Effects of ganglioside biosynthesis inhibition on the expression and phosphorylation of the membrane tyrosine-kinase receptors p185neu/ErbB2 in tumor and normal mammary cell lines.

E. Sottocornola , PhD, I. Colombo, PhD, and B. Berra, Prof

Institute of General Physiology and Biological Chemistry, School of Pharmacy - University of Milan, Milan, Italy

AIM – Gangliosides play an important role in the modulation of membrane receptor tyrosine-kinase activity. So far, no study has been performed in order to define the relation between gangliosides and the oncoprotein p185neu or its normal counterpart erbB2, although their well known involvement in the mammary adenocarcinoma development and mammary gland differentiation. The functional relationship between gangliosides and p185 or erbB2 was evaluated in the tumor (MG1361) and normal (HC11) cell lines, respectively. METHODS – Culture media: for MG1361, complete Williams’ E medium; for HC11 complete RPMI 1640 medium with 5 microg/ml insulin. 10 nM EGF was used to activate the erbB2 receptor. Inhibition of ganglioside biosynthesis was achieved by 30 microM D-PDMP treatment (5 days). Gangliosides were analysed by HP-TLC. p185neu and ErbB2 expression and phosphorylation were evaluated by western blot. RESULTS - Our data indicate that in EGF-stimulated HC11, erbB2 is auto-phosphorylated, but its expression level decreases, as well as the ganglioside GM3 content, with respect to not-stimulated cells. Expression of other ganglioside species remains unchanged. Ganglioside depletion by D-PDMP in EGF-stimulated HC11 does not interfere with the erbB2 auto-phosphorylation ability, but it maintains expression of the receptor at high levels. In MG1361 ganglioside depletion modifies nor p185 expression neither phosphorylation levels. CONCLUSIONS - erbB2 and GM3 could co-localise in the plasma membrane and the presence of GM3 could promote erbB2 degradation. Both hypotheses need further investigation. A possible explanation of the “no effects” in MG1361 may be the constitutive activation of p185neu due to a single amino acid substitution in the protein.

KEY WORDS: glycosphingolipid, MG1361 cell line , HC11 cell line.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Novel Therapies, Part 1.