Structural basis for the regulation of UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells
M. Nomoto et al., Structural basis for the regulation of UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells, CANCER RES, 59(24), 1999, pp. 6214-6222
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.