EXPRESSION OF DE-N-ACETYL-GANGLIOSIDES IN HUMAN-MELANOMA CELLS IS INDUCED BY GENISTEIN OR NOCODAZOLE

Neuraminic acid is the core structure of most known sialic acids. In n atural systems, the amino group at the 5 position of neuraminic acid r esidues is usually assumed to be acylated. Previously, synthetic de-N- acetyl-gangliosides (with free amino groups at the 5 position of neura minic acids) have been shown to modulate cellular proliferation and ty rosine 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 an tibodies against de-N-acetyl-G(M3) and de-N-acetyl-G(D3) that require both the free amino group and the exocyclic side chain of sialic acids for recognition. Cultured human melanoma cells showed low but variabl y detectable levels of reactivity with these antibodies. The ability o f various biologically active molecules to stimulate this reactivity w as explored. Of many compounds tested, only the tyrosine kinase inhibi tor 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 aci d side chains by mild periodate oxidation. High performance thin layer chromatography immune-overlay analysis confirmed the presence of the novel compound de-N-acetyl-G(D3) in these extracts. Several other tyro sine kinase inhibitors tested did not give the same increase in de-N-a cetyl-ganglioside expression. However, the microtubule inhibitor nocod azole caused a similar accumulation of these molecules, particularly i n non-adherent cells expected to be arrested at metaphase. Thus, genis tein may induce de-N-acetyl-ganglioside expression by virtue of its kn own ability to arrest cells in the G(2)M phase, rather than as a gener al consequence of tyrosine kinase inhibition. These studies also provi de a system in which to analyze the enzymatic basis of de-N-acetyl-gan glioside expression and their potential roles as growth regulating mol ecules.