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M Titeux

Publications and source records attributed to M Titeux.

At least 37 records · Page 2Linked to original sources
PubMed1992

Granulocyte-macrophage colony-stimulating factor and erythropoietin act competitively to induce two different programs of differentiation in the human pluripotent cell line UT-7.

The UT-7 cell line was established from a patient with a megakaryoblastic leukemia (Komatsu et al, Cancer Res 51: 341, 1991). Its proliferation is strictly dependent on the presence of hematopoietic growth factors including erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). We investigated the differentiation capacities of this cell line under the action of several growth factors, using immunomarkers, flow cytometry, and ultrastructural techniques. In the presence of GM-CSF and IL-3, eosinophil and basophil promyelocytes were detected, as well as a few cells with erythroid and megakaryocytic (MK) differentiation features. In contrast, Epo induced a marked erythroid differentiation with an increase of glycophorin A expression, accompanied by a few hemoglobinized cells. Differentiation induced by the growth factors took 24 to 48 hours to begin, and increased with cell passages to a plateau at 2 weeks of culture. However, this was not only due to a cell selection because the differential effects of Epo and GM-CSF were observed from a single cell clone and the phenotype could be reversed by opposite growth factors, even after a long period of culture. We subsequently investigated the phenotype of UT-7 in the presence of combinations of Epo, IL-3, and GM-CSF, and showed that GM-CSF and IL-3 act predominantly over Epo. This effect was mediated by a rapid downmodulation of Epo receptors by GM-CSF at messenger RNA and binding sites levels, without a change in receptor affinities. On the other hand, Epo had no effect on number and affinity of GM-CSF receptors. This study shows that UT-7 is a growth factor-dependent pluripotent cell line in which commitment may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors.

Basophils↗
PubMed1990

Expression of platelet glycoprotein Ib by cultured human megakaryocytes: ultrastructural localization and biosynthesis.

Glycoprotein Ib (GPIb), the receptor for von Willebrand factor, is a two-chain member constituent of the platelet/megakaryocytic lineage. Studies on its expression have been hampered by the difficulties in obtaining purified megakaryocytes in a sufficient number. We report a suspension liquid culture procedure that allowed isolation of more than 1 x 10(6) megakaryocytes with a purity ranging from 3% to 88% from the blood of patients with chronic myeloid leukemia, from fetal liver or from normal human bone marrow. GPIb was detected on the plasma membrane of all maturing megakaryocytes and also of promegakaryoblasts devoid of demarcation membranes. GPIb was detected on demarcation membranes of maturing megakaryocytes but was absent from all other organelles, including alpha granules. Biosynthesis of 35S-methionine labeled megakaryocytes showed that GPIb with similar electrophoretic mobility to the platelet molecule was synthesized and that it was also composed of two chains, since its molecular weight shifted in reducing conditions from 170 Kd to 145 Kd. The beta chain remained undetectable after methionine metabolic labeling, but it was immunoprecipitated after 3H-leucine metabolic labeling, confirming that this subunit is devoid of methionine. GPIb was associated with GPIX, as it is in platelets, since anti-GPIb antibodies coprecipitated a 17 Kd polypeptide.

Adult↗
PubMed1988

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody.

A rat monoclonal IgG2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca2+-dependent and Ca2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of Mr 88,000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca2+ and 1 mM Mg2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.

Antibodies, Monoclonal↗
PubMed1988

Uncoupling in the expression of platelet GP IIb/IIIa in human endothelial cells and K562 cells: absence of immunologic crossreactivity between platelet GP IIb and the vitronectin receptor alpha chain.

Platelet glycoproteins (GP) IIb and IIIa exist as noncovalently associated Ca++-dependent heterodimer complexes within the platelet membrane and express the major platelet alloantigens Leka (Baka) and PIA1 (Zwa), which are genetic markers of GP IIb and GP IIIa, respectively. Since heterodimers immunologically related to platelet GP IIb/IIIa have been identified in a number of nucleated cell types, we tested anti-Leka and anti-PIA1 antiserum, polyclonal anti-platelet GP IIb/IIIa IgG, as well as a panel of 28 monoclonal anti-GP IIb, GP IIIa, or complex dependent anti-GP IIb/IIIa antibodies on endothelial cells, peripheral blood mononuclear cells, and the erythroleukemic cells HEL and K562 in order to determine whether nucleated cell GP IIb/IIIa related proteins and platelet GP IIb/IIIa are immunologically related. Using immunofluorescence, immunoblotting, and immunoprecipitation experiments, evidence is presented that (1) the alloantigen Leka is not expressed in endothelial cells of an individual whose platelets are of the Leka/PIA1 phenotype, whereas the PIA1 alloantigen is readily detectable in these cells, (2) that in contrast to HEL cells, which express platelet GP IIb/IIIa and are of the Leka/PIA1 phenotype, platelet GP IIb is immunologically undetectable in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated K562 cells despite the presence of platelet GP IIIa, and (3) that peripheral blood mononuclear cells do not express platelet GP IIb or GP IIIa on their cell surface.

Adult↗
PubMed1987

Expression of blood group A antigen during erythroid differentiation in A1 and A2 subjects.

The expression of blood group A antigen on marrow and blood cells from A1 and A2 subjects was investigated by the binding of Helix pomatia and Dolichos biflorus lectins using immunofluorescence. These two lectins stained BFU-E-derived colonies from A subjects in the early days of culture before the expression of glycophorin. The erythroid origin of these cells was ascertained by the coexpression of two other very early erythroid markers. In bone marrow, the ultrastructural immunogold method revealed that the entire erythroid lineage including proerythroblasts was labeled by HPA, whereas no staining was observed on granulomonocytic cells including myeloblasts. Platelets from A subjects were HPA-labeled and so were platelets from an O subject preincubated in A plasma. Megakaryocytes obtained in CFU-MK-derived colonies were weakly and heterogeneously labeled by the HPA lectin. Cultures from A1 and A2 subjects were the reflection of the genetic differences only when investigations were performed on mature erythroblasts. In contrast, the great majority of immature erythroblasts both from A2 and A1 subjects were equally labeled by both lectins; during further erythroid maturation, binding of both lectins markedly diminished only on A2 erythroblasts. When marrow erythroblasts were investigated at electron microscopic level, heterogeneity of labeling among all stages of maturation was clearly observed in A2 subjects, with staining stronger on immature than on mature erythroblasts. Therefore, the genetic differences between A1 and A2 subjects are revealed during terminal erythroid differentiation.

ABO Blood-Group System↗
PubMed1986

Expression of platelet glycoprotein Ib alpha in HEL cells.

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.

Asialoglycoproteins↗
PubMed1985

Molecular mechanisms regulating the synthesis of transferrin receptors and ferritin in human erythroleukemic cell lines.

The effects of either iron salts or iron chelators on the biosynthesis of transferrin receptors in human erythroleukemic lines was investigated. Addition of these compounds induced a rapid and marked decrease (iron salts) or increase (iron chelators) in the level of transferrin receptors synthesis. Both phenomena were inhibited by actinomycin D. In contrast, the increase in the synthesis of ferritin induced in these cells by addition of iron salts was not inhibited by actinomycin D. These results suggest that iron salts modulate the synthesis of transferrin receptors and ferritin via different molecular mechanisms, of transcriptional and translation type, respectively.

Cell Line↗
PubMed1985

Induction of (2'-5') oligoadenylate synthetase activity during granulocyte and monocyte differentiation.

Variations in the (2'-5') oligoadenylate synthetase (2-5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2-5 A synthetase was enhanced in both cell lines in response to alpha, beta interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2-5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2-5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2-5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2-5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Differentiation of U-937 human monocyte-like cell line by 1 alpha,25-dihydroxyvitamin D3 or by retinoic acid. Opposite effects on insulin receptors.

The monocyte-like human cell line U-937 has been differentiated in vitro by incubation with either 1 alpha,25-dihydroxyvitamin D3 or retinoic acid (RA) plus dibutyryl cyclic AMP (db-cAMP). Both methods were effective in inducing the appearance of maturation markers. Their actions on insulin receptors were the opposite, however; 1 alpha,25-dihydroxyvitamin D3 increased the binding of the hormone, while RA plus db-cAMP decreased the binding. These effects were specific for insulin, since the transferrin receptors were reduced by both methods of differentiation. Thus, the changes in insulin receptors during maturation in vitro depend on the inducing agent and are not causally related to the differentiation process.

Bucladesine↗
PubMed1985

The iron-chelating agent picolinic acid enhances transferrin receptors expression in human erythroleukaemic cell lines.

Picolinic acid, a metal chelating molecule, was administered to human erythroleukaemic cell lines (K 562 and HEL) that were grown in serum-containing media. Picolinic acid inhibited both iron uptake and cell growth. Furthermore, picolinic acid was shown to markedly decrease the level of ferritin in the cells. In spite of the inhibition of cell growth, picolinic acid induced a marked increase in the transferrin-binding capacity of the cells. This phenomenon was due to a two-five-fold enhancement of the rate of transferrin receptor biosynthesis. Other iron-chelating compounds, capable of reducing the level of intracellular iron, also elicited a marked enhancement of the transferrin-binding capacity of the cells. However, the addition of iron, as ferric ammonium citrate, in the culture medium elicited a marked increase in the level of ferritin and a strong decrease in the transferrin-binding capacity of the cells. On the basis of these data we propose that a feed-back mechanism is involved in the regulation of transferrin receptors: when the cells accumulate iron they decrease the number of transferrin receptors in order to prevent further accumulation of iron; when no or low iron is available to the cells, the number of transferrin receptors markedly increases as a compensatory mechanism.

Cell Line↗
PubMed1985

Carbonic anhydrase I is an early specific marker of normal human erythroid differentiation.

The expression of carbonic anhydrase (CA) as a marker of erythroid differentiation was investigated by immunologic and enzymatic procedures. A polyclonal anti-CA antibody was obtained by immunizing rabbits with purified CA I isozyme. This antibody is reactive with CA I but not with CA II. Within blood cells, CA I was only present in erythrocytes, whereas CA II was also detected in platelet lysates by enzymatic assay. Concerning marrow cells, identifiable erythroblasts and some blast cells expressed CA I. Most of the glycophorin A-positive marrow cells were clearly labeled by the anti-CA I antibody. However, rare CA I-positive cells were not reactive with anti-glycophorin A antibodies. We therefore investigated whether these cells were erythroid precursors or progenitors. In cell sorting experiments of marrow cells with the FA6 152 monoclonal antibody, which among hematopoietic progenitors is reactive only with CFU-E and a part of BFU-E, was performed, CA I+ cells were found mainly in the positive fraction. The percentage of CA I+ cells nonreactive with anti-glycophorin A antibodies contained in the two fractions was in the same range as the percentage of erythroid progenitors identified by their capacity to form colonies. In addition, the anti-CA I antibody labeled blood BFU-E-derived colonies as early as day 6 of culture, whereas in similar experiments with the anti-glycophorin A antibodies, they were stained three or four days later. No labeling was observed in CFU-GM- or CFU-MK-derived colonies. The phenotype of the day 6 cells expressing CA I was similar to that of erythroid progenitors (CFU-E or BFU-E): negative for glycophorin A and hemoglobin, and positive for HLA-DR antigen, the antigen identified by FA6 152, and blood group A antigen. Among the cell lines tested, only HEL cells expressed CA I, while K562 was unlabeled by the anti-CA I antibody. In contrast, HEL and K562 cells expressed CA II as detected by a biochemical technique. Synthesis of CA I, as with other erythroid markers such as glycophorin A and hemoglobin, was almost abolished after 12-O-tetradecanoyl-phorbol-13 acetate treatment of HEL cells. In conclusion, CA I appears to be an early specific marker of the erythroid differentiation, expressed by a cell with a similar phenotype as an erythroid progenitor.

Antibody Specificity↗
PubMed1984

Regulation of transferrin receptors in human hematopoietic cell lines.

Cells grown in the presence of ferric ammonium citrate or hemin exhibited a concentration and time-dependent decrease in 125I-transferrin (Trf) binding. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid (an iron chelator) exhibited a marked increase in Trf binding. The decrease or increase in binding activity observed under these different conditions of culture reflected, respectively, a reduction or increase in receptor number rather than an alteration in ligand receptor affinity. Growth of the cells in the presence of saturating concentrations of apotransferrin only induced a slight reduction in receptor number. Investigation of the Trf receptors' turnover and biosynthesis clearly showed that iron and hemin decreased the synthesis of Trf receptors without any modification of the receptor turnover; in contrast, protoporphyrin IX and picolinic acid markedly increased the synthesis of Trf receptors. Our results suggest that hemin, iron, and protoporphyrin IX may represent the main molecules involved in the regulation of Trf receptors.

Antibodies, Monoclonal↗
PubMed1984

The role of iron in the growth of human leukemic cell lines.

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.

Cell Division↗
PubMed1984

Inhibition of transferring binding and iron uptake of hematopoietic cell lines by phorbol esters.

Phorbol esters inhibit cell growth and the binding of transferrin to receptors on K 562, HL 60 and U 937 human leukemic cell lines. Exposure of these cells to 12-0-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C results in a 40% reduction of the specific binding of 125I-transferrin, which is apparent within 15 min. Half-maximal inhibition occurs at about 1 nM. Other tumor promoting phorbol esters also inhibit 125I-transferrin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA reduces the number of transferrin receptors, and does not alter the degradation or the internalization of transferrin. In addition, TPA inhibits iron uptake by these cell lines. These effects are specific, since phorbol esters do not affect either cell growth or the binding of transferrin to Friend erythroleukemia cells and Raji cell line. On the basis of these findings it is suggested that the inhibition of transferrin binding may represent one of the mechanisms by which phorbol esters affect the growth and the differentiation of hematopoietic cell lines.

Biological Transport↗
PubMed1984

Effect of phorbol esters on iron uptake in human hematopoietic cell lines.

We have investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on iron uptake into human hematopoietic cell lines K562, U937, and HL-60. TPA inhibited both cell growth and iron uptake by these cell lines. This effect was rapid, which is typical of phorbol esters which are biologically active, and it occurred at very low concentrations of TPA. This effect of TPA was dependent upon an inhibition of the transferrin-binding capacity as estimated on intact cells. However, experiments with transferrin binding on cell samples dissolved in 1% Triton X-100 showed that TPA-treated cells exhibited a transferrin-binding capacity similar to that of control cells. On the basis of this result, it is suggested that TPA modified a part of transferrin receptors present in the cells; as a result of this modification, these receptors became unavailable for binding transferrin, but they remained physically present in the cell. Other compounds capable of inducing the differentiation of leukemic cells, such as dimethyl sulfoxide, butyrate, retinoic acid, and 1 alpha,25-dihydroxy-vitamin D3, did not acutely inhibit iron uptake. We also investigated the effect of TPA on transferrin receptors in a cellular system in which phorbol esters stimulate cell proliferation. At 16 X 10(-9) M, TPA markedly stimulated the proliferation of T-lymphocytes. However, in spite of this marked stimulation of cell proliferation, TPA-stimulated lymphocytes exhibited a transferrin-binding capacity much inferior to cells stimulated by other mitogens, such as phytohemagglutinin.

Biological Transport↗
PubMed1984

Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts.

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R-PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

Cell Line↗
PubMed1983

Erythroid properties of K562 cells. Effect of hemin, butyrate and TPA induction.

Two sublines of the human leukemia cell line K562 including the original cell line and three clones have been investigated for their erythroid features. All of them produce embryonic and fetal hemoglobins, glycophorin A, spectrin and true acetylcholinesterase, but to a varying extent among the cell lines. The Hb and glycophorin contents were correlated in the different K562 cell lines, whereas acetylcholinesterase was independently expressed from these two other erythroid markers. Hb accumulation is enhanced by exposure of the cells to 100 microM hemin without a significant modification of the expression of the other erythroid markers. Butyrate greatly increased the activity of acetylcholinesterase, slightly enhanced the production of hemoglobin, but did not modify the expression of glycophorin and spectrin. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced an almost complete disappearance of glycophorin, reduced the synthesis of Hb by K562 cells and also abolished the action of hemin on Hb accumulation. Therefore, all the different K562 cell lines exhibit clear erythroid features including acetylcholinesterase. Butyrate or hemin did not induce terminal differentiation of K562 cells, whereas TPA significantly diminished the erythroid phenotype.

Acetylcholinesterase↗