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Increased 2,6 Sialylation of N-Glycans in a Transgenic Mouse Model of Hepatocellular Carcinoma¹

Glycobiologie, Centre de Biophysique Moléculaire, F45071 Orléans Cedex 02, France

Liver cancer is one of the most frequent and lethal malignancies worldwide. Early detection is hampered by the absence of reliable markers. Mice transgenic for the SV40 large T antigen under the control of a liver-specific promoter spontaneously develop well-differentiated hepatocellular carcinomas between 8 to 10 weeks of age. They are excellent models to investigate the alterations of protein expression in the early stages of tumor development and to follow these changes during tumor progression. In the present study, we analyzed the glycosylation changes occurring during tumor development in transgenic mice expressing the SV40 T antigen under the control of the antithrombin III promoter. The analysis of serum and liver glycoproteins by an ELISA type assay, using the lectin from Sambucus nigra (SNA) as a probe, revealed the presence of increased levels of Neu5Ac2,6Galß1,4GlcNAc on N-glycans in the tumor-bearing transgenic mice as compared to controls. On serum glycoproteins the increase in 2,6 sialylation followed tumor progression, reaching up to 10 times control levels. However, significantly higher SNA binding (2-fold) could already be observed on serum glycoproteins from mice exhibiting only microscopically small neoplastic foci. On liver membrane glycoproteins, the increase in 2,6 sialylation was less pronounced, reaching two to three times control values in 6-month-old mice. Western blotting of serum and liver proteins with radiolabeled SNA showed that all glycoproteins that bind the lectin in controls exhibit larger amounts of Neu5Ac2,6Galß1,4GlcNAc on N-glycans in the tumor-bearing mice.

This general increase in 2,6 sialylation on all glycoproteins is due to the increased activity of the galactoside:2,6 sialyltransferase (ST6Gal I), which specifically transfers Neu5Ac residues in 2,6 linkage to Galß1,4GlcNAc units on N-glycans. As for the structures synthesized by the enzyme, the increase of ST6Gal I activity in the serum as well as in liver microsomes of the transgenic mice followed tumor progression. Interestingly, the activity of the galactoside:2,3 sialyltransferase (ST3Gal III), which uses the same acceptor substrate (Galß1,4GlcNAc), was unchanged in the earlier stages of tumor development but decreased in the serum and in liver microsomes from later stages. Using a rat ST6Gal I cDNA as a probe, Northern blots of total RNA extracted from the livers of control and transgenic mice revealed an increased (4-fold) expression of the ST6Gal I gene. The single transcripts detected in both normal and cancerous liver showed identical size.

¹ This work was supported by grants from the Association pour la Recherche sur le Cancer, Biotechnocentre, the Conseil Régional de la Région Centre, and the Ministère de l'Enseignement Supérieur et de la Recherche (to F. P.). D. P. is supported by a fellowship from the Association pour la Recherche sur le Cancer.

² To whom requests for reprints should be addressed, at Glycobiologie, Centre de Biophysique Moléculaire, rue Charles Sadron, F45071 Orléans Cedex 02, France. Phone: (33) 238 25 76 43; Fax: (33) 238 69 00 94; E-mail: piller@cnrs-orleans.fr.

Received 5/19/97. Accepted 7/30/97.

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