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U Testa

Publications and source records attributed to U Testa.

At least 181 records · Page 10Linked to original sources
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

Presence of the Tn antigen on hematopoietic progenitors from patients with the Tn syndrome.

The Tn syndrome is an acquired clonal disorder characterized by the exposure of a normally hidden determinant, the Tn antigen, on the surface of human erythrocytes, platelets, granulocytes, and lymphocytes. Two distinct populations, Tn positive (Tn+) and Tn negative (Tn-), of mature hemopoietic cells are present in Tn patients. To determine whether the Tn antigen is already expressed on erythroid, myeloid, and pluripotent progenitors, light-density mononuclear blood cells from two patients with this syndrome were separated by fluorescent-activated cell sorting and by affinity chromatography into Tn+ and Tn- fractions, using their binding properties to Helix pomatia agglutinin (HPA). Burst-forming-unit erythroid (BFU-E), colony-forming-unit granulocyte/macrophage (CFU-GM), cells were assayed in plasma clot cultures. After 12-14 d of culture, colonies were studied by a double fluorescent labeling procedure. First, a fluorescein-conjugated HPA permitted evaluation of the presence or absence of the Tn antigen at the surface of the cells composing each colony, and second, the binding of a murine monoclonal antibody against either glycophorin A (LICR-LON-R10) or against a myeloid antigen (80H5), revealed by an indirect fluorescent procedure, was used to establish the erythroid or myeloid origin of each cell. The Tn+ fraction obtained by cell sorting gave rise to nearly 100% Tn+ colonies composed exclusively of cells bearing this antigen. The reverse was observed for the Tn- cell fraction. These results demonstrate that in the Tn syndrome, BFU-E, CFU-GM, and CFU-GEMM of the Tn+ clone express the Tn antigen at this early stage of differentiation.

Antigens, Neoplasm↗
PubMed1985

Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold.

Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.

Cytoplasmic Granules↗
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

Target structure for natural killer cells: evidence against a unique role for transferrin receptor.

The transferrin receptor (TfR) was recently proposed as putative natural killer (NK) cell target structure. Here data are presented against this hypothesis and it is shown that low TfR expression and high NK sensitivity can occur concommitantly . K562 cells were studied at various stages of cell proliferation. No change in NK sensitivity could be observed between exponential growth and the plateau phase, whereas TfR expression completely disappeared during the latter. Protein synthesis inhibitors such as cycloheximide (1 microgram/ml, 48 h) and actinomycin D (50 micrograms/ml, 48 h), that abolished the TfR expression at the K562 cell surface, had no effect on NK sensitivity. Similarly, hemin induction (0.1 mM, 5 days) did not change NK susceptibility of K562 cells but considerably diminished TfR expression. Moreover, attempts to block NK sensitivity with anti-TfR monoclonal antibodies were unsuccessful, even when the 42.6 antibody, which is known to bind to the active site of TfR, was used. Finally, no blocking of NK sensitivity could be achieved when K562 cells were preincubated with saturating concentrations of transferrin or when transferrin was added during the NK assay. It therefore seems doubtful that TfR is the unique target structure for NK cells. It remains possible that TfR and NK target structures are often coexpressed on actively dividing cells.

Antibodies, Monoclonal↗
PubMed1984

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.

Beta-Globulins↗
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

Phorbol esters inhibit the binding of low-density lipoproteins (LDL) to U-937 monocytelike cells.

The present study demonstrates that U-937 monocytelike human cells possess specific LDL receptors. 125I-LDL binds at 4 degrees C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I-LDL, whereas HDL are ineffective. At 37 degrees C, LDL are internalized and degraded by a chloroquine-sensitive pathway. Tumor-promoting phorbol esters inhibit the binding of 125I-LDL to its receptor on U-937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, inhibition is 50% at 5 X 10(-9) M of TPA. After removal of phorbol esters, treated cells recover their 125I-LDL-binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.

Cell Line↗
PubMed1984

K562 cells induced to differentiate by phorbol ester tumor promotors resist NK lysis.

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.

Cell Differentiation↗
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

Defect in glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II (HEMPAS).

Congenital dyserythropoietic anaemia type II (HEMPAS) is a hereditary disease believed to be caused by a membrane abnormality of erythroid cells. Since the molecular basis of this membrane abnormality has not yet been defined, membrane glycoproteins of HEMPAS erythrocytes were analysed by cell surface labelling and endo-beta-galactosidase digestion in this study. HEMPAS erythrocytes showed an abnormal glycoprotein profile when cells were labelled by the galactose oxidase/NaB[3H]4 method; Band 3 and Band 4.5 glycoproteins in HEMPAS are labelled but with less intensity although normally these proteins are the major components revealed by the same method. Instead, in HEMPAS, labelled lactosaminoglycans were found as a lower molecular weight glycoconjugate (HEMPAS glycan). HEMPAS glycan was characterized by micelle formation, a monomer molecular weight of 4000, susceptibility to endo-beta-galactosidase and resistance to protease. These characteristics suggest that HEMPAS glycan has the nature of macroglycolipid. Proteins of Band 3 and the glucose transport protein (a component of Band 4.5), which were detected by antibodies showed a slightly decreased molecular weight in HEMPAS erythrocytes compared to those from normal erythrocytes, which was consistent with the decreased glycosylation of these proteins. The results indicate that anomalies in glycosylation occurred specifically in lactosaminoglycan glycoproteins of HEMPAS erythrocytes.

Anemia, Dyserythropoietic, Congenital↗
PubMed1984

Expression of SSEA-I antigen (3-fucosyl-N-acetyl-lactosamine) on normal and leukaemic human haemopoietic cells: modulation by neuraminidase treatment.

Several mouse monoclonal antibodies (MoAbs) considered specific for the myeloid lineage recognize the same carbohydrate structure (3-fucosyl-N-acetyl-lactosamine) which is similar to the murine antigen SSEA-I. We have investigated the expression of this antigen with six different well-characterized murine IgM MoAbs on normal, leukaemic, and cultured cells by immunofluorescence and immunoelectron microscope cytochemistry. The cells were studied before and after neuraminidase treatment since epitopes recognized by these MoAbs may be masked by sialic acid. Among the recognizable normal marrow or blood cells, all these MoAbs specifically labelled the granulocytic lineage from the promyelocyte to the polymorph. After neuraminidase treatment, monocytes became labelled. All the other lineages remained unstained. Several cell lines were studied. Six of eight lymphoblastoid cell lines were stained by these MoABs; reactivity was increased by neuraminidase. One Burkitt cell line and two T cell lines were also found to be positive. These antibodies were tested on leukaemic cells. In acute non-lymphocytic leukaemia they usually labelled promyelocytes, more mature granulocytic and monocytic precursors but did not label myeloblasts; after neuraminidase treatment, these myeloblasts became stained. No labelling was observed on leukaemic proerythroblasts and promegakaryoblast before and after neuraminidase treatment except in one case of promegakaryoblastic leukaemia in which the SSEA-I antigen and platelet peroxidase were expressed in the same cell. In addition, six cases of common acute lymphoblastic leukaemia were studied; the blasts became positive after desialylation. Two examples of T cell acute leukaemia were essentially negative. We conclude, therefore, that the reactivity of haemopoietic cells with these MoAbs alone does not represent a criterion sufficient to sustain their myeloid origin since the SSEA-I antigen may be expressed at the surface of all cell lineages in the early phases of haemopoietic differentiation.

Acute Disease↗
PubMed1984

Congenital dyserythropoietic anaemia type II associated with a new type of G6PD deficiency (G6PD Gabrovizza).

A 6-year-old boy with chronic haemolytic anaemia was found to have glucose 6-phosphate dehydrogenase (G6PD) deficiency and the morphological, ultrastructural and serological features of congenital dyserythropoietic anaemia (CDA) type II. The patient's mother was heterozygous for G6PD deficiency. G6PD from the patient's red cells, upon partial purification and full characterization, was found to be a new variant designated G6PD Gabrovizza. We conclude that two distinct genetic abnormalities coexisted in this patient. We suggest that CDA type II may become clinically more expressed when another abnormality of the erythrocytes coexists.

Anemia, Dyserythropoietic, Congenital↗