g(m3)-ganglioside and Cell-Transformation--Neoplastic

Topics: Animals; Antigens, CD; Cell Transformation, Neoplastic; G(M3) Ganglioside; Gene Expression Regulation; Glycosphingolipids; Humans; Integrins; Lactosylceramides; Membrane Microdomains; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.

Topics: Antigens, CD; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Chromatography, Thin Layer; CSK Tyrosine-Protein Kinase; Electrophoresis, Polyacrylamide Gel; G(M3) Ganglioside; Gangliosides; Humans; Integrin alpha3; Integrin alpha3beta1; Integrins; Kangai-1 Protein; Ligands; Membrane Glycoproteins; Microscopy, Confocal; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Phosphotransferases; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; RNA Interference; src-Family Kinases; Synapses; Tetraspanin 29; Urinary Bladder Neoplasms

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.

Topics: Animals; Antigens, CD; Cell Adhesion; Cell Proliferation; Cell Transformation, Neoplastic; Contact Inhibition; Cricetinae; Fibronectins; G(M3) Ganglioside; Gene Expression Regulation; Glycosphingolipids; Humans; Integrin beta1; Integrins; Lactosylceramides; Laminin; Membrane Microdomains; Mice; Mice, Inbred C57BL; Phenotype; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Tumor Cells, Cultured

A cooperative inhibitory effect of GM3, together with CD9, on haptotactic cell motility was demonstrated by a few lines of study as described below. (i) Haptotactic motility of colorectal carcinoma cell lines SW480, SW620, and HRT18, which express CD9 at a high level, is inhibited by exogenous GM3, but not by GM1. (ii) Motility of gastric cancer cell line MKN74, which expresses CD9 at a low level, was not affected by exogenous GM3. Its motility became susceptible to and inhibited by exogenous GM3, but not GM1, when the CD9 level of MKN74 cells was converted to a high level by transfection with CD9 cDNA. Findings i and ii suggest that haptotactic tumor cell motility is cooperatively inhibited by coexpression of CD9 and GM3. (iii) This possibility was further demonstrated using cell line ldlD 14, and its derivative expressing CD9 through transfection of its gene (termed ldlD/CD9). Both of these cell lines are defective in UDP-Gal 4-epimerase and cannot synthesize GM3 unless cultured in the presence of galactose (Gal(+)), whereas GM3 synthesis does not occur when cells are cultured in the absence of Gal (Gal(-)). Haptotactic motility of parental ldlD cells is low, and shows no difference in the presence and absence of Gal. In contrast, the motility of ldlD/CD9 cells is very high in Gal(-) whereby endogenous GM3 synthesis does not occur, and is very reduced in Gal(+) whereby endogenous GM3 synthesis occurs. (iv) Photoactivatable (3)H-labeled GM3 added to HRT18 cells, followed by UV irradiation, causes cross-linking of GM3 to CD9, as evidenced by (3)H labeling of CD9, which is immunoprecipitated with anti-CD9 antibody. These findings suggest that CD9 is a target molecule interacting with GM3, and that CD9 and GM3 cooperatively downregulate tumor cell motility.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; Cell Migration Inhibition; Cell Transformation, Neoplastic; Chemotaxis; CHO Cells; Clone Cells; Colonic Neoplasms; Cricetinae; Cross-Linking Reagents; Culture Media, Conditioned; G(M3) Ganglioside; Galactose; Humans; Membrane Glycoproteins; Stomach Neoplasms; Tetraspanin 29; Transfection; Tritium; Tumor Cells, Cultured; Ultraviolet Rays

A monoclonal antibody, M2590, that recognizes hematoside (GM3) was used to analyze the immunostaining localization of GM3 of the surface of transformed and non-transformed hamster embryo fibroblasts and B16 melanoma cells. The reactivity of GM3 with the antibody changed markedly depending on the cell density. At the sparse density cells were clearly made visible by the antibody, but at the confluency the accessibility of the antibody to GM3 was greatly decreased. This density dependent change in the reactivity of GM3 was found for both normal and transformed cells. The staining pattern of GM3 was examined in relation to the actin fibers made visible with NBD-Phallacidin during cell spreading. When the cells were still round, the GM3 on microspikes or blebs was highly reactive with the antibody, and by the time cells showed circumferential staining of their actin fibers, GM3 had been distributed over the entire cell surface as punctuated spots. GM3 also was visible in substrate attachment materials (SAM). Trypsin treatment of SAM diminished the reactivity of GM3 with the antibody. The antibody did not inhibit cell attachment or spreading on a substratum coated with fibronectin or laminin.

Topics: Actins; Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Cycle; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Contact Inhibition; Cricetinae; Fibroblasts; G(M3) Ganglioside; Gangliosides; Melanoma, Experimental; Membrane Lipids

When human myeloid and monocytoid leukemic cell lines HL-60 and U937, respectively, were treated with an exogenous sialoglycosphingolipid, ganglioside GM3, in serum-free medium, cell growth was markedly inhibited, and their morphological maturation along a monocytic lineage was observed. In addition to a significant increase in phagocytic and nonspecific esterase activities, marked increase of monocyte-specific surface antigens detectable with monoclonal antibodies such as OKM1 and OKM5 was observed in GM3-fed cells. Other sialoglycosphingolipids with the carbohydrate structure belonging to ganglio-series oligosaccharide, ganglioside GM1 and a brain ganglioside mixture, had no effect on the cell differentiation, showing instead stimulatory actions on the growth of these cell lines. We recently demonstrated that the ganglio-series ganglioside GM3 characteristically increased during macrophage-like cell differentiation of these cell lines. The present results indicate that ganglioside molecular species that specifically increase during monocytic cell differentiation of human myeloid and monocytoid leukemic cell lines may play, in turn, an important role in the differentiation-induction of these cell lines along a monocytic cell lineage.

Topics: Animals; Antibodies, Monoclonal; Cattle; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Dogs; Dose-Response Relationship, Drug; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Monocytes

Topics: 9,10-Dimethyl-1,2-benzanthracene; Cell Communication; Cell Count; Cell Division; Cell Line; Cell Transformation, Neoplastic; G(M3) Ganglioside; Gangliosides; Glucosamine; Methylcholanthrene

Topics: Animals; Avian Sarcoma Viruses; Carbon Radioisotopes; Cell Transformation, Neoplastic; Chick Embryo; Culture Techniques; G(M3) Ganglioside; Gangliosides; Glucosamine; Glycolipids; Mutation; Temperature

Topics: Antibodies; Cell Division; Cell Line; Cell Transformation, Neoplastic; G(M3) Ganglioside; Gangliosides; Glycolipids