neuromedin-n and Pancreatic-Neoplasms

Brief phorbol ester treatment of BON cells results in a persistent release and cellular depletion of immunoreactive chromogranin A (CGA-IR) and neurotensin (NT-IR) cell contents. The purpose of the present study was to characterize the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on the secretion, biosynthesis, and steady-state messenger RNA (mRNA) levels of chromogranin A (CGA) and of a coresident peptide, neurotensin, by a novel human pancreatic carcinoid cell line, called BON. Acute TPA treatment (100 nM, 1 h) of BON cells resulted in 20- and 40-fold elevations in release of CGA-IR and NT-IR, respectively; and a 70-90% depletion of CGA-IR and NT-IR cell contents. TPA treatment also increased the biosynthetic rate of CGA-IR. Steady-state mRNA levels of CGA and NT/N (neurotensin/neuromedin N) were unchanged. Cell contents of CGA-IR and NT-IR were not replenished for a period of up to 6 days; secretion of CGA-IR and NT-IR persisted. In addition, BON cells failed to release CGA in response to stimulation by ionomycin and A23187 several days after acute TPA treatment. Our data indicate that the lack of replenishment of cell contents of CGA-IR and NT-IR is not due to decreases in steady-state CGA-IR and NT-IR mRNA levels, nor is it due to a decrease in biosynthesis of CGA-IR, but it is the result of a loss in the ability of TPA-treated BON cells to store and secrete CGA-IR and NT-IR in a regulated manner. These effects of TPA are mediated through the PKC pathway.

Topics: Analysis of Variance; Blotting, Northern; Calcimycin; Carcinoid Tumor; Cell Division; Cell Line; Chromogranin A; Chromogranins; Dose-Response Relationship, Drug; Gene Expression; Humans; Ionomycin; Kinetics; Methionine; Neurotensin; Pancreatic Hormones; Pancreatic Neoplasms; Peptide Fragments; RNA, Messenger; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.

Topics: Activating Transcription Factor 1; Activating Transcription Factor 2; Adenocarcinoma; Animals; Base Sequence; Binding, Competitive; Cyclic AMP Response Element-Binding Protein; DNA Mutational Analysis; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neurotensin; Pancreatic Neoplasms; Peptide Fragments; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Sequence Deletion; Tissue Distribution; Transcription Factors; Tumor Cells, Cultured

Antisera towards the bioactive peptides, neurotensin (NT, 13 residues) and neuromedin N (NMN, 6 residues), as well as towards three regions of their 147-residue canine precursor were used to identify and to quantitate precursor-derived peptides in extracts of human BON cells. This cell-line, which was obtained from a human pancreatic carcinoid tumor, constitutively expresses NT/NMN mRNA and secretes NT. Quantitation of seven precursor-derived peptides led us to conclude that BON cells display the intestinal pattern of NT/NMN precursor processing, which is primarily characterized by the production of a large molecular (125 amino acid) form of NMN. Four large molecular components, identified by immunochemical analyses and Western blotting, displayed physico-chemical properties which, for the most part, were consistent with the structures predicted from the partially-known human mRNA sequence. However, as shown previously for these peptides in canine gut, the empirically determined M(r) and pI values were slightly higher than those predicted solely from the amino acid content, perhaps due to the presence of additional substituents. These results suggest that BON cells may provide a good in vitro model in which to study the regulation of intestinal NT/NMN precursor processing and the nature of the enzyme(s) involved.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Carcinoid Tumor; Chromatography, High Pressure Liquid; Dogs; Gene Expression; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Molecular Sequence Data; Neurotensin; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Radioimmunoassay; RNA, Messenger; Tumor Cells, Cultured

Neurotensin (NT), an important regulatory hormone of the gut, stimulates growth of the human pancreatic cancer cell line MIA PaCa-2 in vitro. The purpose of our study was to compare the stimulatory effects of NT and neuromedin N (NMN), a structurally related hexapeptide, on the growth of MIA PaCa-2. In addition, the effects of NT on the growth of MIA PaCa-2 xenografts and normal GI tissues were assessed in athymic nude mice. MIA PaCa-2 cells, plated in serum-free media, were treated with either NT (10(-12)-10(-6) M) or NMN (10(-11)-10(-7) M) and cells were counted. For the in vivo study, MIA PaCa-2 cells were inoculated sc into 30 athymic nude mice and then randomized to two groups to receive either NT (600 micrograms kg-1, sc, tid) or vehicle. At sacrifice (day 35), the xenografted tumours, as well as normal host pancreas, jejunum and ileum were removed, weighed, and assayed for DNA, RNA and protein. Both NT and NMN stimulated the growth of MIA PaCa-2 cells in vitro with maximal (approximately 30%) increases occurring with dosages of 10(-9) M. In vivo, NT had a transient effect on xenografted MIA PaCa-2 tumour area with increases noted on days 21 and 25 of the study. Conversely, NT significantly stimulated the growth of jejunum and ileum, with a more pronounced effect noted in the jejunum. NT and NMN have similar growth-stimulatory effects on MIA PaCa-2 cells in vitro, which suggests an interaction through the same receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Division; DNA; Humans; Ileum; Jejunum; Mice; Mice, Inbred BALB C; Mice, Nude; Neurotensin; Organ Size; Pancreas; Pancreatic Neoplasms; Peptide Fragments; Reference Values; RNA; Tumor Cells, Cultured

An acid extract of a human neuroendocrine pancreatic adenoma was found to contain very high concentrations of immunoreactive neurotensin (iNT; approximately 130 mumol/L) as well as immunoreactive neuromedin-N (iNMN; approximately 40 mumol/L), portions of which (iNT, 0.2%; iNMN, 30%) were found in large molecular forms. Processing of the large forms could be mimicked by treatment with pepsin, which increased their immunoreactivity 15- to 20-fold (iNT) and 1- to 2-fold (iNMN), liberating a peptide similar to NMN and 2 fragments of NT [primary product, NT-(4-13)]. Biochemical characterizations using gel electrophoresis, isoelectric focusing, and high pressure liquid chromatography indicated that the large forms were highly basic (pI 8.5-9.5) proteins with a mol wt of about 20K (78% of the total), 45K (8%), and 60K (4%). The 20K protein contained iNT and iNMN in a 1:1 ratio, while a slightly smaller species contained only NMN. These results are in agreement with cDNA studies of canine intestinal mRNA, indicating the presence of a 170-amino acid precursor containing 1 copy each of NT and NMN. They further indicate that within this tumor differential processing of precursor occurred, resulting in a NT to NMN ratio of about 3:1, with additional NMN stored in large molecular forms.

Topics: Adenoma; Adult; Chromatography, Gel; Chromatography, High Pressure Liquid; Electrophoresis; Endocrine System Diseases; Humans; Male; Nervous System Diseases; Neurotensin; Pancreatic Neoplasms; Pepsin A; Peptide Fragments; Protein Precursors; Radioimmunoassay; Tissue Extracts