Erythroid Differentiation Process Research Articles

Effects of succinylacetone on dimethylsulfoxide-mediated induction of heme pathway enzymes in mouse Friend virus-transformed erythroleukemia cells

Heme has been reported to exert a control over its own biosynthesis and to affect the erythroid differentiation process at different sites. In this study, succinylacetone, a powerful inhibitor of δ-aminolevulinic acid dehydrase was used to block heme synthesis and to study the effects of heme depletion on the dimethylsulfoxide (DMSO)-mediated induction of the heme pathway enzymes in Friend virus-transformed erythroleukemia cells. The presence of succinylacetone in the medium during the DMSO treatment (1) potentiates the induction of δ-aminolevulinic acid synthetase (the first enzyme of the pathway) and this effect is reversed by the addition of exogenous hemin; (2) does not affect the induction of δ-aminolevulinic acid dehydrase (the second enzyme); (3) prevents the induction of porphobilinogen deaminase (the third enzyme), since no increase could be detected in either the enzyme activity or the immunoreactive protein and this effect could not be reversed by the addition of exogenous hemin; (4) does not affect the induction of ferrochelatase. The possible role of heme or of intermediate metabolites of the pathway on the induction of these enzymes during the erythroid differentiation process is discussed.

Cation Transport and the Initiation of Mel Cell Differentiation

The murine erythroleukemia (MEL) cell system has served as an important in vitro model for the process of erythroid differentiation for the past decade. Under a controlled set of experimental conditions, MEL cells can be induced to undergo a program of terminal erythroid differentiation which closely resembles the process of normal murine erythropoiesis. MEL cells were originally derived from the spleens of mice infected with Friend virus complex. These virally transformed cells can be grown indefinitely by serial passage in tissue culture medium, but become committed to undergo a program of terminal erythroid differentiation when challenged with dimethylsuIfoxide (DMSO) or a wide variety of other agents.

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells.

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.

Erythropoiesis in ha/ha and sph/sph Mice, Mutants Which Produce Spectrin-Deficient Erythrocytes

AbstractIn order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU–E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU–E concentrations were approximately half. In addition, mutant CFU–E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU–E and BFU–E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.

Erythropoiesis in ha/ha and sph/sph mice, mutants which produce spectrin-deficient erythrocytes

In order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU-E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU-E concentrations were approximately half. In addition, mutant CFU-E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU-E and BFU-E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.

Induction and properties of beta-adrenergic receptors during erythroid differentiation of Friend leukemic cells.

beta-Adrenergic receptors on Friend erythroleukemic cells were identified by the use of 125I-labeled hydroxybenzylpindolol, a potent beta-adrenergic antagonist. Binding of this ligand was saturable and stereospecific. The relative orders of potency of isoproterenol, epinephrine, and norepinephrine to displace bound hydroxybenzylpindolol indicate that the Friend cells have beta 2-adrenergic receptors. After culture for 6 days in the presence of dimethyl sulfoxide or hexamethylene bisacetamide, both undifferentiated and differentiated cells have a similar number of receptors (1500 per cell), but the density of beta receptors on the cell surface increases during the process of erythroid differentiation. Incubation of the Friend cells for 24 hr with high concentrations of butyric acid, dimethyl sulfoxide, or hexamethylenebisacetamide resulted in a striking increase of th number of beta-catecholamine receptors. The induction of beta-adrenergic receptors also occurred in the presence of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate and dexamethasone.

Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells.

The process of erythroid differentiation in mouse Friend leukemia virus transformed cells (T3-C1-2) was examined by following changes in several enzyme activities of the heme biosynthetic pathway and in heme concentration while the cells were undergoing erythroid differentiation after treatment with dimethylsulfoxide. Untreated cells on the one hand, have a limited capacity for spontaneous differentiation. On the other hand, dimethylsulfoxide(DMSO)-treated cells showed an increase in the activities of delta-aminolevulinic acid (ALA) synthetase, ALA dehydratase, uroporphyrinogen-I synthetase, ferrochelatase, and heme concentration by days 1, 1.5, 2, and 4, respectively. The increase of the heme pathway enzymes and heme concentration followed the order of these enzymes or products as they are arranged in the heme biosynthetic pathway. These changes induced by DMSO were effectively inhibited by treatment with actinomycin D, suggesting that continued RNA synthesis is required for the differentiation process. 5-bromo-2'-deoxyuridine (BrdU) (10(-5) M) inhibited the DMSO-induced changes of the heme pathway enzymes. BrdU was most effective when it was present during the first 2 days of cell culture. It gradually lost its inhibitory effect when added after the 3rd day or later. The BrdU-mediated inhibition was completely overcome by the addition of thymidine (7 x 10(-5) M), but not by uridine (7 x 10(-5) M). All these data suggest that a sequential induction of the heme pathway enzyme takes place during erythroid differentiation of Friend leukemia cells, and that the sequential induction of the enzymes may be due to a sequential activation of genes coding for these enzyme activities.