepatoma cells exposed to glucocorticoid hormone in vitro

Glucocorticoid hormone is shown to markedly suppress DNA synthesis in a line of rat hepatoma cells in vitro. In the presence of 300 nM hydrocortisone or 30 nM dexamethasone the incorporation of radioactive thymidine falls to 50% of control levels by 36 hr, and at higher concentrations of hormone inhibition can be noted as early as 12 hr and is nearly complete by 24 hr. This inhibition of radioactive thymidine incorporation reflects a true suppression of DNA synthesis, is accompanied by a corresponding inhibition of cell proliferation, and is readily reversible upon subsequent removal of hormone. In contrast to previously described effects of the glucocorticoid hormones on various cells of lymphoid origin, the inhibition of DNA synthesis in these hepatoma cells is not accompanied by appreciable cell lysis or by degradation of preformed DNA, and even when [(3)H]thymidine incorporation into DNA is inhibited by 90% or more, incorporation of [(14)C]uridine into RNA proceeds with little change. These findings all parallel previous observations on the effects of glucocorticoid hormone on the livers of intact animals and suggest that studies on the mechanism of the inhibition of DNA synthesis in the present more isolated system may lead to a better understanding of the means by which these compounds inhibit liver growth in vivo.Despite the ready suppressibility of DNA synthesis in these hepatoma cells and in two other cell lines of liver origin, none of these cell lines was found to be inducible for tyrosine aminotransferase. The apparent dissociation between two "steroid-sensitive" phenomena is of interest and warrants further investigation.     

Measurement of human serum erythropoietin by erythroid colony-forming technique using fetal mouse liver cells

A quantitative bioassay for serum erythropoietin in anemic patients was established with erythroid colony-forming technique using methyl cellulose. Fetal mouse liver contains many erythropoietin-dependent erythroid colony-forming cells (CFU-E) with the concentration as well as sensitivity to erythropoietin being the highest at 13–14 days of gestation. Linearity in the erythropoietin response curve for the number of colonies from CFU-E in fetal livers of 13–14 days gestation was obtained for erythropoietin concentrations in the culture medium of 3.9 to 125 mU/ml. When newborn liver cells were used instead, the linearity occurred at 31.2 to 250 mU/ml. Measurement of erythropoietin by the erythroid colony-forming technique for sera from patients with various anemias was compared with that of the assay method using radioiron incorporation into heme in the suspension culture of fetal mouse liver cells. There was a good correlation between the values of both assay methods (r = 0.856, p < 0.001). Also, levels of erythropoietin measured by this assay method showed the expected inverse relationship to the hemoglobin concentration in the sera from the patients.     

Hormonal regulation of total RNA and protein biosynthesis in liver cell cultures of rats in the pre- and postnatal period of development

The effects of several hormones on total RNA and protein biosynthesis were examined in primary cultures of liver cells obtained from rat fetuses on 21-22 days of gestation and from 3 week-old weanling rats. The intensity of biosynthesis processes was estimated by the incorporation of labeled precursors in macromolecules. Insulin, cortisol, triiodothyronine (T3) stimulated RNA and protein biosynthesis in both types of cultures. These hormones enhanced total protein biosynthesis in fetal rat liver cells more efficiently than in hepatocytes of weanling rats. Somatotropin (growth hormone--GH) did not change total protein biosynthesis but notably increased RNA synthesis and the production of immunoreactive serum albumin. Experiments on fetal rat liver cell cultures showed that stimulating action of cortisol on RNA synthesis was synergistic in relation to the effects of insulin and GH. It has been concluded that fetal rat liver cell at the end gestation are able to respond adequately to anabolic action of the hormones.     

Hemoglobin switching in sheep and goats: induction of hemoglobin C synthesis in cultures of sheep fetal erythroid cells.

Synthesis of HbF (alpha2gamma2) is replaced by synthesis of Hb A (alpha2betaA2) shortly before birth in sheep homozygous for the betaA globin chain whereas Hb C (alpha2betaC2) is produced transiently during the neonatal period. We have obtained a Hb F-to-Hb C switch by generating erythroid colonies at high erythropoietin concentration in plasma clot cultures of mid-gestation fetal bone marrow or liver. Furthermore, high erythropoietin concentration appeared specifically to activate the gene for betaC and not those for betaA or betaB globin in colonies grown from cells of an animal heterozygous for the betaA and betaB genes. Erythropoietic stress in the form of periodic bleeding or erythropoietin injection in utero did not stimulate production of Hb C (alpha2betaC2) in fetal sheep until shortly before birth, and then only in two of six animals. Thus, factors other than erythropoietin may influence the potential for betaC globin synthesis in vivo in fetal sheep.     

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.     

Actions of insulin and hydrocortisone on macromolecular synthesis in primary epithelial cell cultures from mouse mammary glands.

Monolayer cultures of mammary gland epithelial cells were prepared from the abdominal glands of midpregnancy mice. After collagenase digestion of mammary tissue and separation by differential centrifugation, the isolated epithelial cells were cultured in Eagle's Minimal Essential Medium supplemented with 10% fetal bovine serum and insulin (6 micrograms/ml). Six days later, when the cultures were in log growth and nearly confluent, the effects of insulin and/or hydrocortisone on the rates of RNA, DNA, and protein synthesis were determined in a serum-free medium. At physiological concentrations, insulin enhanced the rates of uptake and incorporation of [3H]uridine into RNA, of [3H]thymidine into DNA, and of [3H]leucine into protein. Hydrocortisone was shown to be biphasic with regard to concentration in attenuating or augmenting insulin's effects on macromolecular synthesis.     

Evidence for the involvement of lutropin-independent RNA synthesis in Leydig cell steroidogenesis.

The effect of incubating purified Leydig cells in Eagle's medium and the subsequent effect of the RNA synthesis inhibitors, actinomycin D and cordycepin, on lutropin-stimulated testosterone synthesis have been investigated. The inhibiting effect was found to be inversely related to the time of preincubation; with cells preincubated for 0, 1, 2 and 3 h with Eagle's medium only, followed by 2-h incubation with lutropin with and without actinomycin D, testosterone synthesis was inhibited by 37 +/- 4, 31 +/- 3, 18 +/- 4 and 14 +/- 3% respectively (means +/- s.e.m., n = 5). In cells that had been preincubated for 3 h there was no significant effect of actinomycin D on testosterone synthesis during the first hour of incubation with lutropin. Thereafter the inhibition increased with time reaching a maximum of 30% after 5 h. The effects of preincubation were not due to endogenous lutropin in the Leydig cells because cells isolated from hypophysectomized rats gave similar results. The inhibition of [3H]uridine incorporation into the Leydig cell RNA was 80 +/- 1% with 8 microgram/ml actinomycin D. Increasing the concentration of this inhibitor to 80 microgram/ml did not significantly increase the inhibition of [3H]uridine incorporation or lutropin-stimulated steroidogenesis in preincubated and non-preincubated cells. With cordycepin the inhibition of both RNA synthesis and lutropin-stimulated testosterone synthesis in non-preincubated cells were the same; with 25.1--251 microgram/ml approx. 30--70% resp. With preincubated cells (3 h), 0--50% inhibition of testosterone synthesis was obtained respectively. The inhibitory effect of actinomycin D oimilar to that obtained with lutropin. These observations suggest that during preincubation and independently of lutropin, synthesis of intermediates, including RNAs required for stimulation of steroidogenesis, takes place and that subsequent stimulation of steroidogenesis by lutropin occurs without further de novo RNA synthesis. These results provide evidence for a permissive role of specific RNA and protein synthesis in the action of lutropin on testosterone synthesis in the Leydig cell.     

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.     

AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation

AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.     

RNA synthesis in the Leydig cells of the mature rat: effect of oestradiol-17 beta

In view of the evidence that there may be an effect of high concentrations of oestradiol on testicular steroidogenic function, we have investigated the effect of this steroid on [3H]uridine incorporation into RNA by testicular cells. Our results have shown that oestradiol in vitro induced a marked dose-dependent inhibition of RNA synthesis by purified Leydig cells. The concentrations of oestradiol tested varied from 2 to 40 mumol/l; these concentrations also impaired net testosterone synthesis in vitro after human chorionic gonadotrophin (hCG) stimulation. Under the effect of oestradiol, the kinetics of [3H]nucleoside incorporation into RNA were impaired early and the inhibition of RNA synthesis was specific for oestrogenic compounds. It was concluded that, in Leydig cells, oestradiol, in addition to its known inhibitory action on the response of testosterone to hCG, triggers a more extensive response that also includes RNA synthesis in vitro.     

In Vitro Assay of Erythropoietin in Fetal Mouse Liver Cultures

S ummary . In a 13 d fetal mouse liver tissue culture system for assay of erythropoietin, stimulation of incorporation of H-thymidine and of ¹²⁵I-iodo-2'-deoxy uridine into DNA and of ⁵⁹Fe iron into haem were compared as metameters of erythropoietin effect. With both DNA and haem tracers, log-dose response curves given by two preparations of human urinary erythopoietin purified to different extents (respectively 2 and 1300IU, by bioassay in vivo , per mg of protein) were essentially identical at low dose levels although at high dose levels the less pure preparation decreased stimulation of both DNA and haem synthesis. Pre-incubation of erythropoietin with an antiserum to human erythropoietin abolished the effects of the hormone on both DNA and haem synthesis.     

Murine endothelial cells support fetal liver erythropoiesis and myelopoiesis via distinct interactions

Endothelial cells are an important component of the haemopoietic microenvironment. To investigate how endothelial cells are involved in haemopoiesis, two established murine endothelial cell lines were assayed in stromal cultures with fetal liver haemopoietic cells. Both endothelial cell lines allowed for the proliferation and differentiation of erythroid and monocyte-macrophage precursors, suggesting that support for haemopoiesis is a general property of endothelial cells.
Erythropoiesis was dependent on the addition of erythropoietin (Epo), whereas myelopoiesis was independent of added Epo. Haemopoietic colonies developed in close contact with the endothelial cells. Erythroid colonies did not develop when transwell filters were used between the stroma and haemopoietic cells, or when conditioned medium was used in place of stromal cells. In contrast, monocyte-macrophage colonies formed in the presence of transwell filters or conditioned medium. Thus close cell contact is necessary for erythropoiesis but not myelopoiesis under these conditions. These results suggest that different molecular mechanisms are used by endothelial cells to support erythroid development and myeloid development in the mouse fetal liver.