Activity of the nucleolar organizers in cultured cells from the bone marrow erythroblastic islets

The erythroblast islands of the bone marrow are the morphofunctional units of erythropoiesis. In this work, the functional state of erythroblast islands' cells of the bone marrow for the first time was defined by estimation of the activity of the nucleolar organizers of erythroid cells in the erythroblast islands cultivated during 24 and 48 hours in presence of various doses of erythropoietin. The findings indicated that an increase in doses of erythropoietin was accompanied by a corresponding increase of the activity of nucleolar organizers in erythrokaryocytes of erythroblast islands. The nucleolar organizers of erythroid cells in cultures of erythroblast islands responded with activation to very small doses of erythropoietin; besides, a proliferative response of erythrokaryocytes was observed after activation of the nucleolar organizers.     

Effects of various concentrations of erythropoietin on cultured erythroblast islands

Proliferative effects of increasing concentrations of erythropoietin were studied in vitro. The maximal amount of erythroblastic islands consisting of residual macrophages and erythroid cells was observed with the doses 0.5 to 1.5 ME/ml of erythropoietin. Smaller doses stimulated the reconstruction of erythroblastic islands.     

Induction of commitment of murine erythroleukemia cells (TSA8) to CFU-E with DMSO

The commitment of novel mouse erythroleukemic (MEL) cells (TSA8) to colony-forming units of erythroid (CFU-E) by dimethylsulfoxide (DMSO) was investigated. After exposure to the inducer in liquid culture, the cells were transferred to a semi-solid culture to examine their ability to form erythroid colonies which were dependent on erythropoietin. Exposure to DMSO for 2 days is optimum for CFU-E type colony formation and colonies induced in this manner are equivalent to CFU-E. The induction occurred in a synchronous manner. Partly stained colonies appeared prior to CFU-E formation and are thought to be a result of asymmetric cell division. Appearance of these partly stained colonies suggested that the number of erythropoietin receptors is important in the complete responsiveness to erythropoietin. TSA8 cells constitute a suitable model system in which to analyse the mechanism of commitment in early erythropoiesis.     

Primitive erythropoiesis of mouse teratocarcinoma stem cells PCC3/A/1 in serum-free medium

Mouse teratocarcinoma stem cells PCC3/A/1 differentiated into various types of cells, such as red cells, when they were grown in serum-free medium containing transferrin and bovine serum albumin on a KCF cell feeder layer. These red cells were stained well with 2,7-diaminofluorene (DAF), and therefore were erythroid cells. They were nucleated and contained embryonic globin chains, immunologically identified with antiembryonic hemoglobin antisera after acid urea Triton X-100 polyacrylamide gel electrophoresis (UT-PAGE). The addition of erythropoietin to the culture medium enhanced the production of both embryonic and adult globin chains. The addition of interleukin-3 also enhanced the production of embryonic globin chains, but not the production of adult globin chains. These results indicated that primitive erythropoiesis of PCC3/A/1 teratocarcinoma cells did not require exogenous addition of any hematopoietic factor such as erythropoietin or interleukin-3. This culture system will be a new model system for investigating the factors regulating the primitive erythropoiesis in yolk sac blood islands.     

Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine erythroleukemia cells (TSA8)

Erythropoietin is a well-known erythroid differentiation and growth factor, but the mechanism of its action is not well understood. In this work, we have examined its mechanism of action on the erythropoietin-responsive murine erythroleukemia cells (TSA8). TSA8 cells become responsive to erythropoietin after induction with DMSO. Stimulatory effects on erythropoietin response are observed with the addition of compounds affecting the cAMP level such as forskolin, phosphodiesterase inhibitor and cholera toxin only in the presence of erythropoietin. cAMP analogues themselves show no stimulatory effect on TSA8 cells, nor does erythropoietin increase cAMP level in the cells. Thus, it is suggested that cAMP does not act as a direct second messenger for signal transduction through erythropoietin receptors, but as a stimulator of the erythropoietin receptor pathway and/or as a second messenger in combination with the receptor pathway. The mechanism for acquisition of responsiveness to growth and differentiation factors of progenitor cells is discussed.     

Different signalling pathways are used in the commitment of murine erythroleukemia cells (TSA8) to differentiate, and in the erythropoietin action on progenitor cells

The murine erythroleukemia (MEL) cell line, TSA8, becomes responsive to erythropoietin after induction with dimethyl sulfoxide (DMSO). We examined the signalling pathways involved in the commitment of TSA8 cells to become the erythroid progenitor cells responsive to erythropoietin, comparing them with the pathway used in an erythropoietin-induced change of the progenitor cells. Amiloride, an inhibitor of the Na+/H+ antiporter, completely blocked the commitment of TSA8 cells to become responsive to erythropoietin at a concentration that did not affect cell proliferation, while it showed no effect on the differentiation or proliferation of the erythroid progenitor cells derived from TSA8 cells by erythropoietin. Ethyleneglycol-bis (beta-aminoethyl ether) N,N,N',N'-tetra acetic acid (EGTA) inhibited the commitment of TSA8 cells to CFU-E-like cells without affecting colony formation. In contrast, EGTA did not inhibit erythropoietin-induced differentiation of the progenitor cells, but did inhibit their proliferation. These results indicate that erythropoietin uses different signalling pathways from those used in the induction of the commitment of TSA8 cells.     

Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis

Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.     

Factors controlling induction of commitment of murine erythroleukemia (TSA8) cells to CFU-E (colony forming unit-erythroid)

On addition of DMSO, the MEL cell line TSA8 becomes committed into erythroid progenitor cells (CFU-E) which can form differentiated colonies in the presence of erythropoietin. To understand the mechanism of cellular commitment, the number and the affinity of the receptors for erythropoietin were estimated. The affinity of the receptors did not change before or after induction. The number of receptors changed depending on the growth phase, but was not dependent on the addition of the inducer. Thus, the presence of the receptors for erythropoietin may be required, but are not essential for responsiveness to erythropoietin. Further examination of the optimum conditions for commitment suggests that the concomitant actions of induced factor(s) with the receptors may control commitment of TSA8 cells to CFU-E.     

Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid differentiation in vitro

During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4′,6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50–60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p?<?0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

Biological effect of human erythropoietin in transgenic mice]

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).     

PARTICLE-INDUCED ERYTHROPOIETIN-INDEPENDENT EFFECTS ON ERYTHROID PRECURSOR CELLS IN MURINE BONE MARROW

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.     

Studies of murine erythroid cell development. Synthesis of heme and hemoglobin

Techniques of cell separation were used to isolate murine erythroid precursors at different states of maturation. Cells were studied before and after short-term incubation in the presence or absence of erythropoietin. Complementary results were obtained by direct examination of the cell fractions and by the short-term culture experiments. Indices of heme synthesis, including incorporation of 59Fe or [2-14C]glycine into heme and activity of delta-aminolevulinic acid synthetase, were already well developed in the least mature cells, chiefly pronormoblasts. Activity then rose moderately in the cell fractions consisting primarily of basophilic and polychromatophilic normoblasts, and fell off with further increases in cell maturity. On short-term culture in the presence of erythropoietin, activity declined with increasing cell maturation except in the least mature fraction where the original level of activity was maintained. By contrast, synthesis of labeled hemoglobin ([3H]leucine) was very low in the least mature cell fractions and rose progressively with increasing cell maturity. The rate of hemoglobin synthesis increase in cells at all stages of maturation when cultured in the presence of erythropoietin. Despite the different patterns observed for heme synthesis and hemoglobin synthesis, both synthetic activities were consistently higher in cells cultured with erythropoietin as compared to controls. These findings suggest that erythropoietin stimulates biochemical differentiation of erythroid precursors at various stages of maturation. They also demonstrate an asynchronism between heme synthesis and hemoglobin syhthesis; heme synthesis is already well developed in the least mature erythroid cells and begins to diminish as the capacity for hemoglobin synthesis continues to rise.     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

The clustering of CpG islands may constitute an important determinant of the 3D organization of interphase chromosomes

We used the 4C-Seq technique to characterize the genome-wide patterns of spatial contacts of several CpG islands located on chromosome 14 in cultured chicken lymphoid and erythroid cells. We observed a clear tendency for the spatial clustering of CpG islands present on the same and different chromosomes, regardless of the presence or absence of promoters within these CpG islands. Accordingly, we observed preferential spatial contacts between Sp1 binding motifs and other GC-rich genomic elements, including the DNA sequence motifs capable of forming G-quadruplexes. However, an anchor placed in a gene/CpG island-poor area formed spatial contacts with other gene/CpG island-poor areas on chromosome 14 and other chromosomes. These results corroborate the two-compartment model of the spatial organization of interphase chromosomes and suggest that the clustering of CpG islands constitutes an important determinant of the 3D organization of the eukaryotic genome in the cell nucleus. Using the ChIP-Seq technique, we mapped the genome-wide CTCF deposition sites in the chicken lymphoid and erythroid cells that were used for the 4C analysis. We observed a good correlation between the density of CTCF deposition sites and the level of 4C signals for the anchors located in CpG islands but not for an anchor located in a gene desert. It is thus possible that CTCF contributes to the clustering of CpG islands observed in our experiments.     

Erythropoietin responses and physical characterization of erythroid progenitor cells in Rauscher virus infected BALB/c mice.

Infection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU-E) increase exponentially up to 800-fold that of normal levels by the third week of infection. In vitro these CFU-E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU-E from normal mice. Numbers of CFU-E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU-E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU-E colonies at concentrations which did not support growth of normal marrow BFU-E. When compared to normal, CFU-E found in RLV-infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU-E were more homogeneous (modal density 1.0695 g/cm3) than CFU-E from normal spleen. Analysis of physical properties of CFU-E and the nonhemoglobinized erythroblast-like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter. Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast-like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU-E.