Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers.
The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.
Dong
Z
Z
Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555, USA.
Wang
X
X
Evers
B M
BM
D000328:Adult; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000096:biosynthesis*; D010446:Peptide Fragments / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D018808:Transcription Factor AP-1; C042090:neuromedin N; D009496:Neurotensin; D003596:Cytosine