neuromedin-n and Colonic-Neoplasms

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.

Topics: Adult; Base Sequence; Colonic Neoplasms; Consensus Sequence; Cytosine; DNA Methylation; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neurotensin; Peptide Fragments; Promoter Regions, Genetic; Transcription Factor AP-1; Tumor Cells, Cultured

The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.

Topics: Aspartic Acid Endopeptidases; Biomarkers, Tumor; Chromatography, High Pressure Liquid; Colonic Neoplasms; Gene Expression; Humans; Neurotensin; Peptide Fragments; Proprotein Convertase 2; Proprotein Convertase 5; Proprotein Convertases; Protein Precursors; Protein Processing, Post-Translational; Radioimmunoassay; RNA, Messenger; RNA, Neoplasm; Serine Endopeptidases; Subtilisins; Tumor Cells, Cultured

Expression of the gene encoding the neurotensin/neuromedin N (NT/N) is developmentally regulated in the gut in a distinctive temporal and spatial fashion. Src kinase, a nonreceptor tyrosine kinase, has been implicated in the growth and differentiation of various tissues; its role in gut differentiation is not known. The purpose of this study was to determine whether the Src signaling pathway plays a role in the activation of the human NT/N promoter.. Caco-2 cells, a human colon cancer cell line that can differentiate to a small bowel phenotype, were transiently transfected with human NT/N promoter fragments linked to luciferase and various amounts of Src expression plasmids or dominant negative Raf; luciferase and beta-galactosidase activities were measured after 48 hours.. Cotransfection of Src resulted in an approximate eightfold increase of NT/N promoter activity; mutation of a proximal activating protein-1/cyclic adenosine monophosphate responsive element site resulted in a dramatic decrease of Src-mediated NT/N induction. Cotransfection with a dominant negative Raf plasmid partially blocked Src-mediated NT/N activation.. Src increases NT/N promoter activity in Caco-2 cells acting, in part, through a proximal AP-1/CRE promoter element. In addition, Src regulation of the NT/N promoter appears to be mediated through a Raf-dependent pathway. We propose that Src may play a role in tissue-specific gene expression in the gut.

Topics: beta-Galactosidase; Colonic Neoplasms; CSK Tyrosine-Protein Kinase; Gene Expression Regulation; Humans; Luciferases; Neurotensin; Peptide Fragments; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras Proteins; Recombinant Fusion Proteins; src-Family Kinases; Transfection; Tumor Cells, Cultured

The stably differentiated human intestinal goblet cell line Cl.16E was used to study the effects of two structurally related regulatory peptides, neurotensin (NT) and neuromedin N (NN), on mucus secretion. NT and NN stimulated rapid release of mucins from filter-grown Cl.16E cells, this effect being dose related with a mean effective dose of 36 nM for NT and 422 nM for NN. The order of potency of NT, three NT fragments corresponding to the NH2-terminal part [NT-(1-11)] or to the COOH-terminal part [NT-(8-13) and NT-(9-13)], and NN in promoting mucin release and in inhibiting 125I-labeled NT binding to Cl.16E cell membranes was identical with NT greater than or equal to NT-(8-13) greater than NN greater than NT-(9-13) much greater than NT-(1-11) supporting the hypothesis that NT and NN stimulate mucin output through interaction with a common NT-preferring receptor. Scatchard analysis of equilibrium binding data showed one population of NT binding sites in Cl.16E cell membranes with the following characteristics: binding capacity (Bmax) was 141 fmol/mg of protein and dissociation constant (Kd) was 1.00 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Binding, Competitive; Calcium; Carbachol; Colon; Colonic Neoplasms; Cyclic AMP; Humans; Kinetics; Molecular Sequence Data; Mucins; Neurotensin; Peptide Fragments; Receptors, Neurotensin; Receptors, Neurotransmitter; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured; Vasoactive Intestinal Peptide