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At least 1,153 records · Page 64Linked to original sources
PubMed1991

Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid-specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

Animals↗
PubMed1991

[Effect of antilymphocyte globulin on growth and erythroid differentiation of K-562 cells].

The influence of antilymphocytic globulin (ALG) on the proliferative activity and erythroid differentiation of erythromyeloid K-562 cells was studied. It has been established that ALG produces a dose-dependent inhibiting effect (20-150 micrograms of ALG/ml) on the proliferative activity of K-562 cells and induces erythroid differentiation of these cells that does not depend on the concentration of ALG used in the test. Pretreatment of K-562 cells with ALG increases their sensitivity to the following growth-inhibiting action of cytosine-arabinoside.

Antilymphocyte Serum↗
PubMed1990

In vitro sensitivity of human hematopoietic progenitor cells to hyperthermia: critical temperature for cells to survive and its application to in vitro purging.

Normal tissue toxicity is a major concern in cancer therapy. Since the hyperthermic killing of normal human hematopoietic progenitor cells including multilineage progenitor cells (CFU-mix) other than committed granulocyte-macrophage progenitor cells (CFU-GM) is not known, we have studied the thermal sensitivity of various types of human hematopoietic progenitor cells: early and late erythroid progenitors (CFU-E and BFU-E), CFU-GM and CFU-mix using semisolid clonogenic assays. When these progenitor cells were exposed at 42-44 degrees C for 2 h, their thermal sensitivity always appeared to be in the order of CFU-E greater than BFU-E greater than CFU-GM greater than CFU-mix; the CFU-mix was the most tolerant to heat damage. In addition, the survival of human hematopoietic progenitor cells decreased exponentially in exposure time- and temperature-dependent manner. At 44 degrees C human hematopoietic progenitor cells including CFU-mix dropped dramatically in number and therefore were unable to form colonies in vitro after exposure for 2 h. This suggests that 44 degrees C may be the critical temperature for the CFU-mix to survive in vitro. Thus temperatures selected for hyperthermia used to purge residual leukemic cells in remission marrow grafts should be 43 degrees C or lower.

Cell Survival↗
PubMed1990

Some new quantitative aspects of fetal erythropoiesis.

Ultrasound guided fetal blood sampling performed for diagnostic purpose has allowed us to determine the evolution of normal hematological parameters i.e. all nucleated cells, red blood cells, platelets, hemoglobin, mean corpuscular volume in 2,680 normal fetuses from the 18th to the 30th week of gestation. Differential blood counts were estimated. We determined normal circulating level of erythropoietic progenitors cells, which is three times higher than in cord blood at birth. Erythropoietin levels were very low and were not modified during the second and the third trimester of pregnancy. All these new data reveal fundamental quantitative differences in the hematopoietic system of fetal on adult subjects.

Blood Cell Count↗
PubMed1989

[Studies on human basophil and eosinophil colonies in human blood mononuclear cell culture: presence of common basophil-eosinophil progenitors].

Human peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers gave rise to three types of colonies that could be distinguished by their unique morphological characteristics; 5.1 +/- 0.9 (mean +/- SE) loose colonies of small cells, 2.1 +/- 0.9 packed colonies of larger cells, and 1.6 +/- 0.7 mixed colonies, were formed when PBMC (2 x 10(5) cells/dish) from 10 healthy volunteers were cultured in the medium containing methylcellulose. Cytochemical analysis with Astra blue-eosin dual staining revealed that all types of colonies consisted of various proportions of basophils, eosinophils, and hybrid (eosinophilic and basophilic) granulocytes which contained both of granules. These hybrid granulocytes were also identified by the ultrastructural features of two kinds of the granules. Relationships between cell numbers added to culture and formed colony numbers indicated colony of the cells to form the colonies. The colonies formed from untreated patients with chronic myelogenous leukemia (CML) during chronic phase were sevenfold of those from healthy volunteers. The colonies formed from treated patients with CML were normal in number. The number was 40 times greater in culture from a patient with basophilic crisis and a patient with myeloid crisis than normal, whereas that from a patient with lymphoid crisis were within normal limit. The number of the colonies from PBMC of patients with eosinophilia were in normal range, whereas those from bone marrow were six times or more than those from PBMC. These findings suggest that PBMC contains common basophil-eosinophil progenitors, and the culture used in this study in considered to be useful in the examination of basophil and eosinophil production from PBMC. Further studies using more purified cell population and other sources of colony stimulating factors such as interleukin-3 (IL-3), IL-4, IL-5, and granulocyte-macrophage colony stimulating factor should be carried out in order to clarify the significance of hybrid granulocytes in basophil and eosinophil proliferation and differentiation.

Adult↗
PubMed1995

Circulating CFU-E during hematopoietic recovery after allogeneic bone marrow transplantation: relationship to erythroid engraftment.

The more mature erythroid progenitor assayable in vitro, the colony-forming unit-erythroid (CFU-E), is normally found in the bone marrow (BM) but is virtually absent from peripheral blood (PB), unlike the more immature progenitor, the burst-forming unit-erythroid (BFU-E). We report on the detection of CFU-E in the PB of six of 18 patients during hematopoietic recovery following allogeneic bone marrow transplantation (BMT); three of six patients with PB CFU-E were under treatment with recombinant human erythropoietin (rhEpo) as well as six of 12 who did not present with PB CFU-E. PB CFU-E were found as early as day 14 following BMT, reached a peak on day 28, and were still detectable on day 60. The presence of PB CFU-E was associated with signs of stimulated erythroid engraftment--an accelerated reticulocyte recovery, an increased number of reticulocytes, higher levels of serum transferrin receptor, and a reduction in transfusional requirements were found in these patients compared to those without PB CFU-E. The numbers of PB and BM BFU-E were similar in the two groups, as well as the numbers of PB and BM CFU-granulocyte/macrophage (CFU-GM) and multipotential CFU (CFU-GEMM); on the other hand, the percentage of BM BFU-E in S phase of the cell cycle was higher in the group of patients with PB CFU-E. While there was no difference between the two groups in serum Epo levels assayed on days 14 and 28 after BMT, patients with PB CFU-E had higher Epo levels in serum samples collected before starting the BMT procedure. These data suggest that the appearance of circulating CFU-E early after BMT is characteristic of a group of patients with an accelerated erythroid engraftment, although the mechanisms leading to the circulation of CFU-E after BMT remain unclear.

Bone Marrow Transplantation↗
PubMed1993

The influence of 4 degrees C storage on proliferative potential of human bone marrow CD34+ cells. Transplantological implications.

The possibility of storage of human bone marrow CD34+ cells at 4 degrees C was investigated for bone marrow transplantational purposes. The cells were placed in Iscove medium supplemented with 20% serum (15% bovine calf serum 5% human AB serum) for three weeks at 4 degrees C. During the storage time, clonogenecity of granulocyto-monocytic (CFU-GM) erythropoietic (BFU-E) and megakaryocytic (CFU-Meg) progenitors was investigated. It was found that it is possible to keep human CD34+ cells at 4 degrees C, at least for a few days before transplantation. At day four of the storage, the number of CFU-GM and BFU-E cells still exceeded 50% of the cells present at day 0. However, we have found that CFU-Meg in comparison to other progenitors are much more sensitive to metabolic storage stress. This enhanced sensitivity of megakaryocytic progenitors could explain, at least partially, the well known phenomenon of retardation of the thrombopoietic recovery in patients undergoing bone marrow transplantations.

Antigens, CD↗
PubMed1994

Stem cell factor improvement of proliferation and maintenance of hemopoietic progenitors in myelodysplastic syndromes.

Human recombinant stem cell factor (rSCF) was tested for its capability of improving the defective growth of hemopoietic progenitors in 28 cases of myelodysplastic syndromes (MDS). In vitro growth and response to rSCF were quite variable. However, in most cases, rSCF stimulated CFU-GM growth induced by rG-CSF, rGM-CSF, rIL-3, 5637 conditioned medium (50-1400% enhancement). rSCF effect was slightly more evident on day 14 CFU-GM and in the presence of rIL-3. BFU-E growth induced by rEPO or rIL-3 + rEPO was enhanced by rSCF in about 50% of cases, in linear correlation with the levels of patients' hemoglobin. rSCF did not increase CFU-E growth, whereas it slightly stimulated CFU-Mk in 33% of the cases. EPO, SCF and, particularly, their combination, enhanced the recovery of normal CFU-E and BFU-E after 7 days of liquid culture. This was less evident in cultures of MDS patients. Conversely, CFU-GM generation in long term liquid cultures, although highly variable, was stimulated by rSCF and, above all, by rSCF + rG-CSF, similarly to what was observed with normal bone marrow samples. SCF seems to enhance in vitro erythropoiesis only in MDS cases presenting without severe anemia. It has little effect on megakaryocytopoiesis, while it seems to be more active on CFU-GM growth and maintenance.

Cell Division↗
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

Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors.

We have studied the effects of recombinant human thrombopoietin (TPO, Mpl ligand) on human hematopoiesis in vitro. TPO alone did not support erythroid burst formation but, in the presence of erythropoietin, it enhanced erythroid burst formation from CD34+ bone marrow and cord blood cells. The burst-promoting activity (BPA) was stronger under 5% serum than 30% serum conditions. The direct nature of BPA effects was documented by replating studies of early erythroid bursts. The BPA of TPO was less than that of interleukin-3 but was comparable with that of granulocyte/macrophage colony-stimulating factor and steel factor. The soluble form of Mpl receptor inhibited burst enhancing effects of TPO, suggesting that the BPA effects of TPO are mediated through the Mpl receptor. These results further delineate the physiologic roles of TPO and may aid in the determination of the clinical usages of TPO.

Adult↗
PubMed1995

Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer.

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.

Animals↗
PubMedDate not supplied

[Structural changes in the chromatin during the cell differentiation of the erythroid series].

Data about the changes in the molecular organization of the cell nucleus on different levels of its structure during erythroid cell differentiation are presented. Changes are observed in DNA methylation and secondary structure. Protein composition of the chromatin and the length of the nucleosomal repeat change. Data about the peculiarities of the chromatin structure of erythroid-specific genes and their association to the nuclear skeleton are presented as well.

Alpha-Globulins↗
PubMed1994

Balance between human peripheral blood CFU-GEMM and CFU-Meg in sustained and acute thrombocytopenia.

Quantitative variations of blood cell progenitors were studied in thrombocytopenic post-chemotherapy patients and in healthy plateletpheresis donors with the aim of better understanding the reasons for their varying presence in peripheral blood. In 6 post-chemotherapy patients with severe thrombocytopenia on days 21-27 after initiation of chemotherapy when white blood cell counts were approximately 2.1-6.0 x 10(9)/l, a balance was observed between the most and least immature progenitors: levels of CFU-Meg were significantly lower than control values whereas levels of CFU-GEMM were 2-fold higher than in controls. Results suggest that an activator distinct from the known poietins may stimulate very immature progenitors. In 15 plateletpheresis donors, numbers of CFU-GEMM and CFU-Meg were greatly increased, respectively 5.5-fold and 10-fold, following plateletpheresis. Data once again indicate a major role of CFU-GEMM in the production of mature blood cells. As the most immature progenitors and thrombocytopoiesis stimulating factor(s) are both present in peripheral blood, such factors may be responsible for the initial engagement of these very early progenitors into a specific cell line.

Acute Disease↗
PubMed1994

Enhancing effects of cyclosporin A on hematopoietic progenitors: possible role of CD8+ T cells as negative regulators.

To elucidate the mechanism by which the administration of cyclosporin A (Cy A) improves a certain type of anemia, Cy A was orally administered to normal mice; hematopoietic activity, natural killer (NK) cell activity, and natural suppressor (NS) cell activity in the bone marrow (BM) were then examined. Hematopoietic colony formation was significantly enhanced by both 7-day and 21-day treatment with Cy A. Cy A did not, however, affect the total cell count or the numbers of macrophages, granulocytes, or B cells in the BM. Neither did Cy A affect the in vitro colony formation of the purified hematopoietic progenitors. These results suggest that Cy A acts on the negative regulators such as NK cells, NS cells, and CD8+ T cells. Since both NK and NS activity were also enhanced by Cy A treatment, these cells were not candidates. CD4+ or CD8+ T cells in the thymus, peripheral blood (PBL), and BM decreased as a result of the treatment. Therefore, to further examine the involvement of CD4+ or CD8+ T cells, hematopoietic colony formation was evaluated using bone marrow cells (BMCs) from mice purged of CD4+ and/or CD8+ cells. A significant enhancement of erythroid and multipotent colonies was observed in the BM of the CD8+ cell-purged mice. On the other hand, a significant enhancement of myeloid colonies was found in the BM of the CD4+ cell-purged mice. These findings suggest that single-positive T cells (particularly CD8+ T cells), which are diminished in number after Cy A treatment, are involved in the negative regulation of hematopoiesis.

Animals↗
PubMed1994

Mutation in the negative regulatory element of the erythropoietin receptor gene in a case of sporadic primary polycythemia.

A 42-year-old Caucasian male with sporadic primary polycythemia has been followed by us for 13 years. During the time of observation, his hemoglobin had been stable, and he has never had an elevated white count or platelet count or any other stigmata of polycythemia vera (PV). Both of his parents, his three children, and all siblings have been hematologically normal. The in vitro culture of erythroid progenitors revealed an absence of autonomous erythropoietin (Epo)-independent erythroid colonies but demonstrated a marked increase in the sensitivity of erythroid progenitors to Epo. We have undertaken a study designed to determine whether a mutation in the Epo receptor (Epo-R) gene could cause the polycythemia phenotype seen in either dominant or recessive primary polycythemia described by us and others, or in polycythemia vera. We have sequenced the cytoplasmic positive and negative regulatory domains of the Epo-R genomic DNA, and a transversion of C to T in nucleotide 6148 was found in one of the patient's chromosomes. This mutation is located in the negative regulatory domain and results in a change from proline to serine (P488S). We have subsequently analyzed more than 40 chromosomes from unrelated normal subjects, as well as autosomal dominant, recessive, and sporadic primary polycythemia and polycythemia vera subjects. In no instance was the same or any other mutation in the Epo-R found. To determine if this Epo-R mutation is a cause of increased sensitivity of erythroid progenitors to erythropoietin, Ba/F3 cells (interleukin-3-dependent murine lymphoid line) were transfected with normal and mutated Epo-R cDNA, rendering the transfected cells viable and able to proliferate in Epo. Transfectants with wild-type and mutant Epo-R cDNA exhibited no difference in the presence of Epo. More recently, we were able to obtain DNA from the seven family members of the propositus and found that the nonpolycythemic mother and one of the siblings have the same Epo-R mutation. We conclude that this first described mutation of Epo-R encountered in humans does not appear on its own to explain the polycythemia phenotype; however, the possibility that it may interact with some other acquired or congenital abnormality in generating the polycythemia phenotype cannot be excluded.

Adult↗
PubMed1993

Retrovirally-produced erythropoietin effectively induces differentiation and proliferation of J2E erythroid cells.

The J2E erythroid cell line terminally differentiates in response to recombinant human erythropoietin (rHu-epo). Here we demonstrate that retrovirally produced monkey erythropoietin (Zen-epo) is also capable of inducing the final maturation steps of J2E cells in a dose-dependant manner. The hormone committed J2E cells to a pathway of accelerated proliferation, haemoglobin synthesis and morphological changes including enucleation. However, only 6 h exposure to Zen-epo was required to initiate maximum haemoglobin synthesis 72 h later. Significantly, the synthesis of haemoglobin was achieved by concomitantly activating globin production, haem synthesis and iron uptake. Methylcellulose assays revealed that the vast majority of clonogenic cells were able to respond to Zen-epo. These data show that retrovirally manufactured erythropoietin is an effective stimulus for the erythropoietic development of J2E cells in vitro.

Animals↗