Structural basis for the regulation of UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells

The UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 (Gal NAc-T3) gene, a member of the Gal NAc transferase gene f amily, is expressed in a tissue-specific manner. To elucidate the function of this gene, we have focused on the molecular mechanism underlying regulat ion of gene expression. We have droned Gal NAc-T3 cDNA and used it to show that Gal NAc-T3 mRNA is expressed in tumor cell lines derived from secretor y epithelial tissue adenocarcinomas but not in cell lines derived from blad der and epidermoid carcinomas. Using a polyclonal antibody to Gal NAc-T3, w e observed protein expression in adenocarcinoma but not non-adenocarcinoma cell lines, and in breast carcinoma cells but not in normal breast tissue, We used Gal NAc-T3 cDNA to isolate three overlapping genomic clones contain ing the 5'-portion of the human Gal NAc-T3 gene, and we sequenced 1.6 kb ar ound the first exon, A transient expression assay using the luciferase gene showed that promoter activity was much higher in MCF-7 cells than in KB ce lls. In vivo footprint experiments showed significant protection of a dista l GC hox, an NRF-1 site, and an AP-2 site in MCF-7 cells. A novel stem and loop structure extending from nucleotide -103 to nucleotide -165 and contig uous to these transcription factor binding sites seemed to be functional in regulating Gal NAc-T3 gene transcription and a KMnO4 footprint experiment showed that this stem and loop structure could be formed in vivo. We also o bserved dimethyl sulfate hypersensitive sites in the untranslated region ar ound nucleotide +50 in MCF-7 but not in KB cells. These findings indicate t hat Gal NAc-T3 gene expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure, and that this regulation is restricted to cell lines derived from epithelial gland a denocarcinomas but not cells derived from nonsecretory epithelial tissue ca rcinomas. In addition, our immunohistochemical results suggest that our ant i-Gal NAc-T3 antibody may be useful fur diagnostic purposes in the early st ages of breast cancer.