concanavalin-a and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.

Topics: Agglutination; Ankyrins; Blood Proteins; Concanavalin A; Cross-Linking Reagents; Dimethyl Adipimidate; Erythrocyte Membrane; Hot Temperature; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Proteins; Potassium Iodide; Protein Binding; Sulfates

Chronic myeloid leukemia (CML) is a clonal disorder of the stem cells which has impaired cell-mediated immune response. Such immune dysfunction may be due to reduced expression of the T-cell receptor (TCR) zeta chain, which is an important component in TCR-mediated signal transduction. In this study, the TCR zeta expression level was detected using a real-time polymerase chain reaction with SYBR Green I technique and relative quantification method. We demonstrated that peripheral blood mononuclear cells (PBMCs) from patients with CML in chronic phase (CML-CP) and CML in complete remission (CML-CR) expressed decreased TCR zeta mRNA levels compared to healthy controls. In addition, TCR zeta chain gene expression was down-regulated in CD4+ T cells from patients with CML-CP. However, the mRNA expression level of the same gene in CD8+ T cells between CML patients and normal individuals did not differ. To confirm that this abnormal zeta chain expression could be corrected, we then stimulated the PBMCs of there patients with interleukin-2, phytohaemagglutinin and concanavalin A. The results showed that it was possible to partially up-regulate zeta chain expression. Taken together, these results indicate that TCR zeta chain expression was decreased in PBMCs and CD4+ T cells from patients with CML and this could be up-regulated partially by using stimulators.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Child; Concanavalin A; Down-Regulation; Female; Gene Expression; Humans; Interleukin-2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes, Mononuclear; Male; Middle Aged; Phytohemagglutinins; Receptors, Antigen, T-Cell; RNA, Messenger; Young Adult

A comparative influence of lectins on the attachment kinetics of erythroid cells (human erythrocytes and erythroleukemia cells K562) to glass formvar has been studied. It is shown that the inhibiting effect of wheat germ agglutinin (WGA) on the adhesion to glass is the same for both types of cells under study, whereas concanavalin A (Con-A) inhibits the erythrocyte adhesion to glass only but it is ineffective for adhesion of K562 cells. Plasmatic membranes of K562 cells are free from band 3 protein contrary to the erythrocyte ones. It is concluded that the inhibition of erythrocyte adhesion by lectins results from their binding to different components of cell membranes; sialic acid residues of glycophorin (WGA) or band 3 protein (Con-A). This conclusion is confirmed by the inhibiting effect of WGA towards attachment of cells K562 to glass and the absence of such an effect of Con-A towards the same cells which do not contain this protein. The most probable form of both cell type attachment to glass is the occurrence of so-called focal contacts. The latter can be easily observed in the case of K562 cells. The intact erythrocyte attachment to formvar occurs slower than to glass. At the same time, the above lectins accelerate the erythrocyte attachment to formvar.

Topics: Cell Adhesion; Concanavalin A; Erythroid Precursor Cells; Glass; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Microscopy, Electron, Scanning; Polyvinyls; Surface Properties; Tumor Cells, Cultured; Wheat Germ Agglutinins

Lectin-receptors on leukocyte and endothelial surfaces are becoming more important in the light of increasing evidence which implicates lectin-carbohydrate interactions in diverse physiological phenomena. This study reports the identification of a major 118 kDa granulocyte surface protein, (Protein 1 a) which binds the lectin wheat germ agglutinin (WGA), and is distinctly different from reported WGA binding granulocyte membrane proteins. Protein 1 a has been isolated from the Triton-soluble and Triton-insoluble lysates of normal individuals and patients with Chronic Myeloid Leukemia (CML) using a combination of differential solubilization, lectin affinity, ion exchange chromatography and HPLC. The protein from the detergent lysates of both normal and CML granulocytes has similar pI values, lectin affinities, and hydrophobicity. However, its solubility in Triton is different in the two cell types. In 71% of CML cases examined, Protein 1 a exhibits decreased Triton solubility suggesting its increased association with the cytoskeleton (CSK). Stimulation of normal granulocytes with WGA leads to the translocation of the soluble form of Protein 1 a to the Triton-insoluble fraction. This cytoskeletal recruitment of Protein 1 a is sustained only under conditions of excess WGA and occupied receptor. The CSK disruptive agent dihydrocytochalasin B (H2CB) releases the insoluble form of the receptor into the Triton-soluble fraction. Investigation of a CSK-involving process such as ligand internalization revealed that CML granulocytes exhibit slower kinetics of internalization of fluorescent WGA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antigens, CD; Concanavalin A; Cytochalasin B; Cytoskeleton; Endocytosis; Female; Granulocytes; Humans; In Vitro Techniques; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukosialin; Male; Polyethylene Glycols; Precipitin Tests; Receptors, Cell Surface; Receptors, Mitogen; Sialoglycoproteins; Solubility; Time Factors; Wheat Germ Agglutinins

Natural killer cells select targets for lysis based on target cell glycoproteins. Compared to controls, K-562 cells treated with kifunensine, an inhibitor of Golgi mannosidase I, accumulate more high mannose-type asparagine-linked oligosaccharide, Man9GlcNAc2, and bind more concanavalin A, an oligomannosyl binding lectin. In addition, natural killer cell lysis of kifunensine-treated cells increases 34% over that of controls. Increased sensitivity to lysis occurs after treatment with other N-glycan processing inhibitors that promote accumulation of high mannose-type glycosides (deoxymannojirimycin and swainsonine). In addition, kifunensine-treated cells form more effector:target conjugates. Monoclonal antibodies to the adhesion molecule LFA-1 and its ligand ICAM-1 reduce lysis of control targets but are less effective in blocking lysis of kifunensine-treated cells. K-562 cells bind anti-ICAM-1 but not anti-LFA-1, and this binding does not change after kifunensine treatment. These data demonstrate conclusively a role for asparagine-linked oligosaccharides in the human natural killer cell:target interaction. The presence of high mannose-type glycans on K-562 cells correlates with increased binding of effectors and a greater susceptibility to lysis. These results support the idea that target cell N-glycosides influence the NK-target interaction mediated by adhesion molecules such as ICAM-1.

Topics: 1-Deoxynojirimycin; Alkaloids; Antibodies, Monoclonal; Carbohydrate Conformation; Carbohydrate Sequence; Cell Adhesion Molecules; Concanavalin A; Cytotoxicity, Immunologic; Glycoproteins; Glycoside Hydrolases; Golgi Apparatus; Humans; Indolizines; Intercellular Adhesion Molecule-1; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Function-Associated Antigen-1; Mannosidases; Molecular Sequence Data; Oligosaccharides; Swainsonine; Tumor Cells, Cultured

This paper reports the isolation and biochemical characterization of a major concanavalin A (Con A)-binding cell surface protein (protein 2, M(r) 75-85 kD) from normal and chronic myeloid leukemic (CML) granulocytes. Our studies show that protein 2 has two differentially glycosylated forms, protein 2a (M(r) 75-85 kD), which does not bind the lectin RCA, and protein 2b (M(r) 80-90 kD), which does. Both molecules show identical retention times on reverse-phase HPLC, irrespective of the cell source. By the procedure used the amount of 2a obtained is about 2.4 times more than that of 2b in normal cells and about 2.6 times more in CML cells. Furthermore, both are approximately 2.4-fold more in CML granulocytes. A polyclonal antibody to protein 2a also immunostains protein 2b. The antibody to protein 2a does not prevent Con A binding but inhibits its internalization. Similarity of the molecules from both the cell types and their increased amounts in CML granulocytes suggest that factors/components other than its structure and amount are responsible for the known defective internalization of Con A by CML granulocytes.

Topics: Blotting, Western; Chromatography, High Pressure Liquid; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glycosylation; Granulocytes; Humans; Immunosorbent Techniques; Lectins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Proteins; Molecular Weight; Plant Lectins

Chronic myelogenous leukaemia (CML) is a haematologic malignancy characterised by excessive growth of myeloid cells and their progenitors. Our studies show that there are several abnormalities in CML red blood cells. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Decreased spectrin tetramer formation observed in several hereditary anaemias has been associated with decreased red cell deformability leading to splenic sequestration. This could also be one of the causes of the severe anaemia observed in CML. Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3.

Topics: Concanavalin A; Cytoskeleton; Erythrocyte Membrane; Hemagglutination; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macromolecular Substances; Microscopy, Electron, Scanning; Spectrin