vasoactive-intestinal-peptide and Adenocarcinoma

HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenocarcinoma; Adenylyl Cyclases; Colonic Neoplasms; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Immunoassay; Membrane Lipids; Protein Kinases; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Recently, we have shown that the expression of receptors for vasoactive intestinal peptide (VIP) on intestinal adenocarcinomas can be used for in vivo targeting of primary or metastatic tumor sites using 123I-labeled VIP. Several other receptors and antigens including the TAG-72 protein have also been implemented for in vivo localization purposes. In this study, we have compared the in vitro and in vivo binding of 123I-VIP and of the 111In-labeled monoclonal antibody (MAb) directed against TAG-72 (OncoScint; 111In-CR-103) in patients with intestinal adenocarcinomas in a single-blinded, prospectively randomized trial.. Twenty patients were administered either 123I-VIP (150-200 MBq; 1 microgram) or 111In-CYT-103 (150 MBq; 1 mg) for one imaging study. After interim analysis demonstrated superior imaging with 123I-VIP, the next 10 patients (accounting for a total of 50 patients) enrolled in this trial underwent both studies in random order to allow for a direct comparison.. In total, 123I-VIP scans were true-positive in 28 of 30 patients (93%) versus 17 of 30 patients administered 111In-CYT-103 (56%). In the subgroup of 10 patients enrolled in the second part of the study, primary intestinal adenocarcinomas were imaged in five of five patients with 123I-VIP and in only two of these patients with 111In-CYT-103. Liver metastases were visualized in five of six patients by 123I-VIP receptor scanning and in four of these patients with 111In-CYT-103. The in vitro results indicated significant binding of 123I-VIP to primary colorectal tumors as well as to HT29 and COLO320 adenocarcinoma cells. In vitro, adenocarcinoma cells also expressed abundant numbers of the TAG-72 antigen.. Intestinal adenocarcinomas co-express VIP receptors and the IAG-72 antigen. Despite significant in vitro binding of both agents, however, the VIP receptor scan is more sensitive in localizing intestinal adenocarcinomas and metastatic spread.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Female; Gastrointestinal Neoplasms; Humans; Indium Radioisotopes; Iodine Radioisotopes; Liver Neoplasms; Male; Middle Aged; Oligopeptides; Pancreatic Neoplasms; Pentetic Acid; Prospective Studies; Radioimmunodetection; Receptors, Vasoactive Intestinal Peptide; Single-Blind Method; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Intestinal adenocarcinomas and various endocrine tumors express large numbers of high-affinity receptors for vasoactive intestinal peptide (VIP). We have evaluated the usefulness of scanning with VIP labeled with iodine-123 for tumor localization in patients with gastrointestinal tumors.. Radioiodinated VIP was purified by high-pressure liquid chromatography and administered as a single intravenous bolus injection (300 pmol [1 microgram]). Scanning with radiolabeled VIP was compared with computed tomography and scanning with somatostatin analogues in 79 patients with colorectal cancer, pancreatic carcinoma, gastric cancer, carcinoid tumor, or insulinoma.. Visualization of gastrointestinal tumors and metastases was obtained with radiolabeled VIP. Binding of the labeled peptide by primary tumors and metastases was visible shortly after the injection and was still demonstrable at 24 hours. In patients with colorectal adenocarcinomas, primary or recurrent tumors were visualized in 10 of 10, liver metastases in 15 of 18, lung metastases in 2 of 3, and lymph-node metastases in 4 of 4. Primary pancreatic adenocarcinomas were visualized by imaging in 10 of 12 patients, and liver metastases were seen in 7 of 7. Primary or recurrent gastric adenocarcinomas were visualized in 5 of 5 patients, and liver metastases were seen in 2 of 2 patients. VIP scans were positive in 9 of 10 patients with carcinoid tumors and in 4 of 4 patients with insulinomas. Some tumors with positive VIP scans were also visualized with somatostatin analogues (4 of 17 colorectal adenocarcinomas, 8 of 9 carcinoids, and 2 of 2 insulinomas). In vitro binding studies confirmed the presence of VIP receptors on gastrointestinal tumors.. Scanning with radiolabeled VIP can visualize intestinal tumors and metastases that express receptors for VIP.

Topics: Adenocarcinoma; Carcinoid Tumor; Colorectal Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Insulinoma; Iodine Radioisotopes; Male; Octreotide; Pancreatic Neoplasms; Radionuclide Imaging; Receptors, Somatostatin; Receptors, Vasoactive Intestinal Peptide; Stomach Neoplasms; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Carcinoma, Neuroendocrine; Cell Differentiation; Colonic Neoplasms; Fatal Outcome; Humans; Leukemia, Hairy Cell; Liver Neoplasms; Male; Middle Aged; Mutation; Proto-Oncogene Proteins B-raf; Vasoactive Intestinal Peptide; Vipoma

Vasoactive intestinal peptide (VIP) is one of the most plentiful neuropeptides in the lung and it has anti-inflammatory effects in the respiratory system. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) regulate immune responses to lipopolysaccharide (LPS). In the present study, we tested the expressions of TREM-1 and TREM-2 in various pulmonary cell lines and/or tissue using an animal model of LPS-induced acute lung injury (ALI), and determined the effects of VIP on expression of the TREM-1 and TREM-2 in lung tissues and cells from ALI mice. We found 1) expression of the TREM-1 mRNA from lung tissues of ALI was significantly increased, whereas the expression of TREM-2 mRNA was decreased in these tissues; 2) TREM-1 mRNA was only expressed in macrophages, while TREM-2 mRNA was detected in HBECs, lung fibroblasts, lung adenocarcinoma cells and macrophages; 3) the ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced lung tissues and macrophages; 4) VIP inhibited expression of the TREM-1 mRNA in a time- and dose-dependent manner in lung cells from LPS-induced ALI mice; however, it increased expression of the TREM-2 mRNA. As a result of these effects, VIP normalized the ratio of TREM-1 to TREM-2 mRNA in these cells. Our results suggest that VIP might exert its anti-inflammatory effect through a mechanism involved in regulation of expression of the TREM-1 and TREM-2 in LPS-induced ALI.

Topics: Acute Lung Injury; Adenocarcinoma; Adenocarcinoma of Lung; Animals; Anti-Inflammatory Agents; Cell Line, Tumor; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Inflammation; Lipopolysaccharides; Lung; Lung Neoplasms; Male; Membrane Glycoproteins; Mice; Myeloid Cells; Neoplasms; Neuroprotective Agents; Receptors, Immunologic; RNA, Messenger; Triggering Receptor Expressed on Myeloid Cells-1; Vasoactive Intestinal Peptide

To investigate the expression of vasoactive intestinal peptide (VIP) in gastric adenocarcinoma, and to evaluate the correlation of VIP level with clinical pathologic parameters.. The level of VIP in sera from gastric adenocarcinoma patients and healthy people was investigated by ELISA. Moreover, the differential gene expression between gastric adenocarcinoma, gastric dysplasia, and the corresponding normal gastric mucosa were determined by RT-PCR. Western Blot was also used to measure the expression of VIP in the gastric adenocarcinoma and the normal gastric mucosa.. The serum level of VIP was (5.794 +/- 0.014) ng/ ml in normal control and was (14.437 +/- 0.825) ng/ml in gastric adenocarcinoma patients, showing significant difference (P < 0.05). Meanwhile,the V/B of gastric adenocarcinoma tissues was greater than that of gastric dysplasia and the corresponding normal gastric mucosa (P <0.01), the values of V/B were 1.5261 +/- 0.3028, 0.9334 +/- 0.2872,and 0.9051 +/- 0.2794, respectively. The values of V/B between normal gastric mucosa and gastric dysplasia were not different significantly (P > 0.05). There were significantly negative correlation between the VIP mRNA expression of the differentiation degree of tumor (P < 0.05). The VIP mRNA expression was higher in gastric adenocarcinoma with lymph node metastasis than that without lymph node matastsis (P < 0.05). The VIP protein expression of the gastric adenocarcinoma tissues was greater than that of normal control.. This findings provide a direct evidence to support the possibility that VIP play a cofactor role in the pathogenesis of gastric adenocarcinoma.

Topics: Adenocarcinoma; Gastric Mucosa; Gene Expression; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Vasoactive Intestinal Peptide

Among U.S. men, prostate cancer (PC) accounts for 29% of all newly diagnosed cancers. A reliable scintigraphic agent to image PC and its metastatic or recurrent lesions and to determine the effectiveness of its treatment will contribute to the management of this disease. All PC overexpresses VPAC1 receptors. This investigation evaluated a probe specific for a (64)Cu-labeled receptor for PET imaging of experimental human PC in athymic nude mice and spontaneously grown PC in transgenic mice.. The probe, TP3939, was synthesized, purified, and labeled with (64)Cu and (99m)Tc. Using a muscle relaxivity assay, biologic activity was assessed and inhibitory concentrations of 50% calculated. Receptor affinity (Kd) for human PC3 cells was determined using (99m)Tc-TP3939 and (64)CuCl(2.) Blood clearance and in vivo stability were studied. After intravenous administration of either (64)Cu-TP3939 or (64)CuCl(2) in PC3 xenografts and in transgenic mice, PET/CT images were acquired. Prostate histology served as the gold standard. Organ distribution studies (percentage injected dose per gram [%ID/g]) in normal prostate were performed. The ratios of tumor to muscle, tumor to blood, normal prostate to muscle, and tumor to normal prostate were determined.. Chemical and radiochemical purities of TP3939 were 96.8% and 98% +/- 2%, respectively. Inhibitory concentrations of 50% and affinity constants were 4.4 x 10(-8) M and 0.77 x 10(-9) M, respectively, for TP3939 and 9.1 x 10(-8) M and 15 x 10(-9) M, respectively, for vasoactive intestinal peptide 28. Binding of (64)CuCl(2) to PC3 was nonspecific. Blood clearance was rapid. In vivo transchelation of (64)Cu-TP3939 to plasma proteins was less than 15%. (64)Cu-TP3939 uptake in PC was 7.48 +/- 3.63 %ID/g at 4 h and 5.78 +/- 0.66 %ID/g at 24 h after injection and was significantly (P < 0.05) greater than with (64)CuCl(2) (4.79 +/- 0.34 %ID/g and 4.03 +/- 0.83 %ID/g at 4 and 24 h, respectively). The ratios of PC to normal prostate at 4 and 24 h were 4 and 2.7, respectively. (64)Cu-TP3939 distinctly imaged histologic grade IV prostate intraepithelial neoplasia in transgenic mice, but (18)F-FDG and CT did not.. Data indicate that TP3939, with its uncompromised biologic activity, delineated xenografts and cases of occult PC that were not detectable with (18)F-FDG. (64)Cu-TP3939 is a promising probe for PET imaging of PC. It may also be useful for localizing recurrent lesions and for determining the effectiveness of its treatment.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Copper Radioisotopes; Humans; In Vitro Techniques; Male; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Transplantation; Organotechnetium Compounds; Positron-Emission Tomography; Prostatic Neoplasms; Radiopharmaceuticals; Receptors, Vasoactive Intestinal Polypeptide, Type I; Tissue Distribution; Transplantation, Heterologous; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a gastrointestinal hormone in the secretin-VIP family. It has been reported that VIP affects some tumor growth, and there is a VIP autocrine regulation in some cancers. However, the effect of VIP on gastric adenocarcinoma is not clear yet. The aim of the present study was to investigate the effect of VIP on gastric adenocarcinoma, especially autocrine regulation of VIP on gastric adenocarcinoma.. VIP mRNA and protein, and its receptor mRNA (VIPR(1) and VIPR(2)) were measured in 15 normal antrum mucosa, 20 gastric adenocarcinoma tissues, and the SGC7901 gastric adenocarcinoma cell line by using reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, or radioimmunoassay methods. The effect of the VIP protein and its antagonist (D-p-Cl-Phe6, Leu17)-VIP on SGC7901 cell growth was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method. The expressions of c-myc mRNA and ornithine decarboxylase (ODC) mRNA in SGC7901 cells before and after the incubated VIP protein and/or its antagonist were also measured by RT-PCR method.. The VIP mRNA expression in gastric adenocarcinoma tissues was significantly higher than that in normal antrum mucosa (P < 0.01). The VIP-positive immunoreactivity cells existed in 40% of gastric adenocarcinoma tissues, but not in normal tissues (P < 0.01). The VIP-positive immunoreactivity nerve fibers were observed in normal tissues, but not in adenocarcinoma tissues (P < 0.01). The expression rate of VIPR(1) mRNA in adenocarcinoma tissues was significantly lower than that in normal tissues, but that of VIPR(2) mRNA in the two kinds of tissues were similar (P > 0.05). In addition, the expression quantity of VIPR(1) mRNA and VIPR(2) mRNA in adenocarcinoma tissues was significantly lower than that in normal tissues (P < 0.05). SGC7901 cells expressed not only VIP mRNA and the VIP protein, but also VIPR(1) and VIPR(2) mRNA. 10(6) SGC7901 cells secreted 13.15 +/- 8.54 pg VIP on average. VIP did not affect the proliferation of SGC7901 cells, but the antagonist stimulated the proliferation of SGC7901 cells from 10(-5) to 10(-8) mol/L concentration incubated for 24-96 h. VIP downregulated the expressions of c-myc and ODC mRNA, but its antagonist upregulated their expressions.. The expression of VIP mRNA upregulates, but the expressions of VIPR mRNA downregulates in gastric adenocarcinoma tissues. The gastric adenocarcinoma tissues contain endocrine cells to secrete VIP, which show malignant specialities. The VIP autocrine regulation exists in SGC7901 cells, and potentially inhibits the proliferation of the cells by downregulating the expressions of c-myc and ODC mRNA. It suggests that VIP may play an important role in the regulation of the growth of gastric cancer cells.

Topics: Adenocarcinoma; Adolescent; Adult; Cell Line, Tumor; Cell Proliferation; Female; Humans; Immunohistochemistry; Male; Middle Aged; Ornithine Decarboxylase; Proto-Oncogene Proteins c-myb; Receptors, Vasoactive Intestinal Polypeptide, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Vasoactive Intestinal Peptide

A 15-mer phosphorothioate antisense oligonucleotide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was labeled with 131I or 125I and the labelled compound was linked to the vasoactive intestinal peptide (VIP) to be bound covalently to a polylysine chain so as to deliver oligonucleotide into tumor cells. The effect of the VIP as carrier on cell uptake of ASON in tissue culture was evaluated in a human colon adenocarcinoma HT29 cell line. The efficacy of VIP-131-ASON on cell growth was evaluated using the MTT assay. Expression of c-myc-encoded protein was measured by flow cytometry. Sense and nosense control Oligonucleotides with VIP carrier were used as control. The results showed that VIP competed effectively with VIP-125I-ASON to bind the HT29 cells. Cell uptake was increased 3-4 fold using the VIP carrier compared to the same dosage of naked DNA. HT29 cells treated with VIP-131I-ASON complexes exhibited 4-fold lower proliferation than those treated with 13I-ASON and 6-fold lower proliferation than those treated with radioiodinated Sense and nosense DNA. Cancer protein expression of HT29 cells treated with VIP-131I-ASON was decreased 2-fold compare with that in 131I-ASON treated cell. The use of a VIP carrier greatly increased 131I-ASON cellular uptake and inhibition of cell proliferation and C-myc cancer protein expressing in HT29 cell by radioiodinated antisense Oligonucleotides.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Carriers; Humans; Iodine Radioisotopes; Isotope Labeling; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-myc; Vasoactive Intestinal Peptide

The aim of this study was to investigate the ultrastructural appearance of mirror biopsies from primary colorectal adenocarcinomas combined with serotonin, vasoactive intestinal polypeptide, bombesin, somatostatin and glucagon expression in order to clarify the histology and immunology of these tissues compared with colorectal adenocarcinomas. The investigation was carried out in 90 cases of mirror biopsies by electron microscope immunocytochemistry. The ultrastructural study revealed that some cases (30%) of mirror biopsies were characterized by the presence of intracellular changes compared to normal tissues. The most significant of them were the presence of swollen abnormal mitochondria and the increased number of the secretory cells. The above enzymic and secretory changes were confirmed by electron immunogold results. Bombesin and VIP appeared to be located in enterochromaffin-like (ECL) endocrine cells primarily responsible for the production of serotonin. Somatostatin was located in D cells while we found numerous glucagon (EG) immunoreactive cells. In conclusion, low somatostatin expression, high glucagon and serotonin expression and the ectopic secretion of VIP and bombesin neuropeptides in mirror biopsies indicate the preneoplastic nature of these tissues and may be useful for the optimal treatment of patients with colorectal adenocarcinoma.

Topics: Adenocarcinoma; Biopsy; Bombesin; Cell Differentiation; Colorectal Neoplasms; Glucagon; Humans; Immunohistochemistry; Microscopy, Electron; Microscopy, Electron, Transmission; Neuropeptides; Serotonin; Somatostatin; Vasoactive Intestinal Peptide

The enteric nerve plexus in the colon was investigated in rats with chemically induced colonic adenocarcinoma. Tissue specimens from the colons of four group rats, namely controls, treated animals without development of colonic macro- or microscopic changes, rats with dysplasia and lymphoid hyperplasia, and rats with colonic adenocarcinoma were studied using immunocytochemistry, and quantified by computerized image analysis. No morphometeric changes were found in the treated rats regarding the myenteric and submucosal ganglia, with the exception of nitric oxide synthase (NOS), where the number of nerve cell bodies/ganglia was reduced in the myenteric ganglia in rats with both lymphoid hyperplasia and dysplasia, and carcinoma. The relative volume density of protein gene product (PGP) 9.5-immunoreactive (IR) nerve fibres was higher in the muscularis propria in rats with lymphoid hyperplasia and dysplasia, and carcinoma. However the relative volume density of PGP 9.5-IR nerve fibres was higher in the submucosa in rats with carcinoma only. The relative volume density of substance P- and VIP-IR nerve fibres was significantly higher in the muscularis propria in rats with colonic carcinoma. The relative volume density of NOS-IR nerve fibres was significantly decreased in both muscularis propria and submucosa in rats with lymphoid hyperplasia and dysplasia, and carcinoma. These findings imply that regulatory signals of the enteric innervation may be involved in the pathogenesis of colorectal cancer.

Topics: Adenocarcinoma; Animals; Colon; Colonic Neoplasms; Enteric Nervous System; Immunohistochemistry; Male; Nerve Fibers; Nerve Tissue Proteins; Nitric Oxide; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Substance P; Thiolester Hydrolases; Ubiquitin Thiolesterase; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is considered to influence cellular proliferation through its action on adenylate cyclase. This study examined VIP in the tumor-neighboring mucosa (TM) and remote normal mucosa (RM) in patients with colorectal carcinoma, and explored its relationship to tumor stage.. Immunohistochemical staining of VIP, using the avidinbiotin peroxidase complex technique, was performed on TM and RM from 55 patients, surgically resected colorectal carcinomas. The VIP immunoreactivity in the lamina propria (LP) of TM and RM was semiquantitatively graded, according to the density of VIP immunoreactive fibrous strands, and correlated with clinical characteristics, pathological findings, and tumor stage.. VIP immunoreactivity in the LP of TM and RM was found mainly as fibrous strands, some of which were nerve fibers. A few pericryptal myofibroblasts also showed VIP immunoreactivity. The VIP immunoreactivity in the LP was significantly greater in TM than in RM. The VIP immunoreactivity in the LP of TM was marginally greater in lesions with distant metastasis. The VIP immunoreactivity in the LP of RM was significantly greater in lesions with deeper wall penetration, in those with lymph node metastasis, and in those at more advanced stages.. These results suggest a possible trophic role of VIP in the progression of colorectal carcinoma, or enhanced VIP secretion secondary to or in parallel with the progression of carcinoma.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Colorectal Neoplasms; Disease Progression; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Neoplasm Staging; Observer Variation; Vasoactive Intestinal Peptide

1. Three human adenocarcinoma cell lines, Colony-24 (Col-24), Col-6 and Col-1 have been studied as confluent epithelial layers able to transport ions vectorially in response to basolateral vasoactive intestinal polypeptide (VIP) and pancreatic polypeptides (PP). 2. Different species PP stimulated responses in Col-24 with Y(4)-like pharmacology. Bovine (b)PP, human (h)PP and porcine (p)PP were equipotent (EC(50) values 3.0--5.0 nM) while rat (r)PP, avian (a)PP and [Leu(31), Pro(34)]PYY (Pro(34)PYY) were significantly less potent. PYY was inactive. The PP pharmacology in Col-1 was comparable with Col-24. However, Col-6 cells were different; pPP had an EC(50) intermediate (22.0 nM) between that of bPP (3.0 nM) and hPP (173.2 nM), with aPP and rPP being at least a further fold less potent. 3. Deamidation of Tyr(36) in bPP (by O-methylation or hydroxylation) or removal of the residue resulted in significant loss of activity in Col-24. 4. GR231118 (1 microM) had no PP-like effects. In Col-24 and Col-1, GR231118 significantly attenuated bPP (30 nM) or hPP (100 nM) responses, but it did not alter bPP responses in Col-6. BIBP3226 and GR231118 both inhibited Y(1)-mediated responses which were only present in Col-6. 5. RT--PCR analysis confirmed the presence of hY(4) receptor mRNA in Col-24 and Col-1 epithelia but a barely visible hY(4) product was observed in Col-6 and we suggest that an atypical Y(4) receptor is expressed in this cell line.

Topics: Adenocarcinoma; Arginine; Colonic Neoplasms; Humans; Neuropeptide Y; Pancreatic Polypeptide; Peptides, Cyclic; Receptors, Neuropeptide Y; Receptors, Somatostatin; Reverse Transcriptase Polymerase Chain Reaction; RNA; Somatostatin; Structure-Activity Relationship; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Human adenocarcinomas of the gastroenteropancreatic system overexpress vasoactive intestinal peptide (VIP) receptors and therefore represent logical diagnostic targets for receptor scintigraphy. Using iodine-123 labelled VIP, the newly employed diagnostic procedure termed VIP receptor scintigraphy (VIP-RS) appears to detect tumour tissue, especially pancreatic metastatic tumours, in almost all cases. So far, however, only a single centre has demonstrated convincing positive results. The aim of this study was to compare the sensitivity and specificity of VIP-RS with those of computer tomography (CT) and transabdominal ultrasound in patients with extensive pancreatic metastatic adenocarcinomas and neuroendocrine tumours. VIP was radiolabelled with carrier-free 123I using the chloramine T-method and preparative high-performance liquid chromatography for purification. Patients with metastatic pancreatic (n=12) and colorectal (n=3) carcinomas (adenocarcinoma: n=13, neuroendocrine tumour: n=2) were studied by VIP-RS, CT, ultrasound and, in one case, also by radioligand receptor autoradiography. Carrier-free radioiodinated VIP of maximum specific radioactivity maintained a high biological activity as determined by cAMP formation in receptor-expressing tumour cell lines. Intravenous injection of 123I-VIP did not cause any side-effects. Biodistribution, determined over 24 h, was high in the lungs and low in abdominal organs. Although all patients had extensive metastatic disease as evidenced by CT and ultrasound, VIP-RS was unable to detect either primaries or metastases in these patients. Only in two patients could a significant uptake of radiolabel be detected in organs directly infiltrated by the primary. To exclude false-negative findings, tumour tissue in one patient with a large primary, undetectable by VIP-RS, was analysed by radioligand receptor autoradiography and shown to be receptor positive. Moreover, in vitro receptor determinations showed that pancreatic carcinomas usually have fewer VIP receptors than the normal tissues to which they metastasize, like the liver. It is concluded that VIP can be radioactively labelled with maximum specific radioactivity while maintaining biological activity. Intravenous administration leads to a biodistribution almost identical to that reported previously. However, in contrast to these reports, very low sensitivity and specificity were observed for the detection of pancreatic cancers. In retrospect, these findings are

Topics: Adenocarcinoma; Adult; Aged; Colorectal Neoplasms; Female; Humans; Iodine Radioisotopes; Liver; Male; Middle Aged; Neuroendocrine Tumors; Pancreatic Neoplasms; Radionuclide Imaging; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) was modified at the C terminus with a spacer and four amino acids to serve as a chelating moiety. The modified peptide, TP 3654, was labeled with Tc-99m and evaluated in normal volunteers, as well as in patients with a history of cancer. Renal clearance (67%) was the primary route of excretion, with approximately 20% of the radioactivity clearing through the hepatobiliary system. No adverse reaction was noted in any of the subjects and all, except one small, of the known lesions as seen by CT, MRI, Tc-99m-MIBI, or mammography were correctly identified within a few minutes of an i.v. injection of approximately 10 mCi of Tc-99m-TP 3654 (specific activity 11.3 x 10(3) Ci/m mol). The scans were in concordance in nine patients. In the remaining two, one with a visible mass in the neck from high grade spindle cell sarcoma and the other with a palpable mass in a breast from ductal epithelial hyperplasia, were localized only with Tc-99m-TP 3654, but not with Tc-99m-MIBI. Both malignancies are known to express VIP receptors. The VIP analog promises to be a nontoxic and reliable agent for imaging cancers in humans that express VIP receptors.

Topics: Adenocarcinoma; Adult; Amino Acid Sequence; Autoradiography; Bone Neoplasms; Breast Neoplasms; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Neoplasms; Organotechnetium Compounds; Osteosarcoma; Radionuclide Imaging; Radiopharmaceuticals; Receptors, Vasoactive Intestinal Peptide; Technetium Tc 99m Sestamibi; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is known to modulate inflammatory reactions, to have trophic effects, and to contribute to diarrhea and has been implicated as an important factor in several inflammatory conditions in the human gut. The aim of the present study was to investigate the effects of irradiation on the expression of VIP in the colon of patients operated on for adenocarcinoma. Some of the patients had received preoperative irradiation (25 Gy) within one week before the operation. Specimens of sigmoideum, 10 cm cranial to the margin of the cancer, were examined, by using antiserum against VIP and immunohistochemistry. There were numerous nerve fibers showing VIP-like immunoreactivity in the damaged mucosa, including the regions showing ulcerations. There was a higher degree of expression of VIP in the ganglion cells in the submucous plexuses in irradiated than nonirradiated patients. The study shows that there is a marked immunohistochemical expression of VIP concomitant with the occurrence of inflammatory and repair processes in the irradiation-damaged human colonic mucosa.

Topics: Adenocarcinoma; Colon; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Vasoactive Intestinal Peptide

Human colonic adenocarcinoma cell lines have conserved several features of the native tissue. Among these is the expression of cell surface receptors for hormones and neurotransmitters that may be involved in the regulation of proliferation and differentiation processes in these cancer cells. Here, we confirm that high-affinity binding sites for the Vasoactive Intestinal Polypeptide (VIP) and for the VIP analogue Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP), were expressed in 4 human colonic adenocarcinoma cell lines, HT29, SW403, DLD-1 and Caco-2, that spontaneously displayed variable phenotypic properties in culture. We demonstrated that after long-term treatments, VIP and PACAP significantly reduced cell proliferation in the 4 cell lines and modulated intracellular cAMP and cGMP levels. Furthermore, conspicuous differences were observed from one cell type to another concerning expression of the receptor subsets or the effects of the neuropeptides on cell growth and on cyclic nucleotides production.

Topics: Adenocarcinoma; Caco-2 Cells; Cell Division; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Growth Inhibitors; HT29 Cells; Humans; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Recent data demonstrated a high sensitivity (>90%) in the visualization of primary/recurrent pancreatic cancer as well as metastases by means of 123I-labeled vasoactive intestinal peptide (VIP). The aim of this study was to investigate the diagnostic value of radioiodinated VIP in patients suffering from adenocarcinoma of the exocrine pancreas.. Sixty consecutive patients (26 women, 34 men; mean age 59 yr) with histologically verified pancreatic cancer were investigated in this study. Twenty-one patients presented with organ-confined malignancy (19 at study entry and 2 during follow-up after initial surgery developed tumor recurrence), while 25 patients had distant metastases along with the local malignancy, and 7 patients had liver metastases after resection of the primary lesion (6 on study entry and 1 during follow-up showed tumor development). In 5 of these patients, abdominal lymph node metastases were present at the time of scanning. Of 10 patients, who had undergone potentially curative surgery for their cancer, 7 remained free of disease during follow-up until death or for at least 6 mo. Iodine-123-VIP (150-200 MBq; approximately 1 microg VIP) was administered to all patients. Scintigraphic results were evaluated as compared to conventional radiologic imaging methods and surgical exploration.. Primary pancreatic tumors were visualized by 123I-VIP in 19/21 patients (90%) with disease confined to the pancreas and in 8/25 patients (32%) suffering both from locoregional and disease metastatic to the liver. The overall 123I-VIP scan sensitivity for primary pancreatic adenocarcinomas was 58% (27/46 scans). Liver metastases were imaged in 29/32 patients (scan sensitivity 90%) and abdominal lymph node metastases in 4/5 patients. In 5 patients, the VIP receptor scan indicated the malignant lesion before CT. In vitro results confirmed specific binding of 123I-VIP to primary pancreatic tumor cells as well as to PANC1 adenocarcinoma cells.. Iodine-123-VIP receptor scanning has the potential to offer additional information to augment diagnostic standard methods and could influence the decision-making process in the treatment of pancreatic cancer.

Topics: Adenocarcinoma; Female; Humans; Iodine Radioisotopes; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Radionuclide Imaging; Receptors, Vasoactive Intestinal Peptide; Sensitivity and Specificity; Vasoactive Intestinal Peptide

The effects of a number of agonists which inhibit intestinal chloride secretion were investigated in Colony-1 (Col-1) cells, a subpopulation derived from the HCA-7 human adenocarcinoma cell line. Neither peptide YY (PYY) or somatostatin 14-28 (SRIF) reduced short-circuit current (SCC) in Col-1 epithelial layers stimulated with vasoactive intestinal polypeptide (VIP), suggesting that their respective receptors are either absent in this cell line, or are not functionally coupled. A second member of the neuropeptide Y family, pancreatic polypeptide (PP), decreased VIP-elevated SCC with an EC50 of 25.6 nM. Maximal PP responses were unaffected by prior addition of PYY, indicating that Col-1 cells may express a PP specific, Y4-like receptor. The alpha 2-adrenoceptor agonist clonidine also attenuated VIP-stimulated SCC (EC50342 nM) through the alpha 2A receptor subtype, since clonidine responses were inhibited by yohimbine and rauwolscine but not altered by previous addition of prazosin. Col-1 cells responded to both apical and basolateral addition of VIP or clonidine; to an extent, this lack of sidedness reflects the ability of drugs to permeate through the Col-1 epithelial layers. Both PP and clonidine also inhibited SCC in unstimulated Col-1 cells or those pretreated with 3-isobutyl-1-methylaxanthine (IBMX) or a submaximal concentration of forskolin, agents which both directly elevate intracellular cAMP. After a maximal concentration of forskolin (10 microM), which increased SCC to a significantly greater extent than either VIP or IBMX, the effects of both agonists were negligible. The absence of PP and clonidine responses under these conditions may have implications for the mechanisms by which these agonists inhibit, chloride secretion in Col-1 epithelia. In addition carbachol reduced SCC stimulated by 10 microM forskolin, in contrast to control carbachol responses which consisted of a rapid decrease followed by a transient elevation in SCC; this observation suggests that Col-1 cells may also be a useful model for studying the interactions between Ca(2+)- and cAMP-dependent mechanisms involved in epithelial ion transport.

Topics: Adenocarcinoma; Adrenergic alpha-Agonists; Chlorides; Clonidine; Colonic Neoplasms; Cyclic AMP; Depression, Chemical; Epithelium; Humans; Pancreatic Polypeptide; Patch-Clamp Techniques; Receptors, Adrenergic, alpha-2; Receptors, Gastrointestinal Hormone; Somatostatin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) appears to be responsible for atropine-resistant, neurally mediated pancreatic ductal bicarbonate secretion and plays a role in both stimulation and inhibition of neoplastic growth in other organs. cDNAs encoding high affinity VIP-1 and VIP-2 receptors have been cloned, and these receptors may be differentiated based on the ability of VIP-1, but not VIP-2, receptors to couple to adenylyl cyclase in response to stimulation with micromolar concentrations of secretin. Recent data from our laboratory suggest expression of a low affinity secretin receptor in seven cell lines derived from human ductal pancreatic adenocarcinomas. In combination with the recent use of (123)I-labeled VIP to successfully image pancreatic adenocarcinomas in humans and the high affinity binding of both VIP and pituitary adenylate cyclase-activating peptides to sections from human pancreatic tumors, these findings suggest that VIP-1 receptors may be expressed on the majority of neoplastic pancreatic duct epithelial cells in vivo. To initially test the hypothesis that expression of VIP-1 receptors plays an important role in the pathophysiology of human ductal pancreatic adenocarcinomas, we used reverse transcription-PCR with Southern blot hybridization to confirm expression of VIP-1 and VIP-2 receptor mRNA in the vast majority of 28 human ductal pancreatic adenocarcinomas. Based on the cellular heterogeneity of these tumors, we also assessed VIP receptor subtype expression in seven well-characterized, secretin-responsive cell lines derived from human ductal pancreatic adenocarcinomas. Only VIP-1 receptor mRNA was detected in all seven secretin-responsive cell lines. A half-maximal increase in intracellular cyclic AMP was obtained with 0.5-5 nM VIP in each of these cell lines, consistent with expression of high affinity VIP receptors. The ability of 1 microM, but not 1 nM, secretin to stimulate intracellular cyclic AMP generation in these cells was consistent with VIP-1 receptor expression. Interestingly, 100 pM, but not 1 microM, VIP stimulated significant growth of VIP-1 receptor-bearing Capan-2 cells both in the absence and presence of serum. Because VIP-1 receptors appear to be expressed in the majority of neoplastic pancreatic duct cell lines and VIP stimulates growth of VIP-1 receptor-bearing Capan-2 cells in vitro, this peptide may well play an important role in the pathophysiology of tumors expressing these receptors in vivo.

Topics: Adenocarcinoma; Blotting, Southern; Cell Division; Cyclic AMP; Female; Humans; Male; Neoplasm Proteins; Pancreas; Pancreatic Neoplasms; Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; RNA, Messenger; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We investigated the effect of neuropeptides, which are vasoactive intestinal polypeptide (VIP), substance P, (SP), neuropeptide Y (NPY), neurokinin A (NKA), somatostatin (SOM), calcitonin gene-related peptide (CGRP), and leucine-enkephalin (L-ENK), on the invasion of murine Colon 26-L5 adenocarcinoma cells through a reconstituted basement membrane (Matrigel) using a Transwell cell culture chamber assay. VIP, SP, NPY, and L-ENK reduced invasive potential of tumor cells in a concentration-dependent manner, whereas SOM, CGRP, and NKA had no effect. Especially, VIP showed the most effective in inhibiting tumor invasion, and achieved 50% reduction at 10(-6) M. A similar effect by VIP was also observed in cell migration to fibronectin. VIP had no effect on the growth of tumor cells at the concentrations ranging from 10(-10) to 10(-6) M. The suppressed ability of the tumor cell motility by VIP (10(-6) M) was practically recovered by co-treatment with 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor. These results indicate that VIP, among the neuropeptides used, could inhibit Matrigel invasion of Colon 26-L5 carcinoma cells through partial suppression of their motility, and the reduction was associated with an intracellular cAMP-mediated pathway.

Topics: Adenocarcinoma; Animals; Biocompatible Materials; Calcitonin Gene-Related Peptide; Cell Division; Cell Movement; Colforsin; Collagen; Colonic Neoplasms; Dideoxyadenosine; Dose-Response Relationship, Drug; Drug Combinations; Enkephalins; Laminin; Leucine; Mice; Neoplasm Invasiveness; Neurokinin A; Neuropeptide Y; Neuropeptides; Proteoglycans; Somatostatin; Substance P; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

To clarify whether advanced colorectal carcinomas possess an amphocrine nature and produce both pancreatic and gut neurohormonal polypeptide and epithelial mucin in comparison with surrounding colorectal mucosa.. Retrospective analysis of paraffin-embedded specimens from 100 cases of colorectal carcinoma (39 colonic and 61 rectal) and surrounding mucosa, with histochemical and immunohistochemical studies.. The immunoreactivity of the carcinomas and surrounding mucosa by the labeled streptavidin-biotin complex method for polyclonal rabbit antibody against human vasoactive intestinal polypeptide, pancreatic polypeptide, and somatostatin was 61%, 59%, and 82% respectively; that of mucosa neighboring immunoreactive tumors was 87%, 85%, and 90%; and that of mucosa neighboring nonimmunoreactive tumors was 67%, 63% and 61%. Double staining for different types of neurohormonal polypeptide revealed that most carcinoma cells had a multiendocrine nature, and a number of neurohormonal polypeptide--positive and epithelial mucin-positive carcinoma cells (amphocrine cells) were found in almost every histological type of carcinoma by double staining for immunoreactivity and periodic acid-Schiff reaction or mucicarmin.. Colorectal carcinomas exhibit not only multiendocrine characteristics, producing various types of neurohormonal polypeptide, but also amphocrine characteristics of different grades, and most tumor-neighboring crypt cells possess those characteristics, too. We concluded that these characteristics of colorectal carcinomas may be related to their origin as multipotential endodermal stem cells.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Pancreas; Pancreatic Polypeptide; Retrospective Studies; Somatostatin; Vasoactive Intestinal Peptide

The response of confluent monolayers of normal and cystic fibrosis (CF) pancreatic epithelial cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin secretion. Mucin secretion was measured as release of M1 antigens by a direct sandwich enzyme immunoassay. Extracellular ATP provoked rapid (< or = 15 min) and strong mucin secretion (+ 480 +/- 35%) by the normal pancreatic cell lines but was not able to induce mucin secretion by the CF cell lines. The order of efficacy of nucleotide agonists with ATP > ADP > AMP > adenosine was that of typical P2-purinergic receptors. ATP induced a rapid and transient intracellular [Ca2+] mobilization in both normal and CF pancreatic epithelial cells. This work demonstrated that CFTR seemed to mediate ATP-dependent mucin secretion.

Topics: Adenocarcinoma; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Adult; Antigens; Calcium; Carbachol; Cell Line; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Humans; Immunoassay; Mucins; Pancreatic Neoplasms; Receptors, Purinergic P2; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The growth of pancreatic cancers may be influenced by certain gut peptides. However, the alteration of gut peptide receptors in the progress of pancreatic carcinogenesis is largely unknown. With storage phosphor autoradiography, this study visualized and characterized receptors for cholecystokinin (CCK), somatostatin (SST), bombesin (BBS), secretin and vasoactive intestinal peptide (VIP) in pancreata of control hamsters (n = 7) and pancreatic preneoplastic lesions (n = 10) or adenocarcinomas (n = 10) of N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters. The specific CCK-A and secretin receptors expressed in normal pancreata were markedly reduced in pancreatic preneoplastic lesions and absent in adenocarcinomas. In the development of pancreatic tumours, the subgroup of SST receptors did not change, but both the affinity and binding capacity declined. In comparison with the binding of VIP to normal pancreata, specific VIP binding was significantly lower in preneoplastic lesions and almost absent in pancreatic adenocarcinomas. No specific binding for BBS was detected in normal pancreas or (pre)neoplastic lesions of hamster pancreas. The reduction or absence of receptors for CCK, secretin, SST and VIP in hamster pancreas with the progress of carcinogenesis suggests that in BOP-treated hamsters, pancreatic adenocarcinomas have, to a large extent, lost the hormone-dependent characteristics of the original tissue.

Topics: Adenocarcinoma; Animals; Carcinogens; Cholecystokinin; Cricetinae; Mesocricetus; Nitrosamines; Pancreas; Pancreatic Neoplasms; Precancerous Conditions; Receptors, Cholecystokinin; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Receptors, Somatostatin; Receptors, Vasoactive Intestinal Peptide; Secretin; Somatostatin; Vasoactive Intestinal Peptide

Recent data suggest that functional receptors (R) for vasoactive intestinal peptide (VIP) are expressed on various tumor cells. The high-level expression of VIPR on tumor cells provided the basis for the successful use of 123I-labeled VIP for the in vivo localization of intestinal adenocarcinomas and endocrine tumors. We here report an update of our imaging results using 123I-VIP (150-200 MBg/1 microgram/patient) in 169 patients. In patients with pancreatic adenocarcinomas without liver metastases, the primary/recurrent tumor was visualized in 16 of 18 patients (89%) and liver metastases were imaged in 15 of 16 patients. In 11 of 12 patients with colorectal adenocarcinomas, the primary/recurrent tumor (92%) was imaged by 123I-VIP. Also, in 21 of 25 patients, liver metastases (84%); in 3 of 6 patients, lung metastases (50%); and in 4 of 5 patients, lymph-node metastases (80%) were imaged by 123I-VIP. In 10 of 10 patients with gastric adenocarcinomas, the primary/recurrent tumor; in 3 of 4 patients, liver metastases; and in 2 of 2 patients, lymph-node metastases were visualized by 123I-VIP. 123I-VIP localized primary intestinal carcinoid tumors in 15 of 17 patients (88%) and 8 of 10 primary insulinomas (80%). We conclude that the 123I-VIPR scintigraphy localizes intestinal adenocarcinomas and endocrine tumors as well as metastatic tumor sites.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; COS Cells; Endocrine Gland Neoplasms; Gastrointestinal Neoplasms; Humans; Iodine Radioisotopes; Molecular Sequence Data; Radioligand Assay; Radionuclide Imaging; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

This study presents the biodistribution, safety and absorbed dose of 123I-VIP administered to 18 patients with intestinal adenocarcinomas or endocrine tumors.. To achieve high-specific activity, 123I-VIP was purified by HPLC. Following intravenous administration of 123I-VIP (172 +/- 17 MBq (4.65 +/- 0.5 mCi); < 300 pmole ( < 1 microgram)/patient), sequential images were recorded during the initial 30 min. Thereafter, whole-body images were acquired in anterior and posterior views at various time points. Dosimetry calculations were performed on the basis of gamma camera data, urine, feces and blood activities.. After injection of labeled peptide, the lung was the primary site of 123I-VIP uptake. Peak lung activity represented 40% +/- 7% of the injected dose at 0.7 hr and declined to 21% +/- 7% at 3.5, to 14% +/- 3% at 7 and to 8% +/- 4% 22 hr postinjection. Radioactivity was excreted into the urine and amounted to 37% +/- 16% of the injected dose within 4, 68% +/- 12% within 8, 82% +/- 16% within 16 and 93% +/- 8% within 24 hr postinjection. The mean effective half-life of 123I-VIP in the lungs was 2.2 and 6 hr in the urinary bladder. The highest radiation absorbed doses were calculated for the lungs [67 muGy/MBq (248 mrad/mCi)], urinary bladder [77 muGy/MBq (284 mrad/mCi)] and thyroid gland [104 muGy/MBq (386 mrad/mCi)]. The effective dose was 28 muSv/MBq (104 mrem/mCi).. HPLC-purified 123I-VIP shows favorable dosimetry and is a safe and promising peptide tracer for localization of tumors expressing receptors for VIP.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Endocrine Gland Neoplasms; Female; Humans; Intestinal Neoplasms; Iodine Radioisotopes; Male; Middle Aged; Radiation Dosage; Receptors, Vasoactive Intestinal Peptide; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Vasoactive Intestinal Peptide

Authors report a case of Verner-Morrison syndrome which occurred in a 75-year-old man. The syndrome was caused by a pancreas VIP-oma with the histological structure of adenocarcinoma. Treatment with somatostatin analogous (octreotid) was effective, but the outcome was lethal due to subsequent pulmonary embolism.

Topics: Adenocarcinoma; Aged; Antineoplastic Agents, Hormonal; Fatal Outcome; Humans; Male; Octreotide; Pancreatic Neoplasms; Syndrome; Vasoactive Intestinal Peptide; Vipoma

The effects of combined administration of vasoactive intestinal peptide (VIP) and the ornithine decarboxylase (ODC) inhibitor, 1,3-diaminopropane (DAP), on development of colon tumors induced by azoxymethane (AOM), on ODC activity of the colon wall, and on the labelling index of colon epithelial cells were investigated in inbred Wistar rats. Rats received weekly subcutaneous injections of AOM for 10 weeks and subcutaneous injections of VIP every other day and drinking water containing DAP (2.5 milligrams) ad libitum until the end of the experiment at week 45. Administration of VIP significantly increased the incidence of colon tumors at week 45. It also resulted in significant increases in colon ODC activity and in the labelling index during administration of AOM, but not after its cessation. Administration of both DAP and VIP significantly reduced the enhanced colon carcinogenesis by VIP. The DAP significantly attenuated the VIP enhancement of colon ODC activity and of the labelling index during AOM administration. These findings indicate that ODC inhibition attenuated enhancement of colon carcinogenesis, and suggest that enhancement of colon carcinogenesis by VIP may be mediated through its polyamine biosynthesis.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Adenoma; Animals; Azoxymethane; Colonic Neoplasms; Diamines; Male; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Random Allocation; Rats; Rats, Wistar; Vasoactive Intestinal Peptide

1. Confluent epithelial layers of a human adenocarcinoma cell line called Colony-6 have been shown to respond to nanomolar concentrations of vasoactive intestinal polypeptide (VIP), peptide YY (PYY), neuropeptide Y (NPY) and somatostatin (Som). 2. The VIP-induced increase in basal short-circuit current (SCC) was attenuated by basolateral application of Som, PYY or NPY, and also by the Y1-receptor agonist [Leu31,Pro34]NPY, as well as pancreatic polypeptide (PP). High concentrations (0.1-3.0 microM) of NPY(2-36) were effective but the C-terminal fragment NPY(13-36) (0.1-1.0 microM) and desamidoNPY (0.6 microM) were not active. A rank order of agonist EC50 values was: PYY > NPY > [Leu31,Pro34]NPY > PP > NPY(2-36) >> NPY (13-36). 3. Receptors for all these peptides were preferentially located within the basolateral domain. Apical addition of PP (1 microM) and Som (100 nM) had no effect upon basal SCC while apical VIP (10 nM) responses were 18%, and apical PYY (100 nM) were 27% the size of respective basolateral controls (100%). 4. Cross-desensitization was observed between [Leu31,Pro34]NPY (1 microM) and both PYY (100 nM) and PP (1 microM) and between PYY and NPY(2-36) (1 microM), but was not significant between PYY (100 nM) and PP (1 microM). We suggest that either these cells express a single new Y-receptor with an unusual phenotype or that two Y-receptor populations exist in Colony-6 cells.

Topics: Adenocarcinoma; Colon; Colonic Neoplasms; Epithelium; Humans; Neuropeptide Y; Pancreatic Polypeptide; Peptide YY; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Neuropeptide Y; Secretory Rate; Sensitivity and Specificity; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

This study has examined the involvement of the Ca(2+)-signalling pathway in the regulation of agonist-stimulated cAMP responses in the human colonic adenocarcinoma cell line, HT29-cl.19A. The muscarinic agonist, carbachol (CCh) stimualted rapid increases in cellular IP3 and cytosolic Ca2+, [Ca2+]i in HT29-cl.19A cells. These were accompanied by a small but significant increase in basal cAMP levels and a marked (3-4-fold) potentiation of both forskolin- (FSK) and VIP-stimulated cAMP generation. Similar effects were observed with two other Ca(2+)-mobilising agonists, neurotensin and ATP. The failure of CCh to elicit potentiation of adenylate cyclase in broken cell preparations indicated an indirect action. Potentiation could be mimicked by the calcium ionophore, ionomycin, and thapsigargin and inhibited 70-90% by depleting intracellular Ca2+ stores suggesting that a rise in [Ca2+]i is the primary mediator of this response. In contrast, increasing [Ca2+]i levels to > 500 nM caused a significant inhibition of FSK-stimulated cAMP generation. The involvement of protein kinase C (PKC) was also assessed. PKC activators phorbol 12,13 dibutyrate (PDB) and 1-oleoyl-2-acetyl glycerol (OAG) potentiated FSK-stimulated cAMP production by 50-70% though PDB markedly inhibited the cAMP response to the receptor-mediated cAMP agonist, VIP. Neither effect could be elicited by the inactive phorbol ester, 4 alpha-phorbol, 12,13 didecanoate (PDD). PKC inhibitors staurosporine and H7 reduced by approximately 25% the CCh-induced potentiation of FSK-stimulated cAMP generation. In conclusion, these results suggest that stimulation of the phosphoinositidase C pathway in HT29-cl.19A colonocytes induces a 'sensitisation' of the adenylate cyclase system resulting in a dramatic amplification of agonist-stimulated cAMP generation. Increases in [Ca2+]i appear to be an important mediator of potentiation though activation of PKC may also play a significant role.

Topics: Adenocarcinoma; Calcium; Calmodulin; Carbachol; Colforsin; Colonic Neoplasms; Cyclic AMP; Drug Synergism; Humans; Phosphoric Diester Hydrolases; Protein Kinase C; Signal Transduction; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Xanthines

Hydrogen peroxide (H2O2) is a reactive oxygen species that can be produced in the digestive tract by inflammatory cells or during reperfusion following ischemia. To evaluate a possible direct effect of H2O2 on epithelial secretory cells, well-differentiated colonic T84 cells were grown to confluence on permeable membranes and studied in Ussing chambers. In this model, where the measured short-circuit current (Isc) reflects electrogenic secretion, we observed that H2O2 stimulated a concentration-dependent and transient secretory response: 5.5 mM H2O2 produced a peak Isc of 12.4 microA/cm2 after 4 min, 2.2 mM H2O2 a peak Isc of 7.9 microA/cm2 after 4 min, and 1.1 mM H2O2 a peak Isc of 5.5 microA/cm2 after 16 min (N = 5). When 97 experiments using 5.5 mM H2O2 were reviewed, the mean peak Isc response was 8.9 +/- 0.5 microA/cm2. A similar secretory response was elicited whether H2O2 was added to the serosal, to the mucosal, or simultaneously to both sides of the T84 cell monolayer. This secretory response reflected transcellular chloride secretion because it was inhibited by the depletion of chloride in the medium and by the suppression of the Na+,K+,2Cl- co-transporter activity necessary for the chloride gradient driving chloride secretion. When T84 cell monolayer resistance was studied, 5.5 mM H2O2 produced a transient decrease in resistance, reflecting transcellular chloride secretion, and a gradual decline in resistance (75% of the initial value after 55 min). The secretory response to H2O2 was increased 2-fold in T84 cells maximally stimulated with 10 nM vasoactive intestinal peptide (VIP), a neuropeptide which acts via cAMP, demonstrating synergism between the two agents. In contrast, the secretory responses produced by H2O2 and carbachol, which acts through the Ca2+ pathway, were additive. A late inhibitory effect of H2O2 was also observed: in cells previously treated with 5.5 mM H2O2, the subsequent secretory responses to either VIP or carbachol were partially inhibited. These secretory effects were specific for the oxidant properties of H2O2 because they were inhibited by 450 U/mL catalase and by 5 mM dithiothreitol, but were unaffected by 50 microM deferoxamine B or Fe3+. H2O2 may be a potential modulator of intestinal or colonic secretion in certain pathologic conditions such as inflammation or ischemia-reperfusion.

Topics: Adenocarcinoma; Carbachol; Catalase; Chlorides; Colonic Neoplasms; Deferoxamine; Dithiothreitol; Electrochemistry; Humans; Hydrogen Peroxide; Iron; L-Lactate Dehydrogenase; Trypan Blue; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; Cell Line; Colonic Neoplasms; Cyclic AMP; Dose-Response Relationship, Drug; Eflornithine; Humans; Kinetics; Ornithine Decarboxylase; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; RNA, Messenger; RNA, Neoplasm; Vasoactive Intestinal Peptide

Fifty exocrine pancreatic adenocarcinomas and 57 benign tumors induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine (BOP) were examined for the presence of argyrophil cells antiinsulin, -glucagon, -somatostatin, -pancreatic polypeptide (PP), -gastrin/CCK, -vasoactive intestinal polypeptide (VIP), and - neuron-specific enolase (NSE) reactive cells. Argyrophil - and antihormone-reactive cells were found in the normal pancreatic ducts and in the acini, as well as in hyperplastic and atypical ducts/ductules, tubular complexes, benign lesions, and in 80% of ductal adenocarcinomas. Insulin and antiNSE-reactive cells were the most common, followed in decreasing frequency by glucagon, somatostatin, and PP cells. Antigastrin-/CCK-and -VIP-reactive cells were found in two cases. Argyrophil cells were present in about 60% of the tumors with Grimelius staining and in 55% of those with Churukian-Schenk staining. Insulin cells were seen in ductal cancer that had grown into a lymph node and in the lymph node metastases of another cancer. A novel finding was the presence of argyrophil and insulin cells within the lumen of some malignant glandular structures. Coexistence of several peptide cells was found in 52% of the cancers. The presence of argyrophil and hormone-producing cells in induced pancreatic ductal/ductular lesions further strengthens the existence of a close developmental relationship between exocrine and endocrine cells of the pancreas.

Topics: Adenocarcinoma; Animals; Cricetinae; Female; Gastrins; Glucagon; Immunohistochemistry; Insulin; Islets of Langerhans; Male; Mesocricetus; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatic Polypeptide; Phosphopyruvate Hydratase; Silver Staining; Somatostatin; Vasoactive Intestinal Peptide

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.

Topics: 1-Methyl-3-isobutylxanthine; Adenocarcinoma; Blood; Cell Division; Colforsin; Colonic Neoplasms; Cyclic AMP; Humans; Insulin; Interphase; Kinetics; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell ("CaCo") antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor responsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-alpha, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Bombesin; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Karyotyping; Male; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Receptors, Bombesin; Receptors, Neurotransmitter; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The HSG-PA human submandibular gland adenocarcinoma cell line has attracted attention recently as a potentially useful cell culture model for studies of salivary duct cell function and regulation. These cells possess a variety of morphological and biochemical markers found in salivary duct cells. Recently, muscarinic cholinergic receptors coupled to inositol intracellular Ca2+ mobilization (He et al., Eur. J. Physiol., 413 (1989) 505-510) and K+ fluxes (Ship et al., Am. J. Physiol., 259 (1990) C340-C348) have been identified in HSG-PA cells. In this study, we report the presence in these cells of functional receptors for two neuropeptides, vasoactive intestinal peptide (VIP) and neurotensin. Receptors for both peptides were labeled in intact cell radioligand binding studies and exhibited pharmacological profiles similar to receptors found in other tissues. There was close agreement between binding Ki values and the ED50 values for stimulation of second messenger production and modulation of K+ efflux, with all values between 1 and 5 nM. Whereas neurotensin stimulated K+ efflux dramatically, VIP alone had no effect but enhanced the response to neurotensin. These studies thus represent the initial documentation of functional receptors for VIP and neurotensin in a cell line of salivary duct cell origin.

Topics: Adenocarcinoma; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; Humans; Neurotensin; Potassium; Receptors, Gastrointestinal Hormone; Receptors, Neurotensin; Receptors, Neurotransmitter; Receptors, Vasoactive Intestinal Peptide; Submandibular Gland Neoplasms; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) stimulated the growth of murine Lewis lung carcinoma cells in culture. The growth promoting effect was dependent on the concentration of VIP. Exposure to VIP for 12 hours followed by removal of the peptide resulted in sustained growth promotion for 4 to 5 days in culture. Synthetic fragments of VIP, i.e., VIP (1-16) and VIP (22-28), and the unrelated peptide neurotensin failed to stimulate the growth of the Lewis lung carcinoma cells. The growth-promoting effect of VIP was also observed in a murine mammary tumor cell line and a human lung adenocarcinoma cell line.

Topics: Adenocarcinoma; Animals; Cell Division; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neurotensin; Peptide Fragments; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP). Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance. In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells. The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells. Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP. After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Topics: Adenocarcinoma; Amylases; Bucladesine; Cytoskeleton; Fluorescent Antibody Technique; Humans; Intermediate Filaments; Parotid Neoplasms; Phenotype; Time Factors; Tumor Cells, Cultured; Tumor Stem Cell Assay; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Adrenocorticotropic Hormone; Biomarkers, Tumor; Bombesin; Gastrins; Glucagon; Hormones; Humans; Immunophenotyping; Lung Neoplasms; Male; Middle Aged; Somatostatin; Vasoactive Intestinal Peptide

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Barium; Barium Compounds; Calcimycin; Cell Line; Chlorides; Colonic Neoplasms; Furosemide; Humans; Kinetics; Mucins; Ouabain; Potassium Channels; Quinine; Stilbenes; Tetraethylammonium; Tetraethylammonium Compounds; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.

Topics: Adenocarcinoma; Calcimycin; Carbachol; Cell Line; Cholera Toxin; Colonic Neoplasms; Cystic Fibrosis; Histamine; Humans; Immunodiffusion; Immunoenzyme Techniques; Intestine, Small; Microscopy, Electron; Mucins; Prostaglandins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Cell Count; Cell Differentiation; Cell Division; Clone Cells; Colonic Neoplasms; Culture Media; Cyclic AMP; Epidermal Growth Factor; HLA Antigens; Humans; Suramin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effect of a synthetic analogue of CCK (Thr4,Nle7CCK-9) on growth of SW-1990 human pancreatic cancer was examined in two experimental models. Nude mice bearing SW-1990 pancreatic cancer xenografts were injected with CCK (5, 15, or 25 micrograms/kg) or vehicle twice daily for 20 days. Animals were then sacrificed and tumor volume, weight, protein, and deoxyribonucleic acid (DNA) content were evaluated. SW-1990 cells were grown in vitro and the effects of CCK, secretin, vasoactive intestinal peptide (VIP), and proglumide (a CCK-receptor antagonist) on cell number and DNA synthesis were determined. The highest dose of CCK, 25 micrograms/kg, significantly increased tumor weight, protein content, and DNA content (P less than 0.005). In vitro, CCK caused significant increases in cell counts of up to 47% at six days and 66% at 12 days compared to control. Graded concentrations of CCK had a biphasic effect on DNA synthesis with significant increases of up to 65% (P less than 0.005). CCK-induced cell proliferation was inhibited by proglumide. Secretin slightly increased cancer cell growth, although not as potently as CCK, VIP or secretin in combination with CCK did not show potentiation. These results indicate that growth of some human pancreatic cancers may be influenced by gastrointestinal peptides, of which CCK is the most potent.

Topics: Adenocarcinoma; Animals; Cholecystokinin; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Male; Mice; Mice, Nude; Organ Size; Pancreas; Pancreatic Neoplasms; Proglumide; Secretin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

It was found that repeated transplantation of ACATOL strain cells resulted in a loss of their sensitivity to growth-stimulating effect of pentagastrin. The effect of gastrin receptor antagonist proglumide, on ACATOL cells growth was inconstant. ACATOL tumor was sensitive to VIP and glucagon in vitro (as shown by adenylate cyclase activity assay), that makes the model promising for selecting potent hormone drugs against colorectal cancer.

Topics: Adenocarcinoma; Adenylyl Cyclases; Animals; Cell Line; Drug Resistance; Drug Screening Assays, Antitumor; Glucagon; Hormones; Intestinal Neoplasms; Intestine, Large; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Pentagastrin; Proglumide; Vasoactive Intestinal Peptide

Metabolic pathways of glucose utilization have been investigated in a human colon adenocarcinoma cell line (HT29) using carbon-13 Nuclear Magnetic Resonance spectroscopy. HT29 cells were adapted to grow on a polystyrene beaded microcarrier and were perfused when attached to the beads in a specially designed NMR cell. Abnormalities in carbohydrate metabolism already observed in several cancer cells were studied in HT29 cells fed with (1-13C)-enriched glucose. The cells were first perfused with a glucose-free medium for 2 h in order to deplete the intracellular store of glycogen, and they were subsequently perfused with a medium containing enriched glucose at an initial concentration of 5.5 mM. Sequential 13C-NMR spectra, recorded at 100.5 MHz (5 min accumulation), show that HT29 cells were able to utilize glucose through the glycolytic pathway while storing glucose as glycogen (glucose was utilized at a rate of 3.9 mumol/mg protein/hr). The glycolytic activity determined by the amount of lactic acid produced was 4.6 microns/mg protein/hr, corresponding to the formation of 1.2 lactic acid per glucose molecule. Glycogen accumulation corresponded to 16 micrograms/mg of protein. Treatment of HT29 with 10 nM vasoactive intestinal peptide (VIP) induced a transient decrease in the level of labelled glycogen to 50% of the initial value. Control level was recovered 12 min after VIP loading.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Glycogen; Glycolysis; Humans; Lactates; Magnetic Resonance Spectroscopy; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Regulation of active K+ influx and Na(+)-K(+)-Cl- cotransport activity in HT-29 cells by vasoactive intestinal peptide (VIP) was investigated. Both active K+ influx, defined as the ouabain-sensitive component, and Na(+)-K(+)-Cl- cotransport, defined as the ouabain-resistant bumetanide-sensitive component, of total K+ uptake were increased by VIP. VIP increased the maximum velocity (Vmax) values for both components with no change in apparent Michaelis constant (Km) values. Three lines of evidence support the role of adenosine 3',5'-cyclic monophosphate (cAMP) as a mediator of the VIP effects. 1) The rank order potencies of VIP and peptide histidineisoleucineamide (PHI) in binding and cAMP production (J. T. Turner, S. B. Jones, and D. B. Bylund, Peptides Fayetteville 7: 849, 1986) and K+ uptake were consistent; 2) alpha 2-adrenergic agonists inhibited both VIP-stimulated cAMP production (J. T. Turner, C. Ray-Prenger, and D. B. Bylund, Mol. Pharmacol. 28: 422, 1985) and K+ uptake; and 3) forskolin, but not dideoxyforskolin, mimicked the effects of VIP on K+ uptake. Because amiloride blocked the VIP-stimulated active K+ component, the VIP effects on active K+ influx may be secondary to a Na(+)-H+ antiporter-mediated increase in cellular Na+ content. Additional experiments indicated that pretreatment of cells with a protein kinase C activator, previously shown to decrease basal Na(+)-K(+)-Cl- cotransport activity and the apparent number of cotransporters in HT-29 cells (C. C. Franklin, J. T. Turner, and H. D. Kim, J. Biol. Chem. 264: 6667, 1989), did not change the magnitude of response of the remaining cotransporters after adenylate cyclase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenocarcinoma; Amiloride; Biological Transport; Bumetanide; Carrier Proteins; Cell Line; Chlorides; Colonic Neoplasms; Humans; Isoquinolines; Kinetics; Norepinephrine; Ouabain; Piperazines; Potassium; Protein Kinase C; Sodium-Potassium-Chloride Symporters; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Yohimbine

We present a case of secondary achalasia due to an adenocarcinoma of the stomach with no tumor infiltration of the esophagus. Immunohistochemical staining revealed a massive infiltration of activated eosinophils in the muscularis of the esophagus with secretion of the highly cytotoxic and neurotoxic eosinophil cationic protein (ECP). Immunohistochemical staining for the neuropeptides VIP and substance P, as well as the histochemical demonstration of AChE, revealed a nearly total absence of all three neurotransmitters/modulators compared to control. The hypothesis is advanced that eosinophil neurotoxicity is the cause of secondary achalasia.

Topics: Adenocarcinoma; Aged; Blood Proteins; Eosinophil Granule Proteins; Esophageal Achalasia; Esophagus; Humans; Immunohistochemistry; Male; Nerve Fibers; Ribonucleases; Stomach Neoplasms; Substance P; Vasoactive Intestinal Peptide

At the maximally effective concentration of 10 nM, VIP induced a marked (12.5-fold stimulation above basal), and sustained increase in short circuit current in the human intestinal epithelial cell line Cl.19A grown on permeable filters and placed in Ussing chambers. Half-maximal increase of Isc was observed for 0.1 nM VIP. This was well correlated with the VIP-stimulated adenylate cyclase activity (ED50:0.07 nM). Binding studies using 125I-VIP indicated that Cl.19A cells express a peptide-specific VIP receptor with a dissociation constant of 0.07 nM. Covalent labeling of receptors followed by SDS-PAGE analysis of membrane proteins resulted in the identification of a 63,000 dalton binding protein in Cl.19A cells.

Topics: Adenocarcinoma; Adenylyl Cyclases; Binding, Competitive; Colonic Neoplasms; Electric Conductivity; Epithelium; Humans; Intestines; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Preincubation with an alpha 2-adrenergic agonist sensitized subsequent forskolin- and vasoactive intestinal peptide-stimulated cyclic AMP production in HT29 cells, a human colonic adenocarcinoma cell line. Preincubation with somatostatin, another agonist negatively coupled to adenylate cyclase, sensitized forskolin-stimulated cyclic AMP production to a lesser extent. alpha 2-Adrenergic agonist preincubation also resulted in desensitization as indicated by a shift to the right in the dose-response curve of a subsequent challenge by an alpha 2-adrenergic agonist. In an effort to elucidate the mechanism for sensitization, we examined protein kinase C and the Na+/H+ antiporter. Whereas these components had marked effects on forskolin stimulation, there was no effect on sensitization. Changes in the concentration of extra-cellular Ca2+ or Mg2+ had no effect on either forskolin stimulation or sensitization. Pertussis toxin pretreatment caused a time-dependent decrease in sensitization, an attenuation of inhibition of cyclic AMP production, and a decrease in subsequent [32P]ADP-ribosylation by pertussis toxin. The time course for these three events was similar, implicating the inhibitory guanine nucleotide regulatory protein in the mechanism for alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production. In addition, pertussis toxin dramatically decreased forskolin-stimulated cyclic AMP production, although with a different time course. These results suggest that the mechanism of sensitization is via an as yet undefined sequence of biochemical events that includes the inhibitory guanine nucleotide regulatory protein, but does not include inhibition of adenylate cyclase nor activation of the Na+/H+ antiporter.

Topics: Adenocarcinoma; Adenylate Cyclase Toxin; Blood Platelets; Carrier Proteins; Cell Line; Colforsin; Colonic Neoplasms; Cyclic AMP; Cycloheximide; Enzyme Activation; Humans; Kinetics; Norepinephrine; Pertussis Toxin; Protein Kinase C; Receptors, Adrenergic, alpha; Sodium; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surface specific binding sites for the vasoactive intestinal peptide (VIP) (KD = 0.5 nM). Their molecular weight was previously estimated to 117 kDa and 64 kDa. This clone underwent functional and structural differentiation when grown in a glucose-free galactose-containing medium. The [125I]VIP binding capacity of cells grown in this medium gradually declined while the cell density increased and reached a value close to zero when cell monolayer was able to form hemicysts. At this time, cells presented numerous tight junctions and desmosomes and a well organized brush border. Binding capacity could be recovered when the post-confluent monolayers were previously disaggregated with EDTA. Neither the affinity for VIP nor the molecular weight of the [125I]VIP cross-linked polypeptides were modified in these cells compared to cells grown in glucose-containing medium. However, surface receptor number of differentiated cells was twice that of undifferentiated cells. Leakproof differentiated cell monolayers grown on permeable substratum produced cAMP in response to VIP only when the peptide was present in the lower chamber of the culture wells. Taking these data altogether, we conclude that the localization of functional VIP receptors is restricted to the basolateral domain in differentiated post-confluent HT29-D4 cells.

Topics: Adenocarcinoma; Adenylyl Cyclases; Cell Differentiation; Cell Line; Colonic Neoplasms; Humans; Microscopy, Electron; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Detergents; Humans; Kinetics; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Solubility; Vasoactive Intestinal Peptide

Recent investigations suggest that although thyroid-stimulating hormone (TSH) is the major regulator of thyroid gland function, it is not the only hormone that regulates the thyroid gland. Another factor regulating thyroid gland function is vasoactive intestinal polypeptide (VIP), which is present in nerves that innervate thyroid blood vessels and follicles. Previous studies have documented that VIP stimulates adenylate cyclase (AC) activity in cultured normal and hyperplastic (Graves) thyroid tissue, thus increasing intracellular cyclic AMP concentration. To our knowledge, however, there is no information concerning the effect of VIP on neoplastic thyroid tissue. Therefore we studied the effect of VIP on normal and neoplastic human thyroid tissue from 10 patients (follicular adenomas, 4; follicular carcinomas, 3; and papillary carcinomas, 3). Adenylate cyclase activity was measured in a 8000 g membrane preparation in the basal state and when incubated with VIP, TSH, and both. VIP increased AC activity in normal tissue and tumor in a dose-dependent manner, with maximal stimulation at 10(-6) mol/L (p less than 0.05). In normal tissue, 10(-6) mol/L VIP and a maximally stimulating concentration of TSH (300 mU/ml) stimulated AC to the same degree. In tumors, although both TSH and VIP increased AC activity, the response to TSH was greater (p less than 0.01). VIP and TSH had an additive effect in normal tissue (p less than 0.05), whereas in tumors the AC response to VIP and TSH was the same as with TSH alone (p = 0.66). This study documents that VIP is a potent stimulator of adenylate cyclase in both normal and neoplastic human thyroid tissues. The magnitude of this effect is similar to that produced by TSH, which implies that VIP plays a physiologically important role in the regulation of the secretion and growth of normal and neoplastic thyroid tissues.

Topics: Adenocarcinoma; Adenoma; Adenylyl Cyclases; Carcinoma, Papillary; Cyclic AMP; Humans; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Vasoactive Intestinal Peptide

We have previously shown that hamster H2T pancreatic ductal cancer has a receptor for vasoactive intestinal peptide (VIP) which is not present on a cell line of human pancreatic ductal cancer (MIA). The purpose of this study was to examine the effect of chronic administration of VIP on the growth of both H2T hamster pancreatic carcinoma and MIA human pancreatic carcinoma in vivo. The growth of H2T was studied in hamsters; a control group of six hamsters received 0.1% bovine serum albumin (BSA) in saline, and two treatment groups of six hamsters each received VIP (1 and 10 nmol/kg), all administered three times a day by i.p. injection for 35 days. Both doses of VIP inhibited the growth of H2T tumor (tumor area, weight, DNA, RNA, and protein content). The growth of MIA was studied in athymic Balb/c mice, one group of 10 received 0.1% BSA and the other 10 received VIP (1 nmol/kg), both three times a day by i.p. injection for 3 months. There was no difference in tumor growth rate between the two groups. Treatment with VIP did not have any effect on body weight or size of the normal pancreas in either the hamsters or the mice. We conclude that the differential response of hamster and human pancreatic cancer to VIP treatment may be due to the presence or absence of VIP receptors.

Topics: Adenocarcinoma; Animals; Body Weight; Cricetinae; Humans; Male; Mesocricetus; Mice; Mice, Inbred BALB C; Organ Size; Pancreatic Neoplasms; Species Specificity; Vasoactive Intestinal Peptide

A neoplastic epithelial cell line initially established in culture from a human parotid gland adenocarcinoma grown in athymic nude mice with a BALB/c genetic background, which has an ultrastructure similar to that of the intercalated duct cell of the salivary glands, was examined for the expression of amylase and vasoactive intestinal polypeptide (VIP). The cultured cells were found by the peroxidase-antiperoxidase (PAP) method to express amylase and ultrastructurally to have secretory granules showing positive immunoreaction with anti-amylase serum. In addition, the cells were found to secrete amylase into the culture medium. The expression of VIP in the cultured cells was observed by the PAP method, immunofluorescent staining method, or immunoelectron microscopy. Moreover, the presence of the polypeptide reactive to antibodies directed against VIP in the cultured cells was confirmed by immunoblotting and radioimmunoassay. These findings indicate that the cells proliferating in culture express both amylase and VIP.

Topics: Adenocarcinoma; Amylases; Animals; Female; Humans; Mice; Mice, Inbred BALB C; Molecular Weight; Parotid Neoplasms; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a neuropeptide with broad tissue distribution. Although its precise function is unknown, it is thought to exert its effect, at least in part, by interacting with cell surface receptors. Nuclear receptors for VIP have now been identified by specific binding of 125I-labeled VIP to nuclei of a human colonic adenocarcinoma cell line (HT29) and by cross-linking of 125I-labeled VIP to its receptor on intact nuclei. In contrast, 125I-labeled transferrin shows only background binding to nuclei but significant binding to intact cells. Purity of the isolated nuclei was further substantiated by electron microscopy. The apparent molecular sizes of the VIP--cross-linked nuclear and cell surface receptors are similar but not identical.

Topics: Adenocarcinoma; Cell Line; Cell Nucleus; Colonic Neoplasms; Humans; Kinetics; Microscopy, Electron; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected by PMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.

Topics: Adenocarcinoma; Adenylyl Cyclases; Binding Sites; Cell Line; Cell Membrane; Colonic Neoplasms; Cyclic AMP; Humans; Phorbol Esters; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.

Topics: Acetylglucosaminidase; Adenocarcinoma; Carrier Proteins; Cell Line; Chromatography, Affinity; Colonic Neoplasms; Glycoproteins; Humans; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Sialic Acids; Vasoactive Intestinal Peptide

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.

Topics: Adenocarcinoma; Binding Sites; Cell Line; Chymotrypsin; Colonic Neoplasms; Cycloheximide; Dose-Response Relationship, Drug; Humans; Kinetics; Methionine; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Humans; Hydrogen-Ion Concentration; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Temperature; Time Factors; Vasoactive Intestinal Peptide

The initial event of VIP action is its interaction with a specific receptor at the surface of a target cell. The understanding of the fine mechanism of action of VIP requires the characterization and of course the purification of the receptor. The understanding of the mechanism which regulates the number of receptor sites at the cell surface, if it occurs, is also a way to characterize the properties of VIP receptor. In this paper we report a number of data which represent several attempts to characterize VIP receptor in a human colonic adenocarcinoma cell (HT 29 cells). We have characterized a monoclonal antibody which partially inhibits 125I-VIP binding to HT 29 cells. We have specifically cross-linked, on intact HT 29 cells, a major polypeptide of Mr-64,000 with 125I-VIP using DTSP or DSS as cross-linking reagents. This polypeptide behaves like a high affinity binding site for VIP. We have demonstrated that VIP is rapidly internalized in HT 29 cells (in less than 10 minutes) and that simultaneously VIP receptors were no more detectable on the cell surface by cross-linking experiments. This suggests that VIP is internalized together with its receptor.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Binding Sites; Cell Line; Colonic Neoplasms; Cross-Linking Reagents; Humans; Immunochemistry; Molecular Weight; Peptides; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) was found to cause a dose-dependent decrease in fructose 2,6-bisphosphatase concomitant with an increase in cyclic AMP in cultured HT29 cancer cells from human colon. The maximum effect was a 41% decrease obtained with 10 nM-VIP, and half-maximum effect was obtained with 0.75 nM-VIP. The effect of 2.5 nM-VIP was almost totally counteracted (i.e. fructose 2,6-bisphosphate concentration was restored) by either adrenaline (1 microM) or the alpha 2-adrenergic agonist UK-14304 (1 microM); the alpha 2-agonist clonidine (1 microM) was less efficient, since the VIP effect was decreased by 72% only. The adrenaline effect was totally antagonized by 1 microM-yohimbine. It is concluded that, in the HT29 cancer cells, the fructose 2,6-bisphosphate-producing system is sensitive to variations of cyclic AMP concentration and is under the dual control of VIP and alpha 2-adrenergic receptors.

Topics: Adenocarcinoma; Brimonidine Tartrate; Cell Line; Clonidine; Colonic Neoplasms; Cyclic AMP; Epinephrine; Fructosediphosphates; Hexosediphosphates; Humans; Quinoxalines; Vasoactive Intestinal Peptide; Yohimbine

We have characterized the alpha 2-adrenergic receptor in membranes from the human colonic adenocarcinoma cell line, HT29, using the recently introduced alpha 2-agonist 5-bromo-6-[2-imidazolin-2-yl-amino]quinoxaline [( 3H]UK-14,304), two other radioligands, and a series of adrenergic agonists and antagonists. We also investigated alpha 2-agonist inhibition of HT29 cell adenylate cyclase and reversal of inhibition by alpha-adrenergic antagonists. [3H] Yohimbine saturation experiments indicated a single class of sites with a KD of 0.61 nM which agreed with the kinetically determined KD of 0.62 nM. Computer analysis of kinetic and saturation experiments with [3H]UK-14,304 revealed two classes of sites. From the saturation data, one site had high affinity for the radioligand (0.14 nM) and comprised 33% of the total number of sites, whereas the other site had lower affinity (6.1 nM). The total number of sites labeled by [3H]UK-14,304 (360 fmol/mg of protein) was approximately equal to the number of sites labeled by [3H]yohimbine (330 fmol/mg), whereas [3H]para-aminoclonidine labeled fewer sites of a single class. Rank order potencies of adrenergic agonists and antagonists obtained from competition binding assays indicated that: the same receptors were labeled by the three radioligands, and the receptors were of the alpha 2 subtype. UK-14,304 and epinephrine inhibited forskolin- and vasoactive intestinal peptide-stimulated adenylate cyclase in a dose-dependent manner up to 32%. Inhibition of the enzyme was reversed by yohimbine and, less potently, by phentolamine and prazosin in a dose-dependent manner. The HT29 cell line appears to be a useful model system for the investigation of the regulation and mechanism of action of alpha 2-adrenergic receptors in human tissues.

Topics: Adenocarcinoma; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Brimonidine Tartrate; Cell Line; Clonidine; Colforsin; Colonic Neoplasms; Dose-Response Relationship, Drug; Guanosine Triphosphate; Humans; Kinetics; Magnesium; Quinoxalines; Radioligand Assay; Receptors, Adrenergic, alpha; Vasoactive Intestinal Peptide; Yohimbine

In the present work, [3H]clonidine was used to characterize alpha 2-adrenoceptors on the human adenocarcinoma cell line HT 29. The effects of alpha 2-adrenergic stimulation on cellular cyclic AMP levels were also investigated. The binding of [3H]clonidine on HT 29 cell membrane preparations was rapid and reversible. Scatchard analysis of the saturation curves indicated the existence of a single class of non-interacting sites with a KD of 1.29 +/- 0.07 nM and a Bmax of 114 +/- 7 fmol/mg of cell membrane protein. The binding sites for [3H]clonidine showed the required specificity for alpha 2-adrenoceptors. The potencies of alpha-adrenergic compounds to displace [3H]clonidine binding ranked as follows: yohimbine greater than phentolamine much greater than prazosin for antagonists and clonidine greater than epinephrine greater than norepinephrine greater than phenylephrine much greater than amidephrine for agonists. When tested on intact cells, epinephrine, norepinephrine and clonidine were found to counteract, in a dose-dependent manner, the increase of cyclic AMP triggered by vasoactive intestinal peptide (VIP). Such inhibitory effects were abolished by the addition of yohimbine but not of prazosin. The physiological amines were the most efficient agonists: both epinephrine and norepinephrine inhibited VIP-induced cyclic AMP accumulation by 50-55% with KD values of 50 nM and 300 nM respectively. Clonidine was a partial agonist only, provoking a weak (25-30%) inhibition of VIP-induced cyclic AMP accumulation even at high concentrations. These results indicate that, like normal colocytes, human colon adenocarcinoma cells HT 29 possess alpha 2-adrenoceptors, the stimulation of which is associated with an inhibition of cyclic AMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Cell Line; Clonidine; Colonic Neoplasms; Cyclic AMP; Humans; Kinetics; Receptors, Adrenergic, alpha; Tritium; Vasoactive Intestinal Peptide

The human colon adenocarcinoma cell-line HT 29 has been shown to possess functional alpha 2-adrenergic receptors. Here, [3H] clonidine was used as radioligand to study the evolution of alpha 2-adrenergic receptivity during the time course of HT 29 cell culture. Scatchard analysis of the saturation curves indicates that the number of [3H]clonidine binding sites increases throughout the 17 day culture period. The maximal number of alpha 2-adrenoceptors is found during the stationary phase of growth, when cell density is high and mitotic rate low. Moreover, the use of adrenaline and clonidine as alpha 2-adrenergic agonists reveals a relationship between the number of receptors and the intensity of the biological effect associated with their stimulation (inhibition of the VIP-induced cyclic AMP accumulation).

Topics: Adenocarcinoma; Cell Line; Cell Membrane; Clonidine; Colonic Neoplasms; Cyclic AMP; Humans; Receptors, Adrenergic, alpha; Tritium; Vasoactive Intestinal Peptide

[125I]Monoiodinated vasoactive intestinal peptide (125I-VIP) was cross-linked with human colonic adenocarcinoma cells (HT29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross-linking reagent. The cross-linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A major polypeptide of Mr = 67 000 was characterized and it behaved like a high-affinity binding site for VIP according to the following data. The concentration of native VIP (0.5 nM) giving half-maximum inhibition of 125I-VIP covalent cross-linking with this polypeptide was very similar to that giving half-maximum displacement of 125I-VIP on HT 29 cells (0.6 nM). Glucagon or insulin was unable to inhibit the labelling of the Mr-67 000 component. In our experimental conditions neither specific 125I-VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr-67 000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor. Preincubation of HT 29 cells with native VIP at 37 degrees C, before 125I-VIP binding and subsequent cross-linking reaction, decreased the labelling of the Mr-67 000 polypeptide up to 80%. Assuming one molecule of 125I-VIP cross-linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr = 64 000, which belongs to the high-affinity VIP binding site of an intestinal human cell line.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Iodine Radioisotopes; Isotope Labeling; Protein Binding; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Growth Hormone-Releasing Hormone; Humans; Kinetics; Peptide PHI; Peptides; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide

Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.

Topics: Adenocarcinoma; Arginase; Cell Line; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cyclic AMP; Humans; Iodine Radioisotopes; Peptide Fragments; Radioisotope Dilution Technique; Structure-Activity Relationship; Trypsin; Vasoactive Intestinal Peptide

We used liposomes made with phospholipids of fatty acid chain length ranging from C12:0 to C16:0 to modify the cAMP dependent protein kinase (PK) activity of HT 29 cells induced by VIP or forskolin. Both VIP and forskolin effects were inhibited in dilauroylphosphatidylcholine (DLPC) treated cells. PK activity was slightly lowered when cells were treated by dimyristoylphosphatidylcholine (DMPC) liposomes. However neither VIP nor forskolin-induced PK activities were affected with dipalmitoylphosphatidylcholine (DPPC) liposomes. Furthermore, the binding of [125I]VIP to DLPC treated cells was drastically lowered whereas no change was observed when cells were incubated with DMPC or DPPC liposomes. On the other hand, the interaction of HT 29 cells with DLPC vesicles provoked a decrease in membrane cholesterol content with subsequent increase in membrane fluidity. These findings provide evidence that, in HT 29 cells, the mechanisms of VIP-receptor interaction and of adenylate cyclase activation is lipid dependent and is regulated by membrane fluidity.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Humans; Kinetics; Liposomes; Lysophosphatidylcholines; Membrane Fluidity; Phosphatidic Acids; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

The time course of internalization of radioiodinated vasoactive intestinal peptide (VIP) in HT29 cells was obtained using the technique of acetic acid removal of cell-surface-bound peptide. Even after 10 min incubation at 37 degrees C, 125I-VIP, initially bound on the HT29 cell surface, was compartmentalized within the cells. During the same time, degraded radioactive material was released by cells in the incubation medium. Localization of internalized 125I-VIP was investigated using two different subcellular fractionation techniques. 10 min after the onset of internalization, 125I-VIP labelling was found in intermediate structures and 10 min later the bulk of the radioactivity was detected in a low-density fraction containing very large lysosomes with a multivesicular aspect. The lysosomotropic agent NH4Cl appeared to inhibit 125I-VIP internalization, degradation and appearance of radiolabelled peptide in the large lysosomes in a time-dependent manner. Moreover, the effect of NH4Cl resulted in an accumulation of radioactive material in fractions containing microsomal structures. On the other hand, bacitracin, together with methylamine, highly enhanced 125I-VIP labelling in a membrane fraction, suggesting that these agents possibly act on a cell surface component of HT29 cells. These results support the conclusion that in HT29 cells, prelysosomal structures and large secondary lysosomes are probably part of the intracellular pathway of internalized VIP.

Topics: Adenocarcinoma; Ammonium Chloride; Bacitracin; Biological Transport; Cell Compartmentation; Cell Fractionation; Cell Line; Humans; Hydrogen-Ion Concentration; Lysosomes; Methylamines; Temperature; Time Factors; Vasoactive Intestinal Peptide

A total of 87 surgical cases of gastric carcinoma including 3 carcinoid tumors were investigated with the methods of silver reaction and immunoperoxidase stain for 8 different brain-gut hormones. Argyrophil (AP) cells were demonstrated in 38 cases (44%), argentaffin (AF) cells in 18 (21%) and endocrine cells in 13 (14%). The occurrence of endocrine cells had no relation with histological types. Glicentin cells were demonstrated in 10 cases, somatostatin in 7, motilin in 3, beta-endorphin in 2 and gastrin in one. Endocrine cells appeared generally in small numbers except one carcinoid tumor which had numerous somatostatin cells. No single cell positive for more than two kinds of hormones could be demonstrated. Two undifferentiated carcinomas looking like carcinoid tumors had argyrophil cells and endocrine cells of either somatostatin or beta-endorphin. These results suggest that carcinoid-like carcinoma or endocrine cell carcinoma may lie on the intermediate state between carcinoma and carcinoid tumor.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Adult; Carcinoid Tumor; Endorphins; Female; Gastrins; Gastrointestinal Hormones; Glucagon; Histocytochemistry; Humans; Male; Microscopy, Electron; Middle Aged; Motilin; Proglucagon; Protein Precursors; Somatostatin; Stomach Neoplasms; Vasoactive Intestinal Peptide; Vasopressins

A primary adenocarcinoma of the nasal cavity with light microscopic, electron microscopic, and immunocytochemical features of an enteric-type carcinoma is presented. The carcinoma contained a variety of dense-core granules similar to those seen in enterochromaffin cells of different functional types. Some granules demonstrated an immunoreactivity with serotonin, cholecystokinin, gastrin, somatostatin and leu-enkephalin antibodies. It is suggested that the endocrine cells in the neoplasm belong to the non-neuroectodermal paraneurone system.

Topics: Adenocarcinoma; Aged; Cholecystokinin; Chromaffin System; Enterochromaffin Cells; Humans; Immunoenzyme Techniques; Male; Microscopy, Electron; Nasal Cavity; Nose Neoplasms; Serotonin; Vasoactive Intestinal Peptide

Gut endocrine cells in a total of 122 intestinal-tract adenocarcinomas induced in inbred Wistar rats by 1,2-dimethylhydrazine dihydrochloride were examined histologically, ultrastructurally, and immunohistochemically for gastrin, somatostatin, vasoactive-intestinal polypeptide (VIP), and glicentin (enteroglucagon). Of the 122 tumors, argyrophil cells were detected in 42 tumors (34.3%) comprising 15 tumors of the well differentiated type and 27 tumors of the poorly differentiated type, including signet-ring-cell carcinomas. Of the 27 tumors of the poorly differentiated type, 12 were regarded as endocrine-cell carcinomas composed of numerous argyrophil or argentaffin cells and mucus-containing cells. Immunohistochemically, 7 of the 12 tumors had glicentin and two of these seven tumors also had gastrin and argentaffin cells synchronously. None of the tumors showed immunoreactivity for somatostatin and VIP. Nine of the 12 tumors metastasized to the lung, pancreas, liver, mesenterium, omentum, and lymph nodes. The metastatic foci of these tumors were also shown to have glicentin and argentaffin cells. Ultrastructurally, four types of endocrine granule were found in the tumor cells and amphicrine cells containing endocrine granules and mucous granules were noted. These endocrine-cell tumors were assumed to develop from totipotent immature cells of endodermal origin.

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Animals; Carcinogens; Chromaffin System; Dimethylhydrazines; Enterochromaffin Cells; Female; Gastrins; Glucagon-Like Peptides; Intestinal Neoplasms; Male; Microscopy, Electron; Neoplasm Metastasis; Neoplasms, Experimental; Rats; Rats, Inbred Strains; Somatostatin; Vasoactive Intestinal Peptide

The cell source of peptide hormone production and the morphological differentiation were investigated in 18 adenocarcinomas of the lung by immunohistochemistry and/or by electron microscopy. These tumors were found by radioimmunoassay of tumor extracts to contain either one or more of 7 peptide hormones, i.e. adrenocorticotropin (ACTH), beta- and gamma-melanocyte stimulating hormones (MSH), somatostatin (SS), vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) and calcitonin (CT). In a combined adeno- and small cell carcinoma, a considerable number of small tumor cells were positively stained for ACTH, beta- and gamma-MSHs and GRP. In a poorly differentiated adenocarcinoma with mucin and CT production, these products were localized in some single cells. Electron microscopy revealed secretory granules indistinguishable from exocrine or endocrine types. In another mucin-positive adenocarcinoma with high SS and CT contents, some tumor cells were stained for SS and/or CT. Two distinct exocrine and endocrine type secretory granules were found in the same cells. In tumors with 100 ng or less of the peptides/g tissue, most tumor cells were not stained for the peptides but a small number showed morphological endocrine differentiation. In conclusion, a considerable proportion of the adenocarcinomas of the lung may show heterogeneous differentiation in both endocrine and exocrine directions.

Topics: Adenocarcinoma; Adrenocorticotropic Hormone; Calcitonin; Cytoplasmic Granules; Gastrin-Releasing Peptide; Histocytochemistry; Hormones; Humans; Lung Neoplasms; Melanocyte-Stimulating Hormones; Microscopy, Electron; Peptide Biosynthesis; Somatostatin; Vasoactive Intestinal Peptide

It is known that the human exocrine pancreas responds to secretin stimulation more than does VIP, a structurally related peptide. We looked for the receptors for those polypeptides in a human pancreatic cancer cell line grown in culture and in nude mice. By analysing the cAMP responses and the 125I-VIP binding we found VIP receptors with a KD of 1.5 10(-9) M. Secretin stimulates the adenylate cyclase through the VIP receptor sites with a KD of 1.7. 10(-6) M. We noted also that during cell proliferation in culture there was about a 5 fold increase of the cAMP response to VIP.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Line; Cyclic AMP; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide

Hybridoma cells have been obtained by fusing RCY 3 Ag 1-2-3 rat myeloma cells with spleen cells from a rat hyperimmunized with human adenocarcinoma cells (HT 29 cell line) grown in serum-free medium. Immunoglobulins secreted by hybridomas were screened for: (i) specific binding to HT 29 cells; (ii) their ability to inhibit the binding of [125I]-vasoactive intestinal peptide (VIP) to HT 29 cells; (iii) their capacity to modulate the cAMP production induced by VIP. The monoclonal antibodies we have obtained from clones 109-10-16 and 109-10-19 compete for the binding of radiolabelled VIP to HT 29 cells and partially inhibit the production of cAMP induced by VIP while they are ineffective in reducing the intracellular level of cAMP attained after stimulation of HT 29 cells by isoproterenol. We never found antibodies which increase the cAMP level in HT 29 cells. The binding of the purified monoclonal antibody 109-10-16 Ig gamma 2c to HT 29 cells was visualized by indirect immunofluorescence and was not present at the surface of all cells. These observations strongly support the hypothesis that the monoclonal antibodies we have characterized interact with an antigenic determinant which belongs to the VIP receptor or at least to a cell surface component closely associated with the receptor.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Cell Line; Cyclic AMP; Humans; Hybridomas; Rats; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Casein Kinases; Cell Line; Colonic Neoplasms; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Gastrointestinal Hormones; Humans; Phosvitin; Protamine Kinase; Protein Kinases; Rectal Neoplasms; Subcellular Fractions; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E1 (PGE1) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10(-9) M VIP promotes a rapid and specific activation of the lower Km cyclic AMP phosphodiesterase (1.7-fold); at 25 degrees C the effect is maintained for more than 15 min, while at 37 degrees C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer (Ka equals 4 x 10(-10) M) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 x 10(-7) M secretin, 10(-5) M isoproterenol and 10(-5) M PGE1; PGE2 and epinephrine are without effect. In HRT 18 cells VIP is less active (Ka equals 2 x 10(-9) M) whereas 10(-6) M PGE1, 10(-6) M PGE2 and 10(-5) M epinephrine are potent inducers of th phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenocarcinoma; Animals; Bucladesine; Catecholamines; Cells, Cultured; Colonic Neoplasms; Cyclic AMP; Enzyme Activation; Gastrointestinal Hormones; Prostaglandins E; Protein Kinases; Rectal Neoplasms; Secretin; Vasoactive Intestinal Peptide

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Cyclic AMP; Gastrointestinal Hormones; Glycogen; Humans; In Vitro Techniques; Phosphorylase a; Vasoactive Intestinal Peptide

This study was undertaken to assess the role of vasoactive intestinal peptide (VIP) in the control of cyclic adenosine 3':5'-monophosphate production in colonic tumor cells. Seven human colorectal adenocarcinoma cell lines in culture were investigated (HT-29, HRT-18, SW-480, Caco-2, CO-115, CO-125, and HCT-8R). These cell-lines had a cyclic adenosine 3':5'-monophosphate production system which was very sensitive to VIP but less so to prostaglandin E1 and/or isoproterenol. Nonintestinal human malignant epithelial cells, such as HeLa (cervix) and Caki-1 and Caki-2 (kidney), by contrast, did not respond to VIP. The dose-response relationships of malignant colorectal cells were compared to those obtained with epithelial cells of normal human colon and showed that: (a) maximal responses were observed with 0.1 micro M VIP in both malignant and normal cells; (b) half-maximal responses were elicited by VIP concentrations in the 0.3 to 2 nM range in malignant cells (1.2 nM in normal cells), thus indicating the high apparent affinity of the cells to VIP; and (c) the magnitudes of the responses (stimulated:basal ratios) were highly variable in malignant cells, ranging from 225 in HT-29 cells to 3.5 in Caco-2 cells, but were more constant, in the order of 25, in normal cells. Secretin, a VIP agonist in intestinal tissue, stimulated cyclic adenosine 3':5'-monophosphate accumulation in all colorectal cells, but with a 1000- to 5000-fold lower potency than did VIP. These results show that the VIP-sensitive adenylate cyclase system operates in malignant as well as in normal colon epithelial cells.

Topics: Adenocarcinoma; Adenylyl Cyclases; Animals; Cell Line; Colonic Neoplasms; Cyclic AMP; Gastrointestinal Hormones; HeLa Cells; Humans; Isoproterenol; Mice; Neoplasms, Experimental; Prostaglandins E; Rectal Neoplasms; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide, secretin, catecholamines and prostaglandin E1 stimulate the accumulation of cyclic AMP in HT 29 cells (see Laburthe, M. et al. (1978) Proc. Natl. Acad. Sci. U.S. 75, 2772-2775). In the present work maximal activation of protein kinases has been obtained at similar or even lower concentrations of the effectors. Maximal stimulation also requires a phosphodiesterase inhibitor. Type I and type II cyclic AMP-dependent protein kinases from basal and stimulated cells have been characterized by DEAE-Sepharose chromatography. Further identidication of the kinase has been carried out by gel electrophoresis and assay of the enzymes in the gel slabs. Comparison of the radioautography patterns of high speed supernatant lysate from basal and stimulated cells shows: First, that one type I and two type II cyclic AMP-dependent protein kinases plus one or two major and two minor cyclic AMP-independent protein kinases are present in HT 29 cells. Second, that all three holoenzymes are fully dissociated upon maximal stimulation, while the activity of the independent kinases appears unchanged.

Topics: Adenocarcinoma; Cells, Cultured; Chromatography, Gel; Colonic Neoplasms; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epinephrine; Glucagon; Humans; Isoproterenol; Prostaglandins E; Protein Kinases; Secretin; Stimulation, Chemical; Theophylline; Vasoactive Intestinal Peptide

Evidence that VIP is the principal humoral mediator of the watery diarrhea syndrome includes: (a) actions of VIP in experimental anaimals parallel the clinical manifestations of the syndrome; (b) infusions of VIP induce watery diarrhea in intestinal loops of dogs and a picture resembling the clinical syndrome in pigs, at circulating levels of the peptide similar to those observed in human disease; (c) most patients with the watery diarrhea syndrome and underlying tumors have elevated plasma levels of VIP; (d) in those patients in whom pre- and postoperative measurements were made, plasma VIP levels fell to the normal range with removal of the tumor and relief of the diarrhea; and (e) extracts of such tumors are rich in VIP-immunoreactivity and VIP-like biologic activity. A few patients with the syndrome have been reported to have normal plasma VIP levels, and it is possible that other humoral agents (such as pancreatic polypeptide, prostaglandins) may contribute to the production of the diarrhea.

Topics: Adenocarcinoma; Adenoma; Adenoma, Islet Cell; Animals; Diarrhea; Dogs; Gastrointestinal Hormones; Humans; Neoplasms; Pancreatic Neoplasms; Swine; Syndrome; Vasoactive Intestinal Peptide

An adenocarcinoma of the second portion of the duodenum in a 26-year-old male is presented. The patient was suffering from pain in the epigastrium. Immunofluorescent studies revealed that it consisted almost exclusively of cells with a distincly positive somatostatin-like immunoreactivity. Ultrastructurally, the cytoplasm of the tumor cells had numerous large round granules (about 400 micrometers) with variable electron density. Most of these cells closely resembled the D cells normally seen in the duodenum and the islets of the pancreas, although a few argyrophil cells could be demonstrated by light microscopy. Radioimmunoassay of extracts of the tumor revealed a large amount of somatostatin (2260 pg/mg); substance P and VIP were detected also. Somatostatinoma has been known to occur in the pancreas, but this seems to be the first somatostatinoma found in the intestine.

Topics: Adenocarcinoma; Adult; Cytoplasmic Granules; Duodenal Neoplasms; Humans; Male; Microscopy, Electron; Somatostatin; Substance P; Vasoactive Intestinal Peptide

Human colonic adenocarcinoma plasma membranes exhibit specific receptors for VIP. Adenylate cyclase activity is stimulated by a VIP concentration as low as 10(-10) mol/1.

Topics: Adenocarcinoma; Adenylyl Cyclases; Cell Membrane; Colonic Neoplasms; Gastrointestinal Hormones; Humans; Receptors, Cell Surface; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3':5'-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3x10(-12) M. Maximum VIP-induced cAMP levels were observed with 10(-9) M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3x10(-10) M VIP. (125)I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10(-10) and 10(-7) M. Half-maximum inhibition of binding was observed with 2x10(-9) M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10(-7) M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E(1) and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10(-8) M; half-maximum stimulation was observed at about 10(-9) M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation.

Topics: Adenocarcinoma; Catecholamines; Cell Line; Colonic Neoplasms; Cyclic AMP; Dose-Response Relationship, Drug; Gastrointestinal Hormones; Hormones; Prostaglandins; Receptors, Cell Surface; Secretin; Vasoactive Intestinal Peptide