g(m1)-ganglioside and Leukemia

Topics: G(M1) Ganglioside; Humans; Leukemia

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms. After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation. To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation ofWEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHl-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.

Topics: Cell Division; Cell Survival; Cholera Toxin; Clone Cells; Cyclic AMP; Drug Resistance, Neoplasm; G(M1) Ganglioside; Humans; Leukemia; Tumor Cells, Cultured

The CT-mediated signaling mechanisms have been widely used as a tool for helping the knowledge of the more complex mechanisms regulating cell growth and proliferation in which gangliosides are involved as receptors and cAMP as second messenger. In the present study we compare the susceptibility of two murine cell lines (SR-4987 stromal cells and L1210 leukemic cells) to inhibitory effect of cholera toxin (CT) on cell growth and correlate their sensitivity to CT with ganglioside content and intracellular cAMP accumulation. The results indicate a very different response of the two cell lines to CT treatment. L1210 cells (which contain GM1a ganglioside) are sensitive to the inhibiting activity of CT (IC50 in the clonogenic assay = 10(-9) M) but no cAMP accumulation was observed after the treatment. SR-4987 cells (which lack GM1a) show a dramatic increase of intracellular cAMP without any inhibition of cell growth following the CT treatment until 10(-8) M. However, after SR4987 cells have incorporated GM1a they became susceptible to CT (with a IC50 value = 10(-11) M). The comparison of these results with our previous studies on WEHI-3B leukemia cells confirms the remarkable heterogeneity of cell sensitivity to the growth inhibition by CT by emphasizing that this inhibition is the final event of very different mechanisms in which CT binding to a specific ganglioside seems to be necessary and sufficient whereas cAMP accumulation may not be coupled with the antiproliferative effect of CT.

Topics: Animals; Cell Membrane; Cholera Toxin; Colforsin; Cyclic AMP; G(M1) Ganglioside; Gangliosides; Leukemia; Mice; Stromal Cells; Tumor Cells, Cultured

Binding of biotinylated fetuin in a solid-phase assay served as activity assay for purification of calcyclin, the product of a cell growth-related cDNA with homologies to Ca(2+)-binding proteins. Asialofetuin failed to bind to calcyclin, emphasizing the importance of sialic acids. Binding of fetuin was most effectively reduced by N-glycolylneuraminic acid within a panel of mostly negatively charged sugars. Bovine submaxillary mucin and the ganglioside GM1, but not asialo-GM1, proved more effective than neoglycoproteins, carrying negatively charged carbohydrate moieties. Extension of N-acetyl-neuraminic acid to its lactosyl derivative increased its inhibitory potency. Among charge-free carbohydrate residues, only N-acetylglucosamine, lactose, and mannose, but not fucose, melibiose, or N-acetylgalactosamine affected fetuin binding, substantiating the inherent selectivity. Chemical modification with group-specific reagents revealed that lysine and arginine residues appear to be involved in ligand binding that is optimal in the presence of Ca2+, but not Zn2+ and stable up to 1 m NaCl. Biotinylation of calcyclin by modification of carboxyl groups facilitated performance of solid-phase assays with calcyclin in solution, yielding similar results with (neo)glycoproteins in relation to assays with immobilized calcyclin, thereby excluding an impact of binding to nitrocellulose on calcyclin's specificity. Subcellular fractionation disclosed the presence of fetuin-binding activity in all fractions, the specific activity decreasing from the nuclear to the particulate cytoplasmic fraction and the cytoplasmic supernatant. Affinity-purified antibodies were employed to detect high levels of calcyclin expression in acute lymphoblastic, myelogenous, and monocytic leukemia cell lines, but not in myeloma or lymphoblastoid cells. In comparison, most cells were nearly devoid of an O-acetylsialic acid-specific protein that is more abundant in various tissue types than calcyclin.

Topics: Acetylglucosamine; alpha-Fetoproteins; Animals; Arginine; Biotin; Calcium; Calcium-Binding Proteins; Carbohydrate Metabolism; Carbohydrates; Cattle; Cell Cycle Proteins; Cell Nucleus; Cytoplasm; G(M1) Ganglioside; Glycoproteins; Humans; Lactose; Leukemia; Lysine; Mannose; Mucins; Myocardium; S100 Calcium Binding Protein A6; S100 Proteins; Submandibular Gland; Tumor Cells, Cultured

Glycosphingolipids of neutrophils, lymphocytes and leukocytes from patients with various types of human leukemia [acute lymphoblastic (ALL), acute unclassified type (AUL), acute myeloblastic (AML), acute monocytic (AMoL), chronic myeloblastic (CML)] and the hypereosinophilic syndrome (HES) were analyzed chemically and immunochemically. No distinct difference was found in the molar ratio of lipid-bound sialic acid to lipid-bound phosphorus in these cells, but a low ratio of cholesterol to lipid-bound phosphorus was found in ALL (3 of 4 cases), AML, CML and AMoL (one of 2 cases). The predominant glycosphingolipid was ceramide dihexoside (CDH) in all cells analyzed, but the amount and the molar ratio of lipid-bound phosphorus to CDH were clearly different in different cell types, indicating that the molar ratio is a useful criterion in the classification of types of leukemia. In addition, molecular diversity of minor glycosphingolipid components was observed in various leukemic cells. Two of the neutral glycosphingolipids in AMoL were tentatively identified as asialo GM1 and Forssman glycolipids by comparing their mobilities on thin-layer chromatography with those of standard glycolipids and by observing the formation of precipitin lines on a double diffusion agar plate with anti-asialo GM1 and anti-Forssman antibodies.

Topics: G(M1) Ganglioside; Globosides; Glycosphingolipids; Humans; Immune Sera; Leukemia; Lymphocytes; Neutrophils

Topics: Adult; Aged; Animals; Antibodies, Monoclonal; Cell Separation; Female; Flow Cytometry; G(M1) Ganglioside; Gangliosides; Humans; Leukemia; Leukemia, Lymphoid; Male; Mice; Middle Aged; Phenotype; Receptors, Antigen, T-Cell; T-Lymphocytes

Cell surface binding fluorescent ligands have been used to distinguish between different types of leukaemic cells and between leukaemic cells and their presumed normal counterparts or progenitors. Binding of these probes was evaluated using the Fluorescence Activated Cell Sorter (FACS) which provides both rapid, objective and quantitative recording of fluorescent signals from individual cells plus physical separation of cells of particular interest. Binding sites for cholera toxin (monosialoganglioside GM1) were found to be normally expressed in chronic leukaemias but greatly diminished or absent in acute leukaemias irrespective of their morphological type. Antibodies specific for the common form of acute lymphoblastic leukaemia (ALL, non-T, non-B) have been produced in rabbits. After extensive absorption and testing these were shown to define a cell surface antigen of non-T, non-B type ALLs. The antigen is absent from other leukaemias with two interesting exceptions--the majority of acute undifferentiated leukaemias express the antigen as do a proportion of chronic granulocytic leukaemias in blast crisis relapse. The anti-ALL antibodies can therefore be used to distinguish different leukaemias and, more significantly, can identify the existence of relatively rare leukaemic cells in the blood of untreated patients and the marrow of treated patients considered to be in remission.

Topics: Antibodies, Neoplasm; Binding Sites; Blood Cells; Cell Membrane; Cell Separation; Cross Reactions; Enterotoxins; Fluorescent Antibody Technique; Fluorescent Dyes; G(M1) Ganglioside; Humans; Leukemia; Leukemia, Lymphoid; Remission, Spontaneous; Vibrio cholerae