There are three major 4-O- and two 6-O-sulfotransferases that add a sulfate group at the C4 and C6 positions of the GalNAc residue, respectively (26, 27). The activities of these sulfotransferases were measured by incubating tissue extracts with the substrates chondroitin [(GlcA-GalNAc)n] or dermatan [(IdoA-GalNAc)n] together with the labeled sulfate donor [35S]-PAPS. The incubations with chondroitin (Fig. 6A) showed a 13-fold up-regulation of 4-O-activity in tumor tissue as compared to normal tissue (p = 0.043), while the 6-O-activity was increased 4-fold (p = 0.08). These assays measure 4- and 6-O-sulfotransferases activities but not DS 4-O-sulfotransferase 1 (D4ST-1) activity, which requires dermatan as substrate. D4ST-1 activity was upregulated in 3 out of 4 patients (Fig. 6B). Moreover, 6-O-sulfation, carried out by C6ST1 (27), was also detected on dermatan and increased in 3 patients out of 4. Next, CS/DS biosynthetic enzymes, beyond the epimerases and O-sulfotransferases, were analyzed using cDNA microarray (Fig. S3; the data obtained have been deposited in the Gene Expression Omnibus (GEO) database, accession number GSE27040). The mRNA levels in ESCC biopsies were compared to a reference pool of
