Induction of colony formation in vitro by human lymphocytes.
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Cell Division↗
Biomedical subjects
E Fibach
Publications and source records attributed to E Fibach.
Control of normal differentiation of myeloid leukemic cells. XI. Induction of a specific requirement for cell viability and growth during the differentiation of myeloid leukemic cells.
Normal hematopoetic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D+ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D+ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D- clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D+ cells, Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D+ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D+ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D+ myeloid leukemic cells is associated with a change in the cell surface membrane.
Agglutination↗
Control of normal differentiation of myeloid leukemic cells. VIII. Induction of differentiation to mature granulocytes in mass culture.
There are three types of myeloid leukemic cells, IR+D, IR+D- and IR-D-. IR+D+ cells were induced to differentiate to granulocytes in mass culture in liquid medium by conditioned medium (CM) from cultures of lungs from mice injected with endotoxin. About 90% of the leukemic cells were induced to differentiate, 50% to mature granulocytes and 40% to intermediate stages. An efficient induction of granulocyte differentiation was also obtained with CM from primary cultures of rat embryo or human spleen and there was a lower activity with CM from various other sources. IR+D- cells were induced to differentiate to about 20% cells with intermediate stages but not to mature granulocytes; IR-D- cells could not be induced to differentiate to intermediate or mature stages. IR+D+ cells were induced to form intermediate stages of granulocyte differentiation, to phagocytose and to attach to the surface of the Petri dish, three days after incubation with CM. Optimum induction of mature granulocytes required six more days incubation with CM. Mature granulocytes induced from leukemic cells showed cytochemical properties and a morphology in the electron microscope similar to that of normal mature granulocytes. These induced granulocytes did not form leukemiac in animals or colonies in agar. The granulocytes induced from the myeloid leukemic cells, therefore, behaved like normal mature granulocytes.
Acid Phosphatase↗
Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization, cellular ATP and associated membrane changes in D+ and D- cells.
Glucose utilization, energy metabolism and associated membrane changes, have been studied in D+ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D- myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D+ than in D- cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D- than in D+ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D+ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D+ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D- cells, were found before most of the D+ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D- cells to differentiate and the acquistion of the metabolic properties of normal granulocytes by differentiating D+ cells, were associated with differences in the organization of the cell surface membrane.
Adenosine Triphosphate↗
Control of normal differentiation of myeloid leukemic cells. V. Normal differentiation in aneuploid leukemic cells and the chromosome banding pattern of D+ and D minus clones.
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Aneuploidy↗
Control of normal differentiation of myeloid leukemic cells. IV. Induction of differentiation by serum from endotoxin treated mice.
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Animals↗
Control of normal differentiation of myeloid leukemic cells to macrophages and granulocytes.
Cells from a myeloid leukemic line in culture can be induced by the differentiation-inducing protein MGI to form colonies with normal differentiation to mature macrophages and granulocytes. This line consisted of clones that can be induced to undergo normal cell differentiation (D(+) clones) and clones (D(-) clones) that were not inducible. D(+) clones were able to undergo differentiation to both macrophages and granulocytes. Normal differentiation was induced even in clones that were no longer diploid. D(+) clones can segregate some D(-) progeny, and D(-) clones can segregate some D(+) progeny. This, therefore, provides a system for studies on the genetic and chemical control of cell differentiation in leukemic cells.
Animals↗
Mobility of carbohydrate-containing structures on the surface membrane and the normal differentiation of myeloid leukemic cells to macrophages and granulocytes.
Clones (D(+)) of a cultured line of myeloid leukemic cells can be induced to undergo normal differentiation to mature macrophages and granulocytes. There are also clones derived from the same cell line (D(-)) that could not be induced to differentiate. The carbohydrate-binding protein concanavalin A was used as a probe to study the mobility of carbohydrate-containing sites on the surface membrane of these cells. Changes in the distribution of concanavalin A binding sites on the surface membrane can be induced by concanavalin A. With the appropriate site mobility, this induction of a new distribution resulted in a concentration of concanavalin A-membrane site complexes on one pole of the cell to form a cap. D(+) and D(-) clones showed 50 and 5% of cells with caps, respectively, although both types of cells bound a similar number of concanavalin A molecules. Treatment of cells with trypsin increased cap formation from 5 to 40% in D(-) cells, but did not change the percentage of cells with caps in D(+) cells. The results show a difference in the mobility of concanavalin A binding sites in these two types of cells and suggest a difference in the fluid state of these carbohydrate-containing structures on the surface membrane. It is suggested that a gain of the ability of myeloid leukemic cells to undergo normal differentiation is associated with an increase in the fluidity of structures on the surface membrane where the concanavalin A sites are located. Differences in fluidity of specific membrane sites may also explain differences in the response of cells to other differentiation-inducing stimuli.
Agglutination↗
Normal differentiation of myeloid leukaemic cells induced by a differentiation-inducing protein.
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Animals↗
Induction of fetal hemoglobin synthesis with recombinant human erythropoietin in anemic patients with heterozygous beta-thalassemia during pregnancy.
OBJECTIVE: Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous beta-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis. METHODS: Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells. RESULTS: RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron. CONCLUSIONS: RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous beta-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated.
Adult↗
Cross evaluation of three flow cytometric F cell counting methods performed by different laboratories.
Three flow cytometric methods of counting F cells were evaluated in the settings of an external laboratory assessment scheme. The laboratories to participate with a different method were located in Oxford (method O), Athens (method A) and Jerusalem, (method J). Two monoclonal anti-gamma chain antibodies were used: monoclonal antibody produced by P. Beverley (Oxford) (BEV) and an antibody provided by Bioatlantic S.A.R.L. (France) (BIO). The specimens tested were mixtures in five predefined ratios of a sample with homozygous deltabeta-thalassemia with 100% F cells with a sample with no F cells. A central independent laboratory prepared and distributed the aliquots (at room temperature) to the participating centers within 2 (O), 3 (A), and 6 (J) days. The performance of the three methods was evaluated by: 1) deviation indices, 2) relative accuracy, as percent difference of the counts from the target values, and 3) bias and linearity by linear regression of the counts versus the target values [parameters: slope (s), y-intercept (y), R squared (Rs), and F ratio]. The highest score of performance was obtained by method A with both monoclonal antibodies.
Erythrocyte Count↗
Techniques for studying stimulation of fetal hemoglobin production in human erythroid cultures.
This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with beta hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with beta hemoglobinopathies.
Benzidines↗
Erythroid differentiation and the cell cycle: some implications from murine foetal and erythroleukemic cells.
On the basis of observations with (1) erythropoietin induced erythroid differentiation of foetal mouse liver proerythroblasts and (2) chemically induced expression of the erythroid program in MELC, it appears that DNA replication plays a critical role in the transition to haemoglobin formation. Erythropoietin acts selectively on proerythroblasts to stimulate first housekeeping RNA species (rRNA, tRNA), then cell proliferation and differentiation. In erythro-leukemia cells expression of the erythroid program is induced by a variety of polar compounds. DNA synthesis appears requisite to this transition to haemoglobin formation, The molecular site of action of inducing compounds is not established but it is suggested that one critical effect is on the structure of chromatin which occurs during DNA replication and results in the transcription of the erythropoietic gene program.
Animals↗