g(m3)-ganglioside and herbimycin

Gangliosides are sialic acid-containing glycosphingolipids and exhibit various physiologic functions. Gangliosides GD1a and GM3 strongly induced interleukin-10 (IL-10) protein secretion and mRNA expression in T cells from normal human subjects while the other gangliosides were ineffective. IL-10 induction by both gangliosides was completely blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A, genistein, and tyrphostin AG 1288, but not by other signal transduction inhibitors. These results suggest that GD1a and GM3 may induce IL-10 production in T cells by regulating the PTK-dependent signaling pathway. These gangliosides may thus act as important immunoregulators via IL-10.

Topics: Benzoquinones; Cell Division; Cell Survival; Cells, Cultured; DNA; Dose-Response Relationship, Drug; G(M3) Ganglioside; Gangliosides; Humans; Interleukin-10; Isoquinolines; Lactams, Macrocyclic; Protein Kinase Inhibitors; Protein Kinases; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; Staurosporine; Sulfonamides; T-Lymphocytes; Time Factors; Transcriptional Activation

Neuraminic acid is the core structure of most known sialic acids. In natural systems, the amino group at the 5 position of neuraminic acid residues is usually assumed to be acylated. Previously, synthetic de-N-acetyl-gangliosides (with free amino groups at the 5 position of neuraminic acids) have been shown to modulate cellular proliferation and tyrosine phosphokinase reactions. While indirect evidence has suggested that traces of these molecules exist naturally in certain tumor cells, further exploration has been hampered by the lack of a system showing consistent expression at an easily detectable level. Using synthetic compounds as antigens, we have developed highly specific monoclonal antibodies against de-N-acetyl-GM3 and de-N-acetyl-GD3 that require both the free amino group and the exocyclic side chain of sialic acids for recognition. Cultured human melanoma cells showed low but variably detectable levels of reactivity with these antibodies. The ability of various biologically active molecules to stimulate this reactivity was explored. Of many compounds tested, only the tyrosine kinase inhibitor genistein induced reactivity in a dose-dependent manner. Antibody reactivity with ganglioside extracts from genistein-treated cells was abolished by chemical re-N-acetylation and/or truncation of sialic acid side chains by mild periodate oxidation. High performance thin layer chromatography immuno-overlay analysis confirmed the presence of the novel compound de-N-acetyl-GD3 in these extracts. Several other tyrosine kinase inhibitors tested did not give the same increase in de-N-acetyl-ganglioside expression. However, the microtubule inhibitor nocodazole caused a similar accumulation of these molecules, particularly in non-adherent cells expected to be arrested at metaphase. Thus, genistein may induce de-N-acetyl-ganglioside expression by virtue of its known ability to arrest cells in the G2M phase, rather than as a general consequence of tyrosine kinase inhibition. These studies also provide a system in which to analyze the enzymatic basis of de-N-acetyl-ganglioside expression and their potential roles as growth regulating molecules.

Topics: Antibodies, Monoclonal; Benzoquinones; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; G(M3) Ganglioside; Genistein; Humans; Isoflavones; Lactams, Macrocyclic; Melanoma; Nocodazole; Protein-Tyrosine Kinases; Quinones; Rifabutin; Spectrometry, Mass, Fast Atom Bombardment; Tumor Cells, Cultured